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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
-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.
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.
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.
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.