Publications

2020
Kinoshita, Noriyuki, et al.Mechanical Stress Regulates Epithelial Tissue Integrity and Stiffness through the FGFR/Erk2 Signaling Pathway during Embryogenesis”. Cell Rep 30.11 (2020): , 30, 11, 3875-3888.e3. Web.Abstract
Physical forces generated by tissue-tissue interactions are a critical component of embryogenesis, aiding the formation of organs in a coordinated manner. In this study, using Xenopus laevis embryos and phosphoproteome analyses, we uncover the rapid activation of the mitogen-activated protein (MAP) kinase Erk2 upon stimulation with centrifugal, compression, or stretching force. We demonstrate that Erk2 induces the remodeling of cytoskeletal proteins, including F-actin, an embryonic cadherin C-cadherin, and the tight junction protein ZO-1. We show these force-dependent changes to be prerequisites for the enhancement of cellular junctions and tissue stiffening during early embryogenesis. Furthermore, Erk2 activation is FGFR1 dependent while not requiring fibroblast growth factor (FGF) ligands, suggesting that cell/tissue deformation triggers receptor activation in the absence of ligands. These findings establish previously unrecognized functions for mechanical forces in embryogenesis and reveal its underlying force-induced signaling pathways.
Greco, Todd M, Michelle A Kennedy, and Ileana M Cristea. “Proteomic Technologies for Deciphering Local and Global Protein Interactions”. Trends Biochem Sci 45.5 (2020): , 45, 5, 454-455. Web.
Hartenian, Ella, et al.RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters”. PLoS Pathog 16.2 (2020): , 16, 2, e1008269. Web.Abstract
In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometry-based global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss.
Hashimoto, Yutaka, et al.Temporal dynamics of protein complex formation and dissociation during human cytomegalovirus infection”. Nat Commun 11.1 (2020): , 11, 1, 806. Web.Abstract
The co-evolution and co-existence of viral pathogens with their hosts for millions of years is reflected in dynamic virus-host protein-protein interactions (PPIs) that are intrinsic to the spread of infections. Here, we investigate the system-wide dynamics of protein complexes throughout infection with the herpesvirus, human cytomegalovirus (HCMV). Integrating thermal shift assays and mass spectrometry quantification with virology and microscopy, we monitor the temporal formation and dissociation of hundreds of functional protein complexes and the dynamics of host-host, virus-host, and virus-virus PPIs. We establish pro-viral roles for cellular protein complexes and translocating proteins. We show the HCMV receptor integrin beta 1 dissociates from extracellular matrix proteins, becoming internalized with CD63, which is necessary for virus production. Moreover, this approach facilitates characterization of essential viral proteins, such as pUL52. This study of temporal protein complex dynamics provides insights into mechanisms of HCMV infection and a resource for biological and therapeutic studies.
2019
Bredrup, Cecilie, et al.A tyrosine kinase-activating variant Asn666Ser in PDGFRB causes a progeria-like condition in the severe end of Penttinen syndrome”. Eur J Hum Genet 27.4 (2019): , 27, 4, 574-581. Web.Abstract
Missense variants located to the "molecular brake" in the tyrosine kinase hinge region of platelet-derived growth factor receptor-β, encoded by PFGFRB, can cause Penttinen-type (Val665Ala) and Penttinen-like (Asn666His) premature ageing syndromes, as well as infantile myofibromatosis (Asn666Lys and Pro660Thr). We have found the same de novo PDGFRB c.1997A>G p.(Asn666Ser) variants in two patients with lipodystrophy, acro-osteolysis and severely reduced vision due to corneal neovascularisation, reminiscent of a severe form of Penttinen syndrome with more pronounced connective tissue destruction. In line with this phenotype, patient skin fibroblasts were prone to apoptosis. Both in patient fibroblasts and stably transduced HeLa and HEK293 cells, autophosphorylation of PDGFRβ was observed, as well as increased phosphorylation of downstream signalling proteins such as STAT1, PLCγ1, PTPN11/SHP2-Tyr580 and AKT. Phosphorylation of MAPK3 (ERK1) and PTPN11/SHP2-Tyr542 appeared unaffected. This suggests that this missense change not only weakens tyrosine kinase autoinhibition, but also influences substrate binding, as both PTPN11 tyrosines (Tyr542 and Tyr580) usually are phosphorylated upon PDGFR activation. Imatinib was a strong inhibitor of phosphorylation of all these targets, suggesting an option for precision medicine based treatment.
Federspiel, Joel D, et al.Conservation and divergence of protein pathways in the vertebrate heart”. PLoS Biol 17.9 (2019): , 17, 9, e3000437. Web.Abstract
Heart disease is the leading cause of death in the western world. Attaining a mechanistic understanding of human heart development and homeostasis and the molecular basis of associated disease states relies on the use of animal models. Here, we present the cardiac proteomes of 4 model vertebrates with dual circulatory systems: the pig (Sus scrofa), the mouse (Mus musculus), and 2 frogs (Xenopus laevis and Xenopus tropicalis). Determination of which proteins and protein pathways are conserved and which have diverged within these species will aid in our ability to choose the appropriate models for determining protein function and to model human disease. We uncover mammalian- and amphibian-specific, as well as species-specific, enriched proteins and protein pathways. Among these, we find and validate an enrichment in cell-cycle-associated proteins within Xenopus laevis. To further investigate functional units within cardiac proteomes, we develop a computational approach to profile the abundance of protein complexes across species. Finally, we demonstrate the utility of these data sets for predicting appropriate model systems for studying given cardiac conditions by testing the role of Kielin/chordin-like protein (Kcp), a protein found as enriched in frog hearts compared to mammals. We establish that germ-line mutations in Kcp in Xenopus lead to valve defects and, ultimately, cardiac failure and death. Thus, integrating these findings with data on proteins responsible for cardiac disease should lead to the development of refined, species-specific models for protein function and disease states.
Hashimoto, Yutaka, Todd M Greco, and Ileana M Cristea. “Contribution of Mass Spectrometry-Based Proteomics to Discoveries in Developmental Biology”. Adv Exp Med Biol 1140 (2019): , 1140, 143-154. Web.Abstract
Understanding multicellular organism development from a molecular perspective is no small feat, yet this level of comprehension affords clinician-scientists the ability to identify root causes and mechanisms of congenital diseases. Inarguably, the maturation of molecular biology tools has significantly contributed to the identification of genetic loci that underlie normal and aberrant developmental programs. In combination with cell biology approaches, these tools have begun to elucidate the spatiotemporal expression and function of developmentally-regulated proteins. The emergence of quantitative mass spectrometry (MS) for biological applications has accelerated the pace at which these proteins can be functionally characterized, driving the construction of an increasingly detailed systems biology picture of developmental processes. Here, we review the quantitative MS-based proteomic technologies that have contributed significantly to understanding the role of proteome regulation in developmental processes. We provide a brief overview of these methodologies, focusing on their ability to provide precise and accurate proteome measurements. We then highlight the use of discovery-based and targeted mass spectrometry approaches in model systems to study cellular differentiation states, tissue phenotypes, and spatiotemporal subcellular organization. We also discuss the current application and future perspectives of MS proteomics to study PTM coordination and the role of protein complexes during development.
Murray, Laura A, et al.Methods for characterizing protein acetylation during viral infection”. Methods Enzymol 626 (2019): , 626, 587-620. Web.Abstract
Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body of work has highlighted the importance of temporal alterations in protein acetylation during infection with a range of human viruses. It has become clear that both cellular and viral proteins are decorated by lysine acetylations, and that these modifications contribute to core host defense and virus replication processes. Further defining the extent and dynamics of protein acetylation events during the progression of an infection can provide an important new perspective on the intricate mechanisms underlying the biology and pathogenesis of virus infections. Here, we provide protocols for identifying, quantifying, and probing the regulation of lysine acetylations during viral infection. We describe the use of acetyl-lysine immunoaffinity purification and quantitative mass spectrometry for assessing the cellular acetylome at different stages of an infection. As an alternative to traditional antibody-mediated western blotting, we discuss the benefits of targeted mass spectrometry approaches for detecting and quantifying site-specific acetylations on proteins of interest. Specifically, we provide a protocol using parallel reaction monitoring (PRM). We further discuss experimental considerations that are specific to studying viral infections. Finally, we provide a brief overview of the types of assays that can be employed to characterize the function of an acetylation event in the context of infection. As a method to interrogate the regulation of acetylation, we describe the Fluor de Lys assay for monitoring the enzymatic activities of deacetylases.
Howard, Timothy R, Bokai Song, and Ileana M Cristea. “Workflows and considerations for investigating protein interactions of viral DNA sensors”. Methods Enzymol 625 (2019): , 625, 309-338. Web.Abstract
DNA sensors are a core component of innate immunity in mammalian cells. In response to pathogen infection, these specialized proteins sense pathogenic DNA from bacteria or viruses and initiate immune signaling cascades. These defense mechanisms rely on the rapid formation and temporal regulation of protein-protein interactions. Similarly, protein interactions underlie virus immune evasion mechanisms, as proteins from diverse viruses associate with and inhibit DNA sensors. Here, we describe experimental protocols for identifying protein interactions of DNA sensors, and discuss considerations for optimal isolation of protein complexes when targeting either endogenous or tagged proteins. Additionally, as viral infections and immune responses are known to induce prominent changes in cellular protein abundances, we provide a workflow for investigating these protein associations in the context of proteome alterations.
Lum, Krystal K, et al.Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA”. mBio 10.4 (2019). Web.Abstract
The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication. The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.
Cristea, Ileana M, et al.Early-Career Scientists Shaping the World”. mSystems 43 (2019). Web.
Cook, Katelyn C, et al.Peroxisome Plasticity at the Virus-Host Interface”. Trends Microbiol 27.11 (2019): , 27, 11, 906-914. Web.Abstract
Peroxisomes are multifunctional organelles with roles in cellular metabolism, cytotoxicity, and signaling. The plastic nature of these organelles allows them to respond to diverse biological processes, such as virus infections, by remodeling their biogenesis, morphology, and composition to enhance specific functions. During virus infections in humans, peroxisomes act as important immune signaling organelles, aiding the host by orchestrating antiviral signaling. However, more recently it was discovered that peroxisomes can also benefit the virus, facilitating virus-host interactions that rewire peroxisomes to support cellular processes for virus replication and spread. Here, we describe recent studies that uncovered this double-edged character of peroxisomes during infection, highlighting mechanisms that viruses have coevolved to take advantage of peroxisome plasticity. We also provide a perspective for future studies by comparing the established roles of peroxisomes in plant infections and discussing the promise of virology studies as a venue to reveal the uncharted biology of peroxisomes.
Hernández Durán, Anna, et al.Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships”. PLoS Biol 17.6 (2019): , 17, 6, e3000316. Web.Abstract
Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)-mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.
Esposito, Mark, et al.Bone vascular niche E-selectin induces mesenchymal-epithelial transition and Wnt activation in cancer cells to promote bone metastasis”. Nat Cell Biol 21.5 (2019): , 21, 5, 627-639. Web.Abstract
How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial-mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal-epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal-epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal-epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.
Federspiel, Joel D, and Ileana M Cristea. “Considerations for Identifying Endogenous Protein Complexes from Tissue via Immunoaffinity Purification and Quantitative Mass Spectrometry”. Methods Mol Biol 1977 (2019): , 1977, 115-143. Web.Abstract
Protein complexes perform key roles in nearly all aspects of biology. Identification of the composition of these complexes offers insights into how different cellular processes are carried out. The use of affinity purification coupled to mass spectrometry has become a method of choice for identifying protein-protein interactions, but has been most frequently applied to cell model systems using tagged and overexpressed bait proteins. Although valuable, this approach can create several potential artifacts due to the presence of a tag on a protein and the higher abundance of the protein of interest (bait). The isolation of endogenous proteins using antibodies raised against the proteins of interest instead of an epitope tag offers a means to examine protein interactions in any cellular or animal model system and without the caveats of overexpressed, tagged proteins. Although conceptually simple, the limited use of this approach has been primarily driven by challenges associated with finding adequate antibodies and experimental conditions for effective isolations. In this chapter, we present a protocol for the optimization of lysis conditions, antibody evaluation, affinity purification, and ultimately identification of protein complexes from endogenous immunoaffinity purifications using quantitative mass spectrometry. We also highlight the increased use of targeted mass spectrometry analyses, such as parallel reaction monitoring (PRM) for orthogonal validation of protein isolation and interactions initially identified via data-dependent mass spectrometry analyses.
Federspiel, Joel D, et al.Hdac4 Interactions in Huntington's Disease Viewed Through the Prism of Multiomics”. Mol Cell Proteomics 18.8 suppl 1 (2019): , 18, 8 suppl 1, S92-S113. Web.Abstract
Huntington's disease (HD) is a monogenic disorder, driven by the expansion of a trinucleotide (CAG) repeat within the huntingtin (Htt) gene and culminating in neuronal degeneration in the brain, predominantly in the striatum and cortex. Histone deacetylase 4 (Hdac4) was previously found to contribute to the disease progression, providing a potential therapeutic target. Hdac4 knockdown reduced accumulation of misfolded Htt protein and improved HD phenotypes. However, the underlying mechanism remains unclear, given its independence on deacetylase activity and the predominant cytoplasmic Hdac4 localization in the brain. Here, we undertook a multiomics approach to uncover the function of Hdac4 in the context of HD pathogenesis. We characterized the interactome of endogenous Hdac4 in brains of HD mouse models. Alterations in interactions were investigated in response to Htt polyQ length, comparing mice with normal (Q20) and disease (Q140) Htt, at both pre- and post-symptomatic ages (2 and 10 months, respectively). Parallel analyses for Hdac5, a related class IIa Hdac, highlighted the unique interaction network established by Hdac4. To validate and distinguish interactions specifically enhanced in an HD-vulnerable brain region, we next characterized endogenous Hdac4 interactions in dissected striata from this HD mouse series. Hdac4 associations were polyQ-dependent in the striatum, but not in the whole brain, particularly in symptomatic mice. Hdac5 interactions did not exhibit polyQ dependence. To identify which Hdac4 interactions and functions could participate in HD pathogenesis, we integrated our interactome with proteome and transcriptome data sets generated from the striata. We discovered an overlap in enriched functional classes with the Hdac4 interactome, particularly in vesicular trafficking and synaptic functions, and we further validated the Hdac4 interaction with the Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex. This study expands the knowledge of Hdac4 regulation and functions in HD, adding to the understanding of the molecular underpinning of HD phenotypes.
Betsinger, Cora N, and Ileana M Cristea. “Mitochondrial Function, Metabolic Regulation, and Human Disease Viewed through the Prism of Sirtuin 4 (SIRT4) Functions”. J Proteome Res 18.5 (2019): , 18, 5, 1929-1938. Web.Abstract
As cellular metabolic hubs, mitochondria are the main energy producers for the cell. These organelles host essential energy producing biochemical processes, including the TCA cycle, fatty acid oxidation, and oxidative phosphorylation. An accumulating body of literature has demonstrated that a majority of mitochondrial proteins are decorated with diverse posttranslational modifications (PTMs). Given the critical roles of these proteins in cellular metabolic pathways and response to environmental stress or pathogens, understanding the role of PTMs in regulating their functions has become an area of intense investigation. A major family of enzymes that regulate PTMs within the mitochondria are sirtuins (SIRTs). Albeit until recently the least understood sirtuin, SIRT4 has emerged as an enzyme capable of removing diverse PTMs from its substrates, thereby modulating their functions. SIRT4 was shown to have ADP-ribosyltransferase, deacetylase, lipoamidase, and deacylase enzymatic activities. As metabolic dysfunction is linked to human disease, SIRT4 levels and activities have been implicated in modulating susceptibility to hyperinsulinemia and diabetes, liver disease, cancer, neurodegeneration, heart disease, aging, and pathogenic infections. Therefore, SIRT4 has emerged as a possible candidate for targeted therapeutics. Here, we discuss the diverse enzymatic activities and substrates of SIRT4 and its roles in human health and disease.
Cristea, Ileana M, and Kathryn S Lilley. “Editorial overview: Untangling proteome organization in space and time”. Curr Opin Chem Biol 48 (2019): , 48, A1-A4. Web.
Hashimoto, Yutaka, et al.Mechanical Force Induces Phosphorylation-Mediated Signaling that Underlies Tissue Response and Robustness in Xenopus Embryos”. Cell Syst 83 (2019): , 8, 3, 226-241.e7. Web.Abstract
Mechanical forces are essential drivers of numerous biological processes, notably during development. Although it is well recognized that cells sense and adapt to mechanical forces, the signal transduction pathways that underlie mechanosensing have remained elusive. Here, we investigate the impact of mechanical centrifugation force on phosphorylation-mediated signaling in Xenopus embryos. By monitoring temporal phosphoproteome and proteome alterations in response to force, we discover and validate elevated phosphorylation on focal adhesion and tight junction components, leading to several mechanistic insights into mechanosensing and tissue restoration. First, we determine changes in kinase activity profiles during mechanoresponse, identifying the activation of basophilic kinases. Pathway interrogation using kinase inhibitor treatment uncovers a crosstalk between the focal adhesion kinase (FAK) and protein kinase C (PKC) in mechanoresponse. Second, we find LIM domain 7 protein (Lmo7) as upregulated upon centrifugation, contributing to mechanoresponse. Third, we discover that mechanical compression force induces a mesenchymal-to-epithelial transition (MET)-like phenotype.
Carabetta, Valerie J, et al.YfmK is an N-lysine acetyltransferase that directly acetylates the histone-like protein HBsu in ”. Proc Natl Acad Sci U S A 116.9 (2019): , 116, 9, 3752-3757. Web.Abstract
N-lysine acetylation is an abundant and dynamic regulatory posttranslational modification that remains poorly characterized in bacteria. In bacteria, hundreds of proteins are known to be acetylated, but the biological significance of the majority of these events remains unclear. Previously, we characterized the acetylome and found that the essential histone-like protein HBsu contains seven previously unknown acetylation sites in vivo. Here, we investigate whether acetylation is a regulatory component of the function of HBsu in nucleoid compaction. Using mutations that mimic the acetylated and unacetylated forms of the protein, we show that the inability to acetylate key HBsu lysine residues results in a more compacted nucleoid. We further investigated the mechanism of HBsu acetylation. We screened deletions of the ∼50 putative GNAT domain-encoding genes in for their effects on DNA compaction, and identified five candidates that may encode acetyltransferases acting on HBsu. Genetic bypass experiments demonstrated that two of these, YfmK and YdgE, can acetylate Hbsu, and their potential sites of action on HBsu were identified. Additionally, purified YfmK was able to directly acetylate HBsu in vitro, suggesting that it is the second identified protein acetyltransferase in We propose that at least one physiological function of the acetylation of HBsu at key lysine residues is to regulate nucleoid compaction, analogous to the role of histone acetylation in eukaryotes.
Cook, Katelyn C, and Ileana M Cristea. “Location is everything: protein translocations as a viral infection strategy”. Curr Opin Chem Biol 48 (2019): , 48, 34-43. Web.Abstract
Protein movement between different subcellular compartments is an essential aspect of biological processes, including transcriptional and metabolic regulation, and immune and stress responses. As obligate intracellular parasites, viruses are master manipulators of cellular composition and organization. Accumulating evidences have highlighted the importance of infection-induced protein translocations between organelles. Both directional and temporal, these translocation events facilitate localization-dependent protein interactions and changes in protein functions that contribute to either host defense or virus replication. The discovery and characterization of protein movement is technically challenging, given the necessity for sensitive detection and subcellular resolution. Here, we discuss infection-induced translocations of host and viral proteins, and the value of integrating quantitative proteomics with advanced microscopy for understanding the biology of human virus infections.
2018
Xu, Linda, et al.Recurrent, Activating Variants in the Receptor Tyrosine Kinase DDR2 Cause Warburg-Cinotti Syndrome”. Am J Hum Genet 103.6 (2018): , 103, 6, 976-983. Web.Abstract
We have investigated a distinct disorder with progressive corneal neovascularization, keloid formation, chronic skin ulcers, wasting of subcutaneous tissue, flexion contractures of the fingers, and acro-osteolysis. In six affected individuals from four families, we found one of two recurrent variants in discoidin domain receptor tyrosine kinase 2 (DDR2): c.1829T>C (p.Leu610Pro) or c.2219A>G (p.Tyr740Cys). DDR2 encodes a collagen-responsive receptor tyrosine kinase that regulates connective-tissue formation. In three of the families, affected individuals comprise singleton adult individuals, and parental samples were not available for verification of the de novo occurrence of the DDR2 variants. In the fourth family, a mother and two of her children were affected, and the c.2219A>G missense variant was proven to be de novo in the mother. Phosphorylation of DDR2 was increased in fibroblasts from affected individuals, suggesting reduced receptor autoinhibition and ligand-independent kinase activation. Evidence for activation of other growth-regulatory signaling pathways was not found. Finally, we found that the protein kinase inhibitor dasatinib prevented DDR2 autophosphorylation in fibroblasts, suggesting an approach to treatment. We propose this progressive, fibrotic condition should be designated as Warburg-Cinotti syndrome.
Lum, Krystal K, et al.Interactome and Proteome Dynamics Uncover Immune Modulatory Associations of the Pathogen Sensing Factor cGAS”. Cell Syst 76 (2018): , 7, 6, 627-642.e6. Web.Abstract
Viral DNA sensing is an essential component of the mammalian innate immune response. Upon binding viral DNA, the cyclic-GMP-AMP synthase (cGAS) catalyzes the production of cyclic dinucleotides to induce type I interferons. However, little is known about how cGAS is homeostatically maintained or regulated upon infection. Here, we define cytoplasmic cGAS interactions with cellular and viral proteins upon herpes simplex virus type 1 (HSV-1) infection in primary human fibroblasts. We compare several HSV-1 strains (wild-type, d109, d106) that induce cytokine responses and apoptosis and place cGAS interactions in the context of temporal proteome alterations using isobaric-labeling mass spectrometry. Follow-up analyses establish a functional interaction between cGAS and 2'-5'-oligoadenylate synthase-like protein OASL. The OAS-like domain interacts with the cGAS Mab21 domain, while the OASL ubiquitin-like domain further inhibits cGAS-mediated interferon response. Our findings explain how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.
Murray, LA, X Sheng, and IM Cristea. “Orchestration of protein acetylation as a toggle for cellular defense and virus replication”. Nat Commun 91 (2018): , 9, 1, 4967. Web.Abstract
Emerging evidence highlights protein acetylation, a prevalent lysine posttranslational modification, as a regulatory mechanism and promising therapeutic target in human viral infections. However, how infections dynamically alter global cellular acetylation or whether viral proteins are acetylated remains virtually unexplored. Here, we establish acetylation as a highly-regulated molecular toggle of protein function integral to the herpesvirus human cytomegalovirus (HCMV) replication. We offer temporal resolution of cellular and viral acetylations. By interrogating dynamic protein acetylation with both protein abundance and subcellular localization, we discover finely tuned spatial acetylations across infection time. We determine that lamin acetylation at the nuclear periphery protects against virus production by inhibiting capsid nuclear egress. Further studies within infectious viral particles identify numerous acetylations, including on the viral transcriptional activator pUL26, which we show represses virus production. Altogether, this study provides specific insights into functions of cellular and viral protein acetylations and a valuable resource of dynamic acetylation events.
Gilbertson, Sarah, et al.Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription”. Elife 7 (2018). Web.Abstract
Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.
Jean Beltran, Pierre M, et al.Infection-Induced Peroxisome Biogenesis Is a Metabolic Strategy for Herpesvirus Replication”. Cell Host Microbe 24.4 (2018): , 24, 4, 526-541.e7. Web.Abstract
Viral proteins have evolved to target cellular organelles and usurp their functions for virus replication. Despite the knowledge of these critical functions for several organelles, little is known about peroxisomes during infection. Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism. Here, we discovered that the enveloped viruses human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) induce the biogenesis of and unique morphological changes to peroxisomes to support their replication. Targeted proteomic quantification revealed a global virus-induced upregulation of peroxisomal proteins. Mathematical modeling and microscopy structural analysis show that infection triggers peroxisome growth and fission, leading to increased peroxisome numbers and irregular disc-like structures. HCMV-induced peroxisome biogenesis increased the phospholipid plasmalogen, thereby enhancing virus production. Peroxisome regulation and dependence were not observed for the non-enveloped adenovirus. Our findings uncover a role of peroxisomes in viral pathogenesis, with likely implications for multiple enveloped viruses.
Sauls, Kimberly, et al.Initiating Events in Direct Cardiomyocyte Reprogramming”. Cell Rep 22.7 (2018): , 22, 7, 1913-1922. Web.Abstract
Direct reprogramming of fibroblasts into cardiomyocyte-like cells (iCM) holds great potential for heart regeneration and disease modeling and may lead to future therapeutic applications. Currently, application of this technology is limited by our lack of understanding of the molecular mechanisms that drive direct iCM reprogramming. Using a quantitative mass spectrometry-based proteomic approach, we identified the temporal global changes in protein abundance that occur during initial phases of iCM reprogramming. Collectively, our results show systematic and temporally distinct alterations in levels of specific functional classes of proteins during the initiating steps of reprogramming including extracellular matrix proteins, translation factors, and chromatin-binding proteins. We have constructed protein relational networks associated with the initial transition of a fibroblast into an iCM. These findings demonstrate the presence of an orchestrated series of temporal steps associated with dynamic changes in protein abundance in a defined group of protein pathways during the initiating events of direct reprogramming.
Cho, Hyunghoon, et al.Principles of Systems Biology, No. 31”. Cell Syst 72 (2018): , 7, 2, 133-135. Web.Abstract
This month: selected work from the 2018 RECOMB meeting, organized by Ecole Polytechnique and held last April in Paris.
Rowland, Elizabeth A, Caroline K Snowden, and Ileana M Cristea. “Protein lipoylation: an evolutionarily conserved metabolic regulator of health and disease”. Curr Opin Chem Biol 42 (2018): , 42, 76-85. Web.Abstract
Lipoylation is a rare, but highly conserved lysine posttranslational modification. To date, it is known to occur on only four multimeric metabolic enzymes in mammals, yet these proteins are staples in the core metabolic landscape. The dysregulation of these mitochondrial proteins is linked to a range of human metabolic disorders. Perhaps most striking is that lipoylation itself, the proteins that add or remove the modification, as well as the proteins it decorates are all evolutionarily conserved from bacteria to humans, highlighting the importance of this essential cofactor. Here, we discuss the biological significance of protein lipoylation, the importance of understanding its regulation in health and disease states, and the advances in mass spectrometry-based proteomic technologies that can aid these studies.
2017
Mirzaei, Mehdi, et al.Age-related neurodegenerative disease associated pathways identified in retinal and vitreous proteome from human glaucoma eyes”. Sci Rep 71 (2017): , 7, 1, 12685. Web.Abstract
ABSTARCT: Glaucoma is a chronic disease that shares many similarities with other neurodegenerative disorders of the central nervous system. This study was designed to evaluate the association between glaucoma and other neurodegenerative disorders by investigating glaucoma-associated protein changes in the retina and vitreous humour. The multiplexed Tandem Mass Tag based proteomics (TMT-MS3) was carried out on retinal tissue and vitreous humour fluid collected from glaucoma patients and age-matched controls followed by functional pathway and protein network interaction analysis. About 5000 proteins were quantified from retinal tissue and vitreous fluid of glaucoma and control eyes. Of the differentially regulated proteins, 122 were found linked with pathophysiology of Alzheimer's disease (AD). Pathway analyses of differentially regulated proteins indicate defects in mitochondrial oxidative phosphorylation machinery. The classical complement pathway associated proteins were activated in the glaucoma samples suggesting an innate inflammatory response. The majority of common differentially regulated proteins in both tissues were members of functional protein networks associated brain changes in AD and other chronic degenerative conditions. Identification of previously reported and novel pathways in glaucoma that overlap with other CNS neurodegenerative disorders promises to provide renewed understanding of the aetiology and pathogenesis of age related neurodegenerative diseases.
Jean Beltran, Pierre M, Katelyn C Cook, and Ileana M Cristea. “Exploring and Exploiting Proteome Organization during Viral Infection”. J Virol 91.18 (2017). Web.Abstract
Viral replication in eukaryotes is a process inherently organized in both space and time. Viral components target subcellular organelles to access host machineries required for replication and spread. Diverse viruses are known to alter organelle shape, composition, function, and dynamics as part of their replication cycles. Here, we highlight recent advances in microscopy and proteomic methods that have helped and will continue to help define mechanisms used by viruses to exploit host proteome organization.
Kennedy, Leslie, et al.Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis”. PLoS Genet 13.9 (2017): , 13, 9, e1007011. Web.Abstract
By the age of 40, one in five adults without symptoms of cardiovascular disease are at risk for developing congestive heart failure. Within this population, dilated cardiomyopathy (DCM) remains one of the leading causes of disease and death, with nearly half of cases genetically determined. Though genetic and high throughput sequencing-based approaches have identified sporadic and inherited mutations in a multitude of genes implicated in cardiomyopathy, how combinations of asymptomatic mutations lead to cardiac failure remains a mystery. Since a number of studies have implicated mutations of the transcription factor TBX20 in congenital heart diseases, we investigated the underlying mechanisms, using an unbiased systems-based screen to identify novel, cardiac-specific binding partners. We demonstrated that TBX20 physically and genetically interacts with the essential transcription factor CASZ1. This interaction is required for survival, as mice heterozygous for both Tbx20 and Casz1 die post-natally as a result of DCM. A Tbx20 mutation associated with human familial DCM sterically interferes with the TBX20-CASZ1 interaction and provides a physical basis for how this human mutation disrupts normal cardiac function. Finally, we employed quantitative proteomic analyses to define the molecular pathways mis-regulated upon disruption of this novel complex. Collectively, our proteomic, biochemical, genetic, and structural studies suggest that the physical interaction between TBX20 and CASZ1 is required for cardiac homeostasis, and further, that reduction or loss of this critical interaction leads to DCM. This work provides strong evidence that DCM can be inherited through a digenic mechanism.
Cristea, Ileana M. “The Host-Pathogen Ecosystem Viewed Through the Prism of Proteomics”. Mol Cell Proteomics 16.4 suppl 1 (2017): , 16, 4 suppl 1, S1-S4. Web.
Crow, Marni S, and Ileana M Cristea. “Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation”. Mol Cell Proteomics 16.4 suppl 1 (2017): , 16, 4 suppl 1, S200-S214. Web.Abstract
The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation.
Jean Beltran, Pierre M, et al.Proteomics and integrative omic approaches for understanding host-pathogen interactions and infectious diseases”. Mol Syst Biol 13.3 (2017): , 13, 3, 922. Web.Abstract
Organisms are constantly exposed to microbial pathogens in their environments. When a pathogen meets its host, a series of intricate intracellular interactions shape the outcome of the infection. The understanding of these host-pathogen interactions is crucial for the development of treatments and preventive measures against infectious diseases. Over the past decade, proteomic approaches have become prime contributors to the discovery and understanding of host-pathogen interactions that represent anti- and pro-pathogenic cellular responses. Here, we review these proteomic methods and their application to studying viral and bacterial intracellular pathogens. We examine approaches for defining spatial and temporal host-pathogen protein interactions upon infection of a host cell. Further expanding the understanding of proteome organization during an infection, we discuss methods that characterize the regulation of host and pathogen proteomes through alterations in protein abundance, localization, and post-translational modifications. Finally, we highlight bioinformatic tools available for analyzing such proteomic datasets, as well as novel strategies for integrating proteomics with other omic tools, such as genomics, transcriptomics, and metabolomics, to obtain a systems-level understanding of infectious diseases.
Greco, Todd M, and Ileana M Cristea. “Proteomics Tracing the Footsteps of Infectious Disease”. Mol Cell Proteomics 16.4 suppl 1 (2017): , 16, 4 suppl 1, S5-S14. Web.Abstract
Every year, a major cause of human disease and death worldwide is infection with the various pathogens-viruses, bacteria, fungi, and protozoa-that are intrinsic to our ecosystem. In efforts to control the prevalence of infectious disease and develop improved therapies, the scientific community has focused on building a molecular picture of pathogen infection and spread. These studies have been aimed at defining the cellular mechanisms that allow pathogen entry into hosts cells, their replication and transmission, as well as the core mechanisms of host defense against pathogens. The past two decades have demonstrated the valuable implementation of proteomic methods in all these areas of infectious disease research. Here, we provide a perspective on the contributions of mass spectrometry and other proteomics approaches to understanding the molecular details of pathogen infection. Specifically, we highlight methods used for defining the composition of viral and bacterial pathogens and the dynamic interaction with their hosts in space and time. We discuss the promise of MS-based proteomics in supporting the development of diagnostics and therapies, and the growing need for multiomics strategies for gaining a systems view of pathogen infection.
Santos, Joana Mendonca, et al.Red Blood Cell Invasion by the Malaria Parasite Is Coordinated by the PfAP2-I Transcription Factor”. Cell Host Microbe 21.6 (2017): , 21, 6, 731-741.e10. Web.Abstract
Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor PfAP2-I, belonging to the Apicomplexan AP2 (ApiAP2) family, that is responsible for regulating the expression of genes involved in RBC invasion. Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of target genes. Although PfAP2-I contains three AP2 DNA-binding domains, only one is required for binding of the target genes during blood stage development. Furthermore, we find that PfAP2-I associates with several chromatin-associated proteins, including the Plasmodium bromodomain protein PfBDP1 and that complex formation is associated with transcriptional regulation. As a key regulator of red blood cell invasion, PfAP2-I represents a potential new antimalarial therapeutic target.
Carabetta, Valerie J, and Ileana M Cristea. “Regulation, Function, and Detection of Protein Acetylation in Bacteria”. J Bacteriol 199.16 (2017). Web.Abstract
-Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-related -acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria.
Rowland, Elizabeth A, et al.Sirtuin Lipoamidase Activity Is Conserved in Bacteria as a Regulator of Metabolic Enzyme Complexes”. mBio 85 (2017). Web.Abstract
Lipoic acid is an essential metabolic cofactor added as a posttranslational modification on several multimeric enzyme complexes. These protein complexes, evolutionarily conserved from bacteria to humans, are core regulators of cellular metabolism. While the multistep enzymatic process of adding lipoyl modifications has been well characterized in , the enzyme required for the removal of these lipoyl moieties (i.e., a lipoamidase or delipoylase) has not yet been identified. Here, we describe our discovery of sirtuins as lipoamidases in bacteria and establish their conserved substrates. Specifically, by using a series of knockout, overexpression, biochemical, , proteomic, and functional assays, we determined the substrates of sirtuin CobB in as components of the pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KDH), and glycine cleavage (GCV) complexes. assays provided direct evidence for this specific CobB activity and its NAD dependence, a signature of all sirtuins. By designing a targeted quantitative mass spectrometry method, we further measured sirtuin-dependent, site-specific lipoylation on these substrates. The biological significance of CobB-modulated lipoylation was next established by its inhibition of both PDH and KDH activities. By restricting the carbon sources available to , we demonstrated that CobB regulates PDH and KDH under several growth conditions. Additionally, we found that SrtN, the sirtuin homolog in Gram-positive , can also act as a lipoamidase. By demonstrating the evolutionary conservation of lipoamidase activity across sirtuin homologs, along with the conservation of common substrates, this work emphasizes the significance of protein lipoylation in regulating central metabolic processes. Here, we demonstrate that sirtuin lipoamidase activity exists in both Gram-positive and Gram-negative bacteria and establishing its conservation from bacteria to humans. Specifically, we discovered that CobB and SrtN act as lipoamidases in and , respectively. Intriguingly, not only is this sirtuin enzymatic activity conserved, but also the lipoylated substrates and functions are conserved, as bacterial sirtuins negatively regulate the lipoylation levels and activities of PDH and KDH. Considering that PDH and KDH regulate two carbon entry points into the tricarboxylic acid cycle, our finding highlights lipoylation as a conserved molecular toggle that regulates central metabolic pathways. Indeed, our findings from tests in which we limited nutrient availability support this. Furthermore, this study illustrates how the integration of technologies from different disciplines provides avenues to uncover enzymatic activities at the core of cellular metabolism regulation.
Pastino, Alexandra K, et al.Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly”. Matrix Biol 59 (2017): , 59, 39-53. Web.Abstract
Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM.
Alfonso-Dunn, Roberto, et al.Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency”. Cell Host Microbe 21.4 (2017): , 21, 4, 507-517.e5. Web.Abstract
The cellular transcriptional coactivator HCF-1 is required for initiation of herpes simplex virus (HSV) lytic infection and for reactivation from latency in sensory neurons. HCF-1 stabilizes the viral Immediate Early (IE) gene enhancer complex and mediates chromatin transitions to promote IE transcription initiation. In infected cells, HCF-1 was also found to be associated with a network of transcription elongation components including the super elongation complex (SEC). IE genes exhibit characteristics of genes controlled by transcriptional elongation, and the SEC-P-TEFb complex is specifically required to drive the levels of productive IE mRNAs. Significantly, compounds that enhance the levels of SEC-P-TEFb also potently stimulated HSV reactivation from latency both in a sensory ganglia model system and in vivo. Thus, transcriptional elongation of HSV IE genes is a key limiting parameter governing both the initiation of HSV infection and reactivation of latent genomes.
EA, Rowland, et al.Sirtuin Lipoamidase Activity Is Conserved in Bacteria as a Regulator of Metabolic Enzyme Complexes”. mBio 85 (2017). Print.
VJ, Carabetta, and Cristea IM. “Regulation, Function, and Detection of Protein Acetylation in Bacteria”. J Bacteriol. 199.16 (2017). Print.
R, Alfonso-Dunn, et al.Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency”. Cell Host Microbe 21.4 (2017): , 21, 4, 507-517. Print.
PM, Jean Beltran, et al.Proteomics and integrative omic approaches for understanding host-pathogen interactions and infectious diseases”. Mol Syst Biol. 13.3 (2017): , 13, 3, 922. Print.
IM, Cristea. “The Host-Pathogen Ecosystem Viewed Through the Prism of Proteomics”. Mol Cell Proteomics 16.4 (2017): , 16, 4, S1-4. Print.
TM, Greco, and Cristea IM. “Proteomics Tracing the Footsteps of Infectious Disease”. Mol Cell Proteomics 16.4 (2017): , 16, 4, S5-14. Print.
MS, Crow, and Cristea IM. “Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation”. Mol Cell Proteomics 16.4 (2017): , 16, 4, S200-214. Print.
2016
Ashford, Paul, et al.HVint: A Strategy for Identifying Novel Protein-Protein Interactions in Herpes Simplex Virus Type 1”. Mol Cell Proteomics 15.9 (2016): , 15, 9, 2939-53. Web.Abstract
Human herpesviruses are widespread human pathogens with a remarkable impact on worldwide public health. Despite intense decades of research, the molecular details in many aspects of their function remain to be fully characterized. To unravel the details of how these viruses operate, a thorough understanding of the relationships between the involved components is key. Here, we present HVint, a novel protein-protein intraviral interaction resource for herpes simplex virus type 1 (HSV-1) integrating data from five external sources. To assess each interaction, we used a scoring scheme that takes into consideration aspects such as the type of detection method and the number of lines of evidence. The coverage of the initial interactome was further increased using evolutionary information, by importing interactions reported for other human herpesviruses. These latter interactions constitute, therefore, computational predictions for potential novel interactions in HSV-1. An independent experimental analysis was performed to confirm a subset of our predicted interactions. This subset covers proteins that contribute to nuclear egress and primary envelopment events, including VP26, pUL31, pUL40, and the recently characterized pUL32 and pUL21. Our findings support a coordinated crosstalk between VP26 and proteins such as pUL31, pUS9, and the CSVC complex, contributing to the development of a model describing the nuclear egress and primary envelopment pathways of newly synthesized HSV-1 capsids. The results are also consistent with recent findings on the involvement of pUL32 in capsid maturation and early tegumentation events. Further, they open the door to new hypotheses on virus-specific regulators of pUS9-dependent transport. To make this repository of interactions readily accessible for the scientific community, we also developed a user-friendly and interactive web interface. Our approach demonstrates the power of computational predictions to assist in the design of targeted experiments for the discovery of novel protein-protein interactions.
Guise, Amanda J, and Ileana M Cristea. “Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5)”. Methods Mol Biol 1436 (2016): , 1436, 47-84. Web.Abstract
As a member of the class IIa family of histone deacetylases, the histone deacetylase 5 (HDAC5) is known to undergo nuclear-cytoplasmic shuttling and to be a critical transcriptional regulator. Its misregulation has been linked to prominent human diseases, including cardiac diseases and tumorigenesis. In this chapter, we describe several experimental methods that have proven effective for studying the functions and regulatory features of HDAC5. We present methods for assessing the subcellular localization, protein interactions, posttranslational modifications (PTMs), and activity of HDAC5 from the standpoint of investigating either the endogenous protein or tagged protein forms in human cells. Specifically, given that at the heart of HDAC5 regulation lie its dynamic localization, interactions, and PTMs, we present methods for assessing HDAC5 localization in fixed and live cells, for isolating HDAC5-containing protein complexes to identify its interactions and modifications, and for determining how these PTMs map to predicted HDAC5 structural motifs. Lastly, we provide examples of approaches for studying HDAC5 functions with a focus on its regulation during cell-cycle progression. These methods can readily be adapted for the study of other HDACs or non-HDAC-proteins of interest. Individually, these techniques capture temporal and spatial snapshots of HDAC5 functions; yet together, these approaches provide powerful tools for investigating both the regulation and regulatory roles of HDAC5 in different cell contexts relevant to health and disease.
Greco, Todd M, and Ileana M Cristea. “The Biochemical Evolution of Protein Complexes”. Trends Biochem Sci 41.1 (2016): , 41, 1, 4-6. Web.Abstract
Over the past decade, it became evident that proteins perform critical functions as components of specialized macromolecular complexes. Here, we discuss a recent study by Wan and colleagues, which highlights the significance of protein complexes by studying their conservation in organisms separated by up to a billion years of evolution.
Waldron, Lauren, et al.The Cardiac TBX5 Interactome Reveals a Chromatin Remodeling Network Essential for Cardiac Septation”. Dev Cell 36.3 (2016): , 36, 3, 262-75. Web.Abstract
Human mutations in the cardiac transcription factor gene TBX5 cause congenital heart disease (CHD), although the underlying mechanism is unknown. We report characterization of the endogenous TBX5 cardiac interactome and demonstrate that TBX5, long considered a transcriptional activator, interacts biochemically and genetically with the nucleosome remodeling and deacetylase (NuRD) repressor complex. Incompatible gene programs are repressed by TBX5 in the developing heart. CHD mis-sense mutations that disrupt the TBX5-NuRD interaction cause depression of a subset of repressed genes. Furthermore, the TBX5-NuRD interaction is required for heart development. Phylogenetic analysis showed that the TBX5-NuRD interaction domain evolved during early diversification of vertebrates, simultaneous with the evolution of cardiac septation. Collectively, this work defines a TBX5-NuRD interaction essential to cardiac development and the evolution of the mammalian heart, and when altered may contribute to human CHD.
Greco, Todd M, Amanda J Guise, and Ileana M Cristea. “Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy”. Methods Mol Biol 1410 (2016): , 1410, 39-63. Web.Abstract
In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability.
Crow, Marni S, et al.Diverse mechanisms evolved by DNA viruses to inhibit early host defenses”. Crit Rev Biochem Mol Biol 51.6 (2016): , 51, 6, 452-481. Web.Abstract
In mammalian cells, early defenses against infection by pathogens are mounted through a complex network of signaling pathways shepherded by immune-modulatory pattern-recognition receptors. As obligate parasites, the survival of viruses is dependent on the evolutionary acquisition of mechanisms that tactfully dismantle and subvert the cellular intrinsic and innate immune responses. Here, we review the diverse mechanisms by which viruses that accommodate DNA genomes are able to circumvent activation of cellular immunity. We start by discussing viral manipulation of host defense protein levels by either transcriptional regulation or protein degradation. We next review viral strategies used to repurpose or inhibit these cellular immune factors by molecular hijacking or by regulating their post-translational modification status. Additionally, we explore the infection-induced temporal modulation of apoptosis to facilitate viral replication and spread. Lastly, the co-evolution of viruses with their hosts is highlighted by the acquisition of elegant mechanisms for suppressing host defenses via viral mimicry of host factors. In closing, we present a perspective on how characterizing these viral evasion tactics both broadens the understanding of virus-host interactions and reveals essential functions of the immune system at the molecular level. This knowledge is critical in understanding the sources of viral pathogenesis, as well as for the design of antiviral therapeutics and autoimmunity treatments.
Luo, Yang, et al.HIV-host interactome revealed directly from infected cells”. Nat Microbiol 17 (2016): , 1, 7, 16068. Web.Abstract
Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen-host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.
Budayeva, Hanna G, and Ileana M Cristea. “Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking”. Mol Cell Proteomics 15.10 (2016): , 15, 10, 3107-3125. Web.Abstract
Human sirtuin 2 (SIRT2) is an NAD-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases.
Viktorovskaya, Olga V, et al.Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements”. PLoS Negl Trop Dis 10.8 (2016): , 10, 8, e0004921. Web.Abstract
BACKGROUND: There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. CONCLUSIONS/SIGNIFICANCE: The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.
Mathias, Rommel A, Todd M Greco, and Ileana M Cristea. “Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators”. Methods Mol Biol 1436 (2016): , 1436, 213-39. Web.Abstract
Recent studies have highlighted the three mitochondrial human sirtuins (SIRT3, SIRT4, and SIRT5) as critical regulators of a wide range of cellular metabolic pathways. A key factor to understanding their impact on metabolism has been the discovery that, in addition to their ability to deacetylate substrates, mitochondrial sirtuins can have other prominent enzymatic activities. SIRT4, one of the least characterized mitochondrial sirtuins, was shown to be the first known cellular lipoamidase, removing lipoyl modifications from lysine residues of substrates. Specifically, SIRT4 was found to delipoylate and modulate the activity of the pyruvate dehydrogenase complex (PDH), a protein complex critical for the production of acetyl-CoA. Furthermore, SIRT4 is well known to have ADP-ribosyltransferase activity and to regulate the activity of the glutamate dehydrogenase complex (GDH). Adding to its impressive range of enzymatic activities are its ability to deacetylate malonyl-CoA decarboxylase (MCD) to regulate lipid catabolism, and its newly recognized ability to remove biotinyl groups from substrates that remain to be defined. Given the wide range of enzymatic activities and the still limited knowledge of its substrates, further studies are needed to characterize its protein interactions and its impact on metabolic pathways. Here, we present several proven protocols for identifying SIRT4 protein interaction networks within the mitochondria. Specifically, we describe methods for generating human cell lines expressing SIRT4, purifying mitochondria from crude organelles, and effectively capturing SIRT4 with its interactions and substrates.
Budayeva, Hanna G, Elizabeth A Rowland, and Ileana M Cristea. “Intricate Roles of Mammalian Sirtuins in Defense against Viral Pathogens”. J Virol 90.1 (2016): , 90, 1, 5-8. Web.Abstract
For a number of years, sirtuin enzymes have been appreciated as effective "sensors" of the cellular environment to rapidly transmit information to diverse cellular pathways. Much effort was placed into exploring their roles in human cancers and aging. However, a growing body of literature brings these enzymes to the spotlight in the field of virology. Here, we discuss sirtuin functions in the context of viral infection, which provide regulatory points for therapeutic intervention against pathogens.
McDonald, Karin R, et al.Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity”. PLoS Genet 12.9 (2016): , 12, 9, e1006238. Web.Abstract
Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.
Jean Beltran, Pierre M, Rommel A Mathias, and Ileana M Cristea. “A Portrait of the Human Organelle Proteome In Space and Time during Cytomegalovirus Infection”. Cell Syst 34 (2016): , 3, 4, 361-373.e6. Web.Abstract
The organelles within a eukaryotic host are manipulated by viruses to support successful virus replication and spread of infection, yet the global impact of viral infection on host organelles is poorly understood. Integrating microscopy, subcellular fractionation, mass spectrometry, and functional analyses, we conducted a cell-wide study of organelles in primary fibroblasts throughout the time course of human cytomegalovirus (HCMV) infection. We used label-free and isobaric-labeling proteomics to characterize nearly 4,000 host and 100 viral proteins, then classified their specific subcellular locations over time using machine learning. We observed a global reorganization of proteins across the secretory pathway, plasma membrane, and mitochondria, including reorganization and processing of lysosomal proteins into distinct subpopulations and translocations of individual proteins between organelles at specific time points. We also demonstrate that MYO18A, an unconventional myosin that translocates from the plasma membrane to the viral assembly complex, is necessary for efficient HCMV replication. This study provides a comprehensive resource for understanding host and virus biology during HCMV pathogenesis.
Lum, Krystal K, and Ileana M Cristea. “Proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection”. Expert Rev Proteomics 13.3 (2016): , 13, 3, 325-40. Web.Abstract
The integration of proteomic methods to virology has facilitated a significant breadth of biological insight into mechanisms of virus replication, antiviral host responses and viral subversion of host defenses. Throughout the course of infection, these cellular mechanisms rely heavily on the formation of temporally and spatially regulated virus-host protein-protein interactions. Reviewed here are proteomic-based approaches that have been used to characterize this dynamic virus-host interplay. Specifically discussed are the contribution of integrative mass spectrometry, antibody-based affinity purification of protein complexes, cross-linking and protein array techniques for elucidating complex networks of virus-host protein associations during infection with a diverse range of RNA and DNA viruses. The benefits and limitations of applying proteomic methods to virology are explored, and the contribution of these approaches to important biological discoveries and to inspiring new tractable avenues for the design of antiviral therapeutics is highlighted.
Giguère, Sophie SB, et al.The Proteomic Profile of Deleted in Breast Cancer 1 (DBC1) Interactions Points to a Multifaceted Regulation of Gene Expression”. Mol Cell Proteomics 15.3 (2016): , 15, 3, 791-809. Web.Abstract
Deleted in breast cancer 1 (DBC1) has emerged as an important regulator of multiple cellular processes, ranging from gene expression to cell cycle progression. DBC1 has been linked to tumorigenesis both as an inhibitor of histone deacetylases, HDAC3 and sirtuin 1, and as a transcriptional cofactor for nuclear hormone receptors. However, despite mounting interest in DBC1, relatively little is known about the range of its interacting partners and the scope of its functions. Here, we carried out a functional proteomics-based investigation of DBC1 interactions in two relevant cell types, T cells and kidney cells. Microscopy, molecular biology, biochemistry, and mass spectrometry studies allowed us to assess DBC1 mRNA and protein levels, localization, phosphorylation status, and protein interaction networks. The comparison of DBC1 interactions in these cell types revealed conserved regulatory roles for DBC1 in gene expression, chromatin organization and modification, and cell cycle progression. Interestingly, we observe previously unrecognized DBC1 interactions with proteins encoded by cancer-associated genes. Among these interactions are five components of the SWI/SNF complex, the most frequently mutated chromatin remodeling complex in human cancers. Additionally, we identified a DBC1 interaction with TBL1XR1, a component of the NCoR complex, which we validated by reciprocal isolation. Strikingly, we discovered that DBC1 associates with proteins that regulate the circadian cycle, including DDX5, DHX9, and SFPQ. We validated this interaction by colocalization and reciprocal isolation. Functional assessment of this association demonstrated that DBC1 protein levels are important for regulating CLOCK and BMAL1 protein oscillations in synchronized T cells. Our results suggest that DBC1 is integral to the maintenance of the circadian molecular clock. Furthermore, the identified interactions provide a valuable resource for the exploration of pathways involved in DBC1-associated tumorigenesis.
Carabetta, Valerie J, et al.Temporal Regulation of the Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth”. mSystems 13 (2016). Web.Abstract
-Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. IMPORTANCE: The past decade highlighted -lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in . To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth.
Song, Ren, et al.Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion”. mBio 71 (2016): , 7, 1, e02145-15. Web.Abstract
UNLABELLED: Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β) or gamma interferon (IFN-γ) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion. IMPORTANCE: Neurons are highly polarized cells with long axonal processes that connect to distant targets. PNS axons that innervate peripheral tissues are exposed to various situations that follow infection, inflammation, and damage of the tissue. After viral infection in the periphery, axons represent potential front-line barriers to PNS infection and damage. Indeed, most viral infections do not spread to the PNS, yet the mechanisms responsible are not well studied. We devised an experimental system to study how axons respond to inflammatory cytokines that would be produced by infected tissues. We found that axons respond differentially to type I and type II interferons. The response to type I interferon (IFN-β) is a rapid axon-only response. The response to type II interferon (IFN-γ) involves long-distance signaling to the PNS cell body. These responses to two interferons erect an efficient and rapid barrier to PNS infection.
Diner, Benjamin A, et al.Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection”. mBio 76 (2016). Web.Abstract
The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. IMPORTANCE: How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.
BA, Diner, et al.Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection”. mBio 76 (2016). Print.
PM, Jean Beltran, Mathias RA, and Cristea IM. “A Portrait of the Human Organelle Proteome In Space and Time during Cytomegalovirus Infection”. Cell Syst. 34 (2016): , 3, 4, 361-373. Print.
MS, Crow, et al.Diverse mechanisms evolved by DNA viruses to inhibit early host defenses.”. Crit Rev Biochem Mol Biol. 51.6 (2016): , 51, 6, 452-481. Print.
HG, Budayeva, and Cristea IM. “Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking”. Mol Cell Proteomics 15.10 (2016): , 15, 10, 3107-3125. Print.
RA, Mathias, Greco TM, and Cristea IM. “Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators”. Methods Mol Biol 1436.213-39 (2016). Print.
AJ, Guise, and Cristea IM. “Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5)”. Methods Mol Biol 1436 (2016): , 1436, 47-84. Print.
TM, Greco, and Cristea IM. “The Biochemical Evolution of Protein Complexes.”. Trends Biochem Sci. 41.1 (2016): , 41, 1, 4-6. Web. PubMedAbstract

Over the past decade, it became evident that proteins perform critical functions as components of specialized macromolecular complexes. Here, we discuss a recent study by Wan and colleagues, which highlights the significance of protein complexes by studying their conservation in organisms separated by up to a billion years of evolution. 

L, Waldron, et al.The Cardiac TBX5 Interactome Reveals a Chromatin Remodeling Network Essential for Cardiac Septation.”. Dev Cell 36.3 (2016): , 36, 3, 262-75. Web. PubMedAbstract

Human mutations in the cardiac transcription factor gene TBX5 cause congenital heart disease (CHD), although the underlying mechanism is unknown. We report characterization of the endogenous TBX5 cardiac interactome and demonstrate that TBX5, long considered a transcriptional activator, interacts biochemically and genetically with the nucleosome remodeling and deacetylase (NuRD) repressor complex. Incompatible gene programs are repressed by TBX5 in the developing heart. CHD mis-sense mutations that disrupt the TBX5-NuRD interaction cause depression of a subset of repressed genes. Furthermore, the TBX5-NuRD interaction is required for heart development. Phylogenetic analysis showed that the TBX5-NuRD interaction domain evolved during early diversification of vertebrates, simultaneous with the evolution of cardiac septation. Collectively, this work defines a TBX5-NuRD interaction essential to cardiac development and the evolution of the mammalian heart, and when altered may contribute to human CHD. 

TM, Greco, Guise AJ, and Cristea IM. “Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.”. Methods Mol Biol (2016). Web. PubMedAbstract

In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. 

KK, Lum, and Cristea IM. “Proteomic approaches to uncovering virus-host protein interactions during the progression of viral infection.”. Expert Rev Proteomics 13.3 (2016): , 13, 3, 325-40. Web. PubMedAbstract

The integration of proteomic methods to virology has facilitated a significant breadth of biological insight into mechanisms of virus replication, antiviral host responses and viral subversion of host defenses. Throughout the course of infection, these cellular mechanisms rely heavily on the formation of temporally and spatially regulated virus-host protein-protein interactions. Reviewed here are proteomic-based approaches that have been used to characterize this dynamic virus-host interplay. Specifically discussed are the contribution of integrative mass spectrometry, antibody-based affinity purification of protein complexes, cross-linking and protein array techniques for elucidating complex networks of virus-host protein associations during infection with a diverse range of RNA and DNA viruses. The benefits and limitations of applying proteomic methods to virology are explored, and the contribution of these approaches to important biological discoveries and to inspiring new tractable avenues for the design of antiviral therapeutics is highlighted. 

Song, R, et al.Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion.”. MBio 71 (2016). Web. PubMedAbstract

nfection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β) or gamma interferon (IFN-γ) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion.

IMPORTANCE:

Neurons are highly polarized cells with long axonal processes that connect to distant targets. PNS axons that innervate peripheral tissues are exposed to various situations that follow infection, inflammation, and damage of the tissue. After viral infection in the periphery, axons represent potential front-line barriers to PNS infection and damage. Indeed, most viral infections do not spread to the PNS, yet the mechanisms responsible are not well studied. We devised an experimental system to study how axons respond to inflammatory cytokines that would be produced by infected tissues. We found that axons respond differentially to type I and type II interferons. The response to type I interferon (IFN-β) is a rapid axon-only response. The response to type II interferon (IFN-γ) involves long-distance signaling to the PNS cell body. These responses to two interferons erect an efficient and rapid barrier to PNS infection.

2015
Diner, Benjamin A, and Ileana M Cristea. “Blowing Off Steam: Virus Inhibition of cGAS DNA Sensing”. Cell Host Microbe 18.3 (2015): , 18, 3, 270-2. Web.Abstract
Detection of viral DNA is essential for eliciting mammalian innate immunity. However, viruses have acquired effective mechanisms for blocking host defense. Indeed, in this issue of Cell Host & Microbe, Wu et al. (2015) discover a herpesviral strategy for inhibiting the prominent host sensor of viral DNA, cGAS.
Orzalli, Megan H, et al.cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection”. Proc Natl Acad Sci U S A 112.14 (2015): , 112, 14, E1773-81. Web.Abstract
Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.
Hahn, Jeanette, et al.ComGA-RelA interaction and persistence in the Bacillus subtilis K-state”. Mol Microbiol 97.3 (2015): , 97, 3, 454-71. Web.Abstract
The bistably expressed K-state of Bacillus subtilis is characterized by two distinct features; transformability and arrested growth when K-state cells are exposed to fresh medium. The arrest is manifested by a failure to assemble replisomes and by decreased rates of cell growth and rRNA synthesis. These phenotypes are all partially explained by the presence of the AAA(+) protein ComGA, which is also required for the binding of transforming DNA to the cell surface and for the assembly of the transformation pilus that mediates DNA transport. We have discovered that ComGA interacts with RelA and that the ComGA-dependent inhibition of rRNA synthesis is largely bypassed in strains that cannot synthesize the alarmone (p)ppGpp. We propose that the interaction of ComGA with RelA prevents the hydrolysis of (p)ppGpp in K-state cells, which are thus trapped in a non-growing state until ComGA is degraded. We show that some K-state cells exhibit tolerance to antibiotics, a form of type 1 persistence, and we propose that the bistable expression of both transformability and the growth arrest are bet-hedging adaptations that improve fitness in the face of varying environments, such as those presumably encountered by B. subtilis in the soil.
Rowles, Daniell L, et al.DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection”. Proteomics 15.12 (2015): , 15, 12, 1968-82. Web.Abstract
Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.
Diner, Benjamin A, Krystal K Lum, and Ileana M Cristea. “The emerging role of nuclear viral DNA sensors”. J Biol Chem 290.44 (2015): , 290, 44, 26412-21. Web.Abstract
Detecting pathogenic DNA by intracellular receptors termed "sensors" is critical toward galvanizing host immune responses and eliminating microbial infections. Emerging evidence has challenged the dogma that sensing of viral DNA occurs exclusively in sub-cellular compartments normally devoid of cellular DNA. The interferon-inducible protein IFI16 was shown to bind nuclear viral DNA and initiate immune signaling, culminating in antiviral cytokine secretion. Here, we review the newly characterized nucleus-originating immune signaling pathways, their links to other crucial host defenses, and unique mechanisms by which viruses suppress their functions. We frame these findings in the context of human pathologies associated with nuclear replicating DNA viruses.
Diner, Benjamin A, et al.The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA”. Mol Syst Biol 11.1 (2015): , 11, 1, 787. Web.Abstract
The human PYHIN proteins, AIM2, IFI16, IFIX, and MNDA, are critical regulators of immune response, transcription, apoptosis, and cell cycle. However, their protein interactions and underlying mechanisms remain largely uncharacterized. Here, we provide the interaction network for all PYHIN proteins and define a function in sensing of viral DNA for the previously uncharacterized IFIX protein. By designing a cell-based inducible system and integrating microscopy, immunoaffinity capture, quantitative mass spectrometry, and bioinformatics, we identify over 300 PYHIN interactions reflective of diverse functions, including DNA damage response, transcription regulation, intracellular signaling, and antiviral response. In view of the IFIX interaction with antiviral factors, including nuclear PML bodies, we further characterize IFIX and demonstrate its function in restricting herpesvirus replication. We discover that IFIX detects viral DNA in both the nucleus and cytoplasm, binding foreign DNA via its HIN domain in a sequence-non-specific manner. Furthermore, IFIX contributes to the induction of interferon response. Our results highlight the value of integrative proteomics in deducing protein function and establish IFIX as an antiviral DNA sensor important for mounting immune responses.
Diner, Benjamin A, et al.Interactions of the Antiviral Factor Interferon Gamma-Inducible Protein 16 (IFI16) Mediate Immune Signaling and Herpes Simplex Virus-1 Immunosuppression”. Mol Cell Proteomics 14.9 (2015): , 14, 9, 2341-56. Web.Abstract
The interferon-inducible protein IFI16 has emerged as a critical antiviral factor and sensor of viral DNA. IFI16 binds nuclear viral DNA, triggering expression of antiviral cytokines during infection with herpesviruses. The knowledge of the mechanisms and protein interactions through which IFI16 exerts its antiviral functions remains limited. Here, we provide the first characterization of endogenous IFI16 interactions following infection with the prominent human pathogen herpes simplex virus 1 (HSV-1). By integrating proteomics and virology approaches, we identified and validated IFI16 interactions with both viral and host proteins that are involved in HSV-1 immunosuppressive mechanisms and host antiviral responses. We discover that during early HSV-1 infection, IFI16 is recruited to sub-nuclear puncta and subsequently targeted for degradation. We observed that the HSV-1 E3 ubiquitin ligase ICP0 is necessary, but not sufficient, for the proteasom e-mediated degradation of IFI16 following infection. We substantiate that this ICP0-mediated mechanism suppresses IFI16-dependent immune responses. Utilizing an HSV-1 strain that lacks ICP0 ubiquitin ligase activity provided a system for studying IFI16-dependent cytokine responses to HSV-1, as IFI16 levels were maintained throughout infection. We next defined temporal IFI16 interactions during this immune signaling response. We discovered and validated interactions with the viral protein ICP8 and cellular ND10 nuclear body components, sites at which HSV-1 DNA is present during infection. These interactions may be critical for IFI16 to bind to nuclear viral DNA. Altogether, our results provide critical insights into both viral inhibition of IFI16 and interactions that can contribute to IFI16 antiviral functions.
Koyuncu, Orkide O, et al.The number of alphaherpesvirus particles infecting axons and the axonal protein repertoire determines the outcome of neuronal infection”. mBio 62 (2015). Web.Abstract
UNLABELLED: Infection by alphaherpesviruses invariably results in invasion of the peripheral nervous system (PNS) and establishment of either a latent or productive infection. Infection begins with long-distance retrograde transport of viral capsids and tegument proteins in axons toward the neuronal nuclei. Initial steps of axonal entry, retrograde transport, and replication in neuronal nuclei are poorly understood. To better understand how the mode of infection in the PNS is determined, we utilized a compartmented neuron culturing system where distal axons of PNS neurons are physically separated from cell bodies. We infected isolated axons with fluorescent-protein-tagged pseudorabies virus (PRV) particles and monitored viral entry and transport in axons and replication in cell bodies during low and high multiplicities of infection (MOIs of 0.01 to 100). We found a threshold for efficient retrograde transport in axons between MOIs of 1 and 10 and a threshold for productive infection in the neuronal cell bodies between MOIs of 1 and 0.1. Below an MOI of 0.1, the viral genomes that moved to neuronal nuclei were silenced. These genomes can be reactivated after superinfection by a nonreplicating virus, but not by a replicating virus. We further showed that viral particles at high-MOI infections compete for axonal proteins and that this competition determines the number of viral particles reaching the nuclei. Using mass spectrometry, we identified axonal proteins that are differentially regulated by PRV infection. Our results demonstrate the impact of the multiplicity of infection and the axonal milieu on the establishment of neuronal infection initiated from axons. IMPORTANCE: Alphaherpesvirus genomes may remain silent in peripheral nervous system (PNS) neurons for the lives of their hosts. These genomes occasionally reactivate to produce infectious virus that can reinfect peripheral tissues and spread to other hosts. Here, we use a neuronal culture system to investigate the outcome of axonal infection using different numbers of viral particles and coinfection assays. We found that the dynamics of viral entry, transport, and replication change dramatically depending on the number of virus particles that infect axons. We demonstrate that viral genomes are silenced when the infecting particle number is low and that these genomes can be reactivated by superinfection with UV-inactivated virus, but not with replicating virus. We further show that viral invasion rapidly changes the profiles of axonal proteins and that some of these axonal proteins are rate limiting for efficient infection. Our study provides new insights into the establishment of silent versus productive alphaherpesvirus infections in the PNS.
Mathias, Rommel A, Amanda J Guise, and Ileana M Cristea. “Post-translational modifications regulate class IIa histone deacetylase (HDAC) function in health and disease”. Mol Cell Proteomics 14.3 (2015): , 14, 3, 456-70. Web.Abstract
Class IIa histone deacetylases (HDACs4, -5, -7, and -9) modulate the physiology of the human cardiovascular, musculoskeletal, nervous, and immune systems. The regulatory capacity of this family of enzymes stems from their ability to shuttle between nuclear and cytoplasmic compartments in response to signal-driven post-translational modification. Here, we review the current knowledge of modifications that control spatial and temporal histone deacetylase functions by regulating subcellular localization, transcriptional functions, and cell cycle-dependent activity, ultimately impacting on human disease. We discuss the contribution of these modifications to cardiac and vascular hypertrophy, myoblast differentiation, neuronal cell survival, and neurodegenerative disorders.
Lin, Kah-Wai, et al.Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length”. Nat Commun 6 (2015): , 6, 8290. Web.Abstract
Almost 400 genes affect yeast telomere length, including Est1, which is critical for recruitment and activation of telomerase. Here we use mass spectrometry to identify novel telomerase regulators by their co-purification with the telomerase holoenzyme. In addition to all known subunits, over 100 proteins are telomerase associated, including all three subunits of the essential Cdc48-Npl4-Ufd1 complex as well as three E3 ubiquitin ligases. The Cdc48 complex is evolutionarily conserved and targets ubiquitinated proteins for degradation. Est1 levels are ∼40-fold higher in cells with reduced Cdc48, yet, paradoxically, telomeres are shorter. Furthermore, Est1 is ubiquitinated and its cell cycle-regulated abundance is lost in Cdc48-deficient cells. Deletion of the telomerase-associated E3 ligase, Ufd4, in cdc48-3 cells further increases Est1 abundance but suppresses the telomere length phenotype of the single mutant. These data argue that, in concert with Ufd4, the Cdc48 complex regulates telomerase by controlling the level and activity of Est1.
Crow, Marni S, Aaron Javitt, and Ileana M Cristea. “A proteomics perspective on viral DNA sensors in host defense and viral immune evasion mechanisms”. J Mol Biol 427.11 (2015): , 427, 11, 1995-2012. Web.Abstract
The sensing of viral DNA is an essential step of cellular immune response to infections with DNA viruses. These human pathogens are spread worldwide, triggering a wide range of virus-induced diseases, and are associated with high levels of morbidity and mortality. Despite similarities between DNA molecules, mammalian cells have the remarkable ability to distinguish viral DNA from their own DNA. This detection is carried out by specialized antiviral proteins, called DNA sensors. These sensors bind to foreign DNA to activate downstream immune signaling pathways and alert neighboring cells by eliciting the expression of antiviral cytokines. The sensing of viral DNA was shown to occur both in the cytoplasm and in the nucleus of infected cells, disproving the notion that sensing occurred by simple spatial separation of viral and host DNA. A number of omic approaches, in particular, mass-spectrometry-based proteomic methods, have significantly contributed to the constantly evolving field of viral DNA sensing. Here, we review the impact of omic methods on the identification of viral DNA sensors, as well as on the characterization of mechanisms involved in host defense or viral immune evasion.
Cristea, Ileana M, and David Graham. “Virology meets Proteomics”. Proteomics 15.12 (2015): , 15, 12, 1941-2. Web.
2014
Castellana, Michele, et al.Enzyme clustering accelerates processing of intermediates through metabolic channeling”. Nat Biotechnol 32.10 (2014): , 32, 10, 1011-8. Web.Abstract
We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.
Greco, Todd M, Benjamin A Diner, and Ileana M Cristea. “The Impact of Mass Spectrometry-Based Proteomics on Fundamental Discoveries in Virology”. Annu Rev Virol 11 (2014): , 1, 1, 581-604. Web.Abstract
In recent years, mass spectrometry has emerged as a core component of fundamental discoveries in virology. As a consequence of their coevolution, viruses and host cells have established complex, dynamic interactions that function either in promoting virus replication and dissemination or in host defense against invading pathogens. Thus, viral infection triggers an impressive range of proteome changes. Alterations in protein abundances, interactions, posttranslational modifications, subcellular localizations, and secretion are temporally regulated during the progression of an infection. Consequently, understanding viral infection at the molecular level requires versatile approaches that afford both breadth and depth of analysis. Mass spectrometry is uniquely positioned to bridge this experimental dichotomy. Its application to both unbiased systems analyses and targeted, hypothesis-driven studies has accelerated discoveries in viral pathogenesis and host defense. Here, we review the contributions of mass spectrometry-based proteomic approaches to understanding viral morphogenesis, replication, and assembly and to characterizing host responses to infection.
Jean Beltran, Pierre M, and Ileana M Cristea. “The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics”. Expert Rev Proteomics 11.6 (2014): , 11, 6, 697-711. Web.Abstract
Viruses have coevolved with their hosts, acquiring strategies to subvert host cellular pathways for effective viral replication and spread. Human cytomegalovirus (HCMV), a widely-spread β-herpesvirus, is a major cause of birth defects and opportunistic infections in HIV-1/AIDS patients. HCMV displays an intricate system-wide modulation of the human cell proteome. An impressive array of virus-host protein interactions occurs throughout the infection. To investigate the virus life cycle, proteomics has recently become a significant component of virology studies. Here, we review the mass spectrometry-based proteomics approaches used in HCMV studies, as well as their contribution to understanding the HCMV life cycle and the virus-induced changes to host cells. The importance of the biological insights gained from these studies clearly demonstrate the impact that proteomics has had and can continue to have on understanding HCMV biology and identifying new therapeutic targets.
Budayeva, Hanna G, and Ileana M Cristea. “A mass spectrometry view of stable and transient protein interactions”. Adv Exp Med Biol 806 (2014): , 806, 263-82. Web.Abstract
Through an impressive range of dynamic interactions, proteins succeed to carry out the majority of functions in a cell. These temporally and spatially regulated interactions provide the means through which one single protein can perform diverse functions and modulate different cellular pathways. Understanding the identity and nature of these interactions is therefore critical for defining protein functions and their contribution to health and disease processes. Here, we provide an overview of workflows that incorporate immunoaffinity purifications and quantitative mass spectrometry (frequently abbreviated as IP-MS or AP-MS) for characterizing protein-protein interactions. We discuss experimental aspects that should be considered when optimizing the isolation of a protein complex. As the presence of nonspecific associations is a concern in these experiments, we discuss the common sources of nonspecific interactions and present label-free and metabolic labeling mass spectrometry-based methods that can help determine the specificity of interactions. The effective regulation of cellular pathways and the rapid reaction to various environmental stresses rely on the formation of stable, transient, and fast-exchanging protein-protein interactions. While determining the exact nature of an interaction remains challenging, we review cross-linking and metabolic labeling approaches that can help address this important aspect of characterizing protein interactions and macromolecular assemblies.
Guise, Amanda J, et al.Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5)”. Proteomics 14.19 (2014): , 14, 19, 2156-66. Web.Abstract
Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin-6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves this transcriptional repressive function. Class IIa HDAC shuttling is controlled, in part, by phosphorylations flanking the nuclear localization signal (NLS). Furthermore, we have reported that phosphorylation within the NLS by the kinase Aurora B modulates the localization and function of the class IIa HDAC5 during mitosis. While we identified numerous additional HDAC5 phosphorylations, their regulatory functions remain unknown. Here, we studied phosphorylation sites within functional HDAC5 domains, including the deacetylation domain (DAC, Ser755), nuclear export signal (NES, Ser1108), and an acidic domain (AD, Ser611). We have generated phosphomutant cell lines to investigate how absence of phosphorylation at these sites impacts HDAC5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative MS, we have defined the interactions and HDAC5-containing complexes mediated by site-specific phosphorylation and quantified selected changes using parallel reaction monitoring. These results expand the current understanding of HDAC regulation, and the functions of this critical family of proteins within human cells.
Miteva, Yana V, and Ileana M Cristea. “A proteomic perspective of Sirtuin 6 (SIRT6) phosphorylation and interactions and their dependence on its catalytic activity”. Mol Cell Proteomics 13.1 (2014): , 13, 1, 168-83. Web.Abstract
Sirtuin 6 (SIRT6), a member of the mammalian sirtuin family, is a nuclear deacetylase with substrate-specific NAD(+)-dependent activity. SIRT6 has emerged as a critical regulator of diverse processes, including DNA repair, gene expression, telomere maintenance, and metabolism. However, our knowledge regarding its interactions and regulation remains limited. Here, we present a comprehensive proteomics-based analysis of SIRT6 protein interactions and their dependence on SIRT6 catalytic activity. We also identify evolutionarily conserved SIRT6 phosphorylations, including four within a proline-rich disordered region, and show that the conserved S338 phosphorylation can modulate selected SIRT6 interactions. By integrating molecular biology tools, microscopy, immunoaffinity purifications, label-free quantitative mass spectrometry, and bioinformatic analyses, we have established the first large-scale SIRT6 interaction network. Relative protein abundances and gene ontology functional assessment highlighted proteins involved in transcription regulation, chromatin organization, nuclear transport, telomerase function, and RNA processing. Independent immunoisolations under increased stringency distinguished the most stable SIRT6 interactions. One prominent interaction with Ras-GTPase-activating protein-binding protein 1 (G3BP1) was further validated by microscopy, reciprocal purifications, and isolations in different cell types and of endogenous SIRT6. Interestingly, a subset of specific interactions, including G3BP1, were significantly reduced or abolished in isolations of catalytically deficient SIRT6 mutant, revealing previously unknown interplay between SIRT6 activity and its associations. Overall, our study reveals putative means of regulation of SIRT6 functions via interactions and modifications, providing an important resource for future studies on the molecular mechanisms underlying sirtuin functions.
Amin, Nirav M, et al.Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)”. Development 141.4 (2014): , 141, 4, 962-73. Web.Abstract
The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.
Mathias, Rommel A, et al.Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity”. Cell 159.7 (2014): , 159, 7, 1615-25. Web.Abstract
Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.
Tsai, Yuan-Chin, Todd M Greco, and Ileana M Cristea. “Sirtuin 7 plays a role in ribosome biogenesis and protein synthesis”. Mol Cell Proteomics 13.1 (2014): , 13, 1, 73-83. Web.Abstract
It has been shown that SIRT7 regulates rDNA transcription and that reduced SIRT7 levels inhibit tumor growth. This anti-tumor effect could be due to reduced Pol I activity and perturbed ribosome biogenesis. In this study, using pulse labeling with RNA and amino acid analogs, we found that SIRT7 knockdown efficiently suppressed both RNA and protein synthesis. Surprisingly, SIRT7 knockdown preferentially inhibited protein synthesis over rDNA transcription, whereas the levels of both were reduced to similar extents following Pol I knockdown. Using an affinity purification mass spectrometry approach and functional analyses of the resulting SIRT7 interactome, we identified and validated SIRT7 interactions with proteins involved in ribosomal biogenesis. Indeed, SIRT7 co-fractionated with monoribosomes within a sucrose gradient. Using reciprocal isolations, we determined that SIRT7 interacts specifically with mTOR and GTF3C1, a component of the Pol III transcription factor TFIIIC2 complex. Further studies found that SIRT7 knockdown triggered an increase in the levels of LC3B-II, an autophagosome marker, suggesting a link between SIRT7 and the mTOR pathway. Additionally, we provide several lines of evidence that SIRT7 plays a role in modulating Pol III function. Immunoaffinity purification of SIRT7-GFP from a nuclear fraction demonstrated specific SIRT7 interaction with five out of six components of the TFIIIC2 complex, but not with the TFIIIA or TFIIIB complex, the former of which is required for Pol III-dependent transcription of tRNA genes. ChIP assays showed SIRT7 localization to the Pol III targeting genes, and SIRT7 knockdown triggered a reduction in tRNA levels. Taken together, these data suggest that SIRT7 may regulate Pol III transcription through mTOR and the TFIIIC2 complex. We propose that SIRT7 is involved in multiple pathways involved in ribosome biogenesis, and we hypothesize that its down-regulation may contribute to an antitumor effect, partly through the inhibition of protein synthesis.
Koyuncu, Emre, et al.Sirtuins are evolutionarily conserved viral restriction factors”. mBio 56 (2014). Web.Abstract
UNLABELLED: The seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD(+)-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue in Escherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity. IMPORTANCE: We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.
2013
Mann, Jessica M, et al.Complex formation and processing of the minor transformation pilins of Bacillus subtilis”. Mol Microbiol 90.6 (2013): , 90, 6, 1201-15. Web.Abstract
Transformation in most bacteria is dependent on orthologues of Type 2 secretion and Type 4 pilus system proteins. In each system, pilin proteins (major and minor) are required to make the pilus structure and are essential to the process, although the precise roles of the minor pilins remain unclear. We have explored protein-protein interactions among the competence minor pilins of Bacillus subtilis through in vitro binding studies, immunopurification and mass spectrometry. We demonstrate that the minor pilins directly interact, and the minor pilin ComGG interacts with most of the known proteins required for transformation. We find that ComGG requires other ComG proteins for its stabilization and for processing by the pre-pilin peptidase. These observations, C-terminal mutations in ComGG that prevent processing and the inaccessibility of pre-ComGG to externally added protease suggest a model in which pre-ComGG must be associated with other minor pilins for processing to take place. We propose that ComGG does not become a transmembrane protein until after processing. These behaviours contrast with that of pre-ComGC, the major pilin, which is accessible to externally added protease and requires only the peptidase to be processed. The roles of the pilins and of the pilus in transformation are discussed.

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