Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.

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.

HTT-OMNI: A Web-based Platform for Huntingtin Interaction Exploration and Multi-omics Data Integration.

Huntington's disease (HD) is a progressive neurological disorder that is caused by polyglutamine expansion of the huntingtin (HTT) protein. With the hope to uncover key modifiers of disease, a focus of the field of HD research has been on characterizing HTT-interacting proteins (HIPs) and the effect of the HTT polyglutamine expansion on the cellular omics landscape. However, while hundreds of studies have uncovered over 3000 potential HIPs to date, a means to interrogate these complementary interaction and omics datasets does not exist. The lack of a unified platform for exploring this breadth of potential HIPs and associated omics data represents a substantial barrier toward understanding the impact of HTT polyQ expansion and identifying interactions proximal to HD pathogenesis. Here, we describe the development of a web-based platform called HTT-OMNI (HTT OMics and Network Integration). This application facilitates the visualization and exploration of ∼3400 potential HTT interactors (from the HINT database) and their associated polyQ-dependent omics measurements, such as transcriptome and proteome abundances. Additionally, HTT-OMNI allows for the integration of user-generated datasets with existing HIPs and omic measurements. We first demonstrate the utility of HTT-OMNI for filtering existing HTT PPIs based on a variety of experimental metadata parameters, highlighting its capacity to select for HIPs detected in specific model organisms and tissues. Next, we leverage our application to visualize the relationships between HTT PPIs, genetic disease modifiers, and their multiomic landscape. Finally, we generate and analyze a previously unreported dataset of HTT PPIs, aimed at defining tissue-specific HTT interactions and the polyQ-dependent modulation of their relative stabilities in the cortex and striatum of HD mouse models.

Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships.

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.

The DNA Sensor cGAS is Decorated by Acetylation and Phosphorylation Modifications in the Context of Immune Signaling.

The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared with the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses-HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection.

YfmK is an Nε-lysine acetyltransferase that directly acetylates the histone-like protein HBsu in Bacillus subtilis.

Nε-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 Bacillus subtilis 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 B. subtilis 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 B. subtilis 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.

Hdac4 Interactions in Huntington's Disease Viewed Through the Prism of Multiomics.

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.

The Biochemical Evolution of Protein Complexes.

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.

Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis.

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.

Proteomics Tracing the Footsteps of Infectious Disease.

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.

Contribution of Mass Spectrometry-Based Proteomics to Discoveries in Developmental Biology.

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.

Systematic discovery of structural elements governing stability of mammalian messenger RNAs.

Decoding post-transcriptional regulatory programs in RNA is a critical step towards the larger goal of developing predictive dynamical models of cellular behaviour. Despite recent efforts, the vast landscape of RNA regulatory elements remains largely uncharacterized. A long-standing obstacle is the contribution of local RNA secondary structure to the definition of interaction partners in a variety of regulatory contexts, including--but not limited to--transcript stability, alternative splicing and localization. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (for example, human cardiac troponin T) or affects other aspects of RNA biology. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence. Here we present a computational framework based on context-free grammars and mutual information that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behaviour. By applying this framework to genome-wide human mRNA stability data, we reveal eight highly significant elements with substantial structural information, for the strongest of which we show a major role in global mRNA regulation. Through biochemistry, mass spectrometry and in vivo binding studies, we identified human HNRPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1, also known as HNRNPA2B1) as the key regulator that binds this element and stabilizes a large number of its target genes. We created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach could also be used to reveal the structural elements that modulate other aspects of RNA behaviour.

The Proteomic Profile of Deleted in Breast Cancer 1 (DBC1) Interactions Points to a Multifaceted Regulation of Gene Expression.

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.

Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT).

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.

The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA.

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.

Sirtuin 7 plays a role in ribosome biogenesis and protein synthesis.

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.

DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

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.

A proteomic perspective of inbuilt viral protein regulation: pUL46 tegument protein is targeted for degradation by ICP0 during herpes simplex virus type 1 infection.

Much like the host cells they infect, viruses must also regulate their life cycles. Herpes simples virus type 1 (HSV-1), a prominent human pathogen, uses a promoter-rich genome in conjunction with multiple viral trans-activating factors. Following entry into host cells, the virion-associated outer tegument proteins pUL46 and pUL47 act to increase expression of viral immediate-early (α) genes, thereby helping initiate the infection life cycle. Because pUL46 has gone largely unstudied, we employed a hybrid mass spectrometry-based approach to determine how pUL46 exerts its functions during early stages of infection. For a spatio-temporal characterization of pUL46, time-lapse microscopy was performed in live cells to define its dynamic localization from 2 to 24 h postinfection. Next, pUL46-containing protein complexes were immunoaffinity purified during infection of human fibroblasts and analyzed by mass spectrometry to investigate virus-virus and virus-host interactions, as well as post-translational modifications. We demonstrated that pUL46 is heavily phosphorylated in at least 23 sites. One phosphorylation site matched the consensus 14-3-3 phospho-binding motif, consistent with our identification of 14-3-3 proteins and host and viral kinases as specific pUL46 interactions. Moreover, we determined that pUL46 specifically interacts with the viral E3 ubiquitin ligase ICP0. We demonstrated that pUL46 is partially degraded in a proteasome-mediated manner during infection, and that the catalytic activity of ICP0 is responsible for this degradation. This is the first evidence of a viral protein being targeted for degradation by another viral protein during HSV-1 infection. Together, these data indicate that pUL46 levels are tightly controlled and important for the temporal regulation of viral gene expression throughout the virus life cycle. The concept of a structural virion protein, pUL46, performing nonstructural roles is likely to reflect a theme common to many viruses, and a better understanding of these functions will be important for developing therapeutics.

A complex of YlbF, YmcA and YaaT regulates sporulation, competence and biofilm formation by accelerating the phosphorylation of Spo0A.

Bacillus subtilis has adopted a bet-hedging strategy to ensure survival in changing environments. From a clonal population, numerous sub-populations can emerge, expressing different sets of genes that govern the developmental processes of sporulation, competence and biofilm formation. The master transcriptional regulator Spo0A controls the entry into all three fates and the production of the phosphorylated active form of Spo0A is precisely regulated via a phosphorelay, involving at least four proteins. Two proteins, YmcA and YlbF were previously shown to play an unidentified role in the regulation of biofilm formation, and in addition, YlbF was shown to regulate competence and sporulation. Using an unbiased proteomics screen, we demonstrate that YmcA and YlbF interact with a third protein, YaaT to form a tripartite complex. We show that all three proteins are required for proper establishment of the three above-mentioned developmental states. We show that the complex regulates the activity of Spo0A in vivo and, using in vitro reconstitution experiments, determine that they stimulate the phosphorelay, probably by interacting with Spo0F and Spo0B. We propose that the YmcA-YlbF-YaaT ternary complex is required to increase Spo0A~P levels above the thresholds needed to induce development.

Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.