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.

Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition.

Intelligent data acquisition (IDA) strategies, such as a real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). In this work, we introduce inSeqAPI, an instrument application programing interface (iAPI) program that enables construction of novel data acquisition algorithms. First, we analyze biotinylated cysteine peptides from ABPP experiments to demonstrate that a real-time search method within inSeqAPI performs similarly to an equivalent vendor method. Then, we describe PairQuant, a method within inSeqAPI designed for the hyperplexing approach that utilizes protein-level isotopic labeling and peptide-level TMT labeling. PairQuant allows for TMT analysis of 36 conditions in a single sample and achieves ∼98% coverage of both peptide pair partners in a hyperplexed experiment as well as a 40% improvement in the number of quantified cysteine sites compared with non-RTS acquisition. We applied this method in the ABPP study of ligandable cysteine sites in the nucleus leading to an identification of additional druggable sites on protein- and DNA-interaction domains of transcription regulators and on nuclear ubiquitin ligases.

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases.

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor-related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose-response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.

Primate cerebrospinal fluid CHI3L1 reflects brain TREM2 agonism.

INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.

Muscle specific kinase (MuSK) activation preserves neuromuscular junctions in the diaphragm but is not sufficient to provide a functional benefit in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking.

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-ß1 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.

Intricate Roles of Mammalian Sirtuins in Defense against Viral Pathogens.

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.

A mass spectrometry view of stable and transient protein interactions.

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.

Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression.

IMPORTANCE: This study expands the growing understanding that protein acetylation is a highly regulated molecular toggle of protein function in both host anti-viral defense and viral replication. We describe a pro-viral role for the human enzyme SIRT2, showing that its deacetylase activity supports HCMV replication. By integrating quantitative proteomics, flow cytometry cell cycle assays, microscopy, and functional virology assays, we investigate the temporality of SIRT2 functions and substrates. We identify a pro-viral role for the SIRT2 deacetylase activity via regulation of CDK2 K6 acetylation and the G1-S cell cycle transition. These findings highlight a link between viral infection, protein acetylation, and cell cycle progression.

Monitoring protein communities and their responses to therapeutics.

Most therapeutics are designed to alter the activities of proteins. From metabolic enzymes to cell surface receptors, connecting the function of a protein to a cellular phenotype, to the activity of a drug and to a clinical outcome represents key mechanistic milestones during drug development. Yet, even for therapeutics with exquisite specificity, the sequence of events following target engagement can be complex. Interconnected communities of structural, metabolic and signalling proteins modulate diverse downstream effects that manifest as interindividual differences in efficacy, adverse effects and resistance to therapy. Recent advances in mass spectrometry proteomics have made it possible to decipher these complex relationships and to understand how factors such as genotype, cell type, local environment and external perturbations influence them. In this Review, we explore how proteomic technologies are expanding our understanding of protein communities and their responses to large- and small-molecule therapeutics.

Transcriptional Elongation of HSV Immediate Early Genes by the Super Elongation Complex Drives Lytic Infection and Reactivation from Latency.

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.

Sirtuins are evolutionarily conserved viral restriction factors.

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.

Histone deacetylases in herpesvirus replication and virus-stimulated host defense.

Emerging evidence highlights a critical role for protein acetylation during herpesvirus infection. As prominent modulators of protein acetylation, histone deacetylases (HDACs) are essential transcriptional and epigenetic regulators. Not surprisingly, viruses have evolved a wide array of mechanisms to subvert HDAC functions. Here, we review the mechanisms underlying HDAC regulation during herpesvirus infection. We next discuss the roles of acetylation in host defense against herpesvirus infection. Finally, we provide a perspective on the contribution of current mass spectrometry-based "omic" technologies to infectious disease research, offering a systems biology view of infection.