Proteins directly control almost all cellular processes and researchers in many biological areas routinely use mass spectrometry for the characterization of proteins. Amongst a growing list of ...available quantitative proteomic techniques, Stable Isotope Labeling by Amino acids in Culture (SILAC) remains one of the most simple, accurate, and robust techniques for cultured cellular systems. SILAC enables strict quantitative peptide measurements, thus removing false positives and facilitates large-scale kinetics of entire proteomes. In this, chapter we describe an optimized labeling strategy and experimental design for SILAC workflows for characterizing the components downstream of cell death stimuli.
Sperm develop from puberty in the seminiferous tubules, inside the blood‐testis barrier to prevent their recognition as “non‐self” by the immune system, and it is widely assumed that human ...sperm‐specific proteins cannot access the circulatory or immune systems. Sperm‐specific proteins aberrantly expressed in cancer, known as cancer‐testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell‐derived and sperm‐specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is “outside” the blood‐testis barrier. From TIF, the proteins can access the circulatory‐ and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm‐specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood‐based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex‐specific and male‐fertility‐related responses.
BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent ...monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.
T cell receptors (TCRs) recognize antigens presented by major histocompatibility complex (MHC) and MHC class I-like molecules. We describe a diverse population of human γδ T cells isolated from ...peripheral blood and tissues that exhibit autoreactivity to the monomorphic MHC-related protein 1 (MR1). The crystal structure of a γδTCR-MR1-antigen complex starkly contrasts with all other TCR-MHC and TCR-MHC-I-like complex structures. Namely, the γδTCR binds underneath the MR1 antigen-binding cleft, where contacts are dominated by the MR1 α3 domain. A similar pattern of reactivity was observed for diverse MR1-restricted γδTCRs from multiple individuals. Accordingly, we simultaneously report MR1 as a ligand for human γδ T cells and redefine the parameters for TCR recognition.
Pathogenic Acinetobacter species, including Acinetobacter baumannii and Acinetobacter nosocomialis, are opportunistic human pathogens of increasing relevance worldwide. Although their mechanisms of ...drug resistance are well studied, the virulence factors that govern Acinetobacter pathogenesis are incompletely characterized. Here we define the complete secretome of A. nosocomialis strain M2 in minimal medium and demonstrate that pathogenic Acinetobacter species produce both a functional type I secretion system (T1SS) and a contact-dependent inhibition (CDI) system. Using bioinformatics, quantitative proteomics, and mutational analyses, we show that Acinetobacter uses its T1SS for exporting two putative T1SS effectors, an Repeats-in-Toxin (RTX)-serralysin-like toxin, and the biofilm-associated protein (Bap). Moreover, we found that mutation of any component of the T1SS system abrogated type VI secretion activity under nutrient-limited conditions, indicating a previously unrecognized cross-talk between these two systems. We also demonstrate that the Acinetobacter T1SS is required for biofilm formation. Last, we show that both A. nosocomialis and A. baumannii produce functioning CDI systems that mediate growth inhibition of sister cells lacking the cognate immunity protein. The Acinetobacter CDI systems are widely distributed across pathogenic Acinetobacter species, with many A. baumannii isolates harboring two distinct CDI systems. Collectively, these data demonstrate the power of differential, quantitative proteomics approaches to study secreted proteins, define the role of previously uncharacterized protein export systems, and observe cross-talk between secretion systems in the pathobiology of medically relevant Acinetobacter species.
The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during ...mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleBCR in C. rodentium) that modifies conserved arginine residues in death domain-containing host proteins with N-acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection in vitro, or C. rodentium infection in vivo. NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and C. rodentium infection caused rapid and preferential modification of Arg117 in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleBCR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.
Signaling via the intracellular pathogen receptors nucleotide‐binding oligomerization domain‐containing proteins NOD1 and NOD2 requires receptor interacting kinase 2 (RIPK2), an adaptor kinase that ...can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG‐tagged RIPK2 knock‐in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice, we were able to generate a detailed map of post‐translational modifications on RIPK2. Similar to other reports, we did not detect ubiquitination of RIPK2 lysine 209 during NOD2 signaling. However, using site‐directed mutagenesis we identified a new regulatory region on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP and downstream signaling outcomes.
Synopsis
Polyubiquitination on multiple lysine residues of RIPK2 is required for anti‐bacterial responses mediated by the pathogen recognition receptor NOD2. This study identifies a regulatory region on the C‐lobe of the kinase domain that controls binding of the E3 ligase XIAP, RIPK2 ubiquitination and downstream signalling events.
RIPK2 is phosphorylated and ubiquitinated on multiple residues in the kinase, intermediate and CARD domain during NOD2 signaling.
Mutagenesis of single modified sites has little impact on RIPK2 function.
K209 and I212 form a regulatory region on the C‐lobe of the kinase that controls XIAP binding and NOD2 signaling.
Polyubiquitination on multiple lysine residues of RIPK2 is required for anti‐bacterial responses mediated by the pathogen recognition receptor NOD2. This study identifies a regulatory region on the C‐lobe of the kinase domain that controls binding of the E3 ligase XIAP, RIPK2 ubiquitination and downstream signalling events.
Coactivator‐associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues of target proteins critical for health and disease. CARM1 has recently emerged as an ...important regulator of skeletal muscle biology. The purpose of this study was to further investigate CARM1 biology in skeletal muscle. For our first experiment, we utilized 3‐month‐old male and female mice (n = 3/sex) that were housed and cared for in accordance with Canadian Council for Animal Care guidelines. Mice were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; Epizyme, Inc.) or vehicle via oral gavage BID for 2, 4, or 8 days. Tissues were collected 6 hours following the final dose and muscle mass was recorded. Western blotting was performed to evaluate protein expression in the tibialis anterior (TA) muscle and liver. To assess CARM1 activity, we investigated the arginine methylation status (i.e., the methylated form of the protein relative to its total amount) of known CARM1 substrates BAF155 and PABP1, as well as the myocellular level of arginine methylated proteins preferentially marked by CARM1. We observed similar outcomes between males and females, so the data were collapsed by sex (n = 6). CARM1 inhibition significantly reduced BAF155 methylation status in the TA muscle by 70‐80% between 2‐8 days of treatment. Muscle PABP1 methylation status was also attenuated (p < 0.05) by 60‐65% after 4 and 8 days of exposure. Similarly, arginine methylation of CARM1‐specific substrates across all muscle proteins was reduced by ~50% (p < 0.05) between 2‐8 days of CARM1 inhibitor administration. The attenuation of BAF155 and PABP1 methylation status, as well as arginine methylated CARM1‐specific substrates, was greater in liver as compared to muscle. Next, we completed additional experiments to assess the effect of pharmacological CARM1 inhibition on skeletal muscle function. After 11 days of CARM1 inhibitor treatment, we observed a sex‐specific reduction (p < 0.05) in exercise capacity and muscular endurance such that muscle function in female mice was unaffected by CARM1 suppression. Collectively, these results suggest that global, pharmacological CARM1 inhibition preferentially impacts liver enzyme activity over muscle. Furthermore, CARM1 inhibitor treatment may affect muscle performance with males being more sensitive to the functional outcomes of CARM1 suppression than females. Future work in our laboratory will examine the mechanisms and functional consequences of sex‐based differences in CARM1 biology within skeletal muscle.
Thermal proteome profiling (TPP) is a powerful tool for drug target deconvolution. Recently, data-independent acquisition mass spectrometry (DIA-MS) approaches have demonstrated significant ...improvements to depth and missingness in proteome data, but traditional TPP (a.k.a. CEllular Thermal Shift Assay "CETSA") workflows typically employ multiplexing reagents reliant on data-dependent acquisition (DDA). Herein, we introduce a new experimental design for the Proteome Integral Solubility Alteration via label-free DIA approach (PISA-DIA). We highlight the proteome coverage and sensitivity achieved by using multiple overlapping thermal gradients alongside DIA-MS, which maximizes efficiencies in PISA sample concatenation and safeguards against missing protein targets that exist at high melting temperatures. We demonstrate our extended PISA-DIA design has superior proteome coverage as compared to using tandem-mass tags (TMT) necessitating DDA-MS analysis. Importantly, we demonstrate our PISA-DIA approach has the quantitative and statistical rigor using A-1331852, a specific inhibitor of BCL-xL. Due to the high melt temperature of this protein target, we utilized our extended multiple gradient PISA-DIA workflow to identify BCL-xL. We assert our novel overlapping gradient PISA-DIA-MS approach is ideal for unbiased drug target deconvolution, spanning a large temperature range whilst minimizing target dropout between gradients, increasing the likelihood of resolving the protein targets of novel compounds.
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