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BACKGROUND: Gender-affirming surgery (GAS) can be an important part of comprehensive care for transgender and gender diverse (TGD) individuals, but this care is not provided by the Department of Veterans Affairs (VA) because of an exclusion in the medical benefits package. OBJECTIVE: To describe the receipt of GAS by veterans and assess the associations between key sociodemographic characteristics and receipt of chest ("top") and genital ("bottom") surgeries. DESIGN: Cross-sectional national survey (the GendeR Affirming Care Evaluation (GRACE)), among TGD Veterans conducted between September 2022 and July 2023. PARTICIPANTS: A total of 6653 Veterans (54% response rate) completed the survey. MAIN MEASURES: Self-reported "top" and "bottom" GAS were key outcomes. Covariates included gender identity, sex assigned at birth, age, race, ethnicity, income, employment status, education, relationship status, sexual orientation, and geographic region. KEY RESULTS: Among all respondents, 39% had ≥ 1 GAS. Among the 4430 veterans interested in top surgery, 38% received it; 23% of 3911 veterans interested in bottom surgery had received it. In multivariable models, older age (50 + vs. 18-39) was associated with higher receipt of top and bottom surgery while nonbinary gender identity (vs. binary gender identity), lower household income (< $50,000 vs. > $75,000), less education (less than a college graduate vs. Master's degree or more), sexual orientations other than heterosexual, and residing in a region other than the Pacific were associated with lower receipt of top and bottom surgery. Individuals assigned male (vs. assigned female) at birth had lower receipt of top surgery and higher receipt of bottom surgery. CONCLUSIONS: GAS receipt was low and there were important disparities by gender, sex, income, education, sexual orientation, and region. By removing the exclusion to providing GAS, VA could reduce barriers to accessing GAS and decrease disparities among TGD veterans.
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OBJECTIVES: To examine the associations of two measures of minority stress, non-affirmation minority stress and internalized transphobia, with subjective cognitive decline (SCD) among transgender and gender diverse (TGD) veterans. METHOD: We administered a cross-sectional survey from September 2022 to July 2023 to TGD veterans. The final analytic sample included 3,152 TGD veterans aged ≥45 years. We used a generalized linear model with quasi-Poisson distribution to calculate prevalence ratios (PR) and 95% confidence intervals (CIs) measuring the relationship between non-affirmation minority stress and internalized transphobia and past-year SCD. RESULTS: The mean age was 61.3 years (SD = 9.7) and the majority (70%) identified as trans women or women. Overall, 27.2% (n = 857) reported SCD. Adjusted models revealed that TGD veterans who reported experiencing non-affirmation minority stress or internalized transphobia had greater risk of past-year SCD compared to those who did not report either stressor (aPR: 1.09, 95% CI: 1.04-1.15; aPR: 1.19, 95% CI: 1.12-1.27). CONCLUSION: Our findings demonstrate that proximal and distal processes of stigma are associated with SCD among TGD veterans and underscore the need for addressing multiple types of discrimination. Above all, these results indicate the lasting sequelae of transphobia and need for systemic changes to prioritize the safety and welfare of TGD people.
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γ-Secretase plays a pivotal role in the production of neurotoxic amyloid ß-peptides (Aß) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H(+)-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aß production.
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Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/imunologia , Proteínas de Membrana/metabolismo , Presenilina-2/metabolismo , Serina Endopeptidases/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mutação , Presenilina-2/genética , Ligação Proteica/genética , Serina Endopeptidases/genética , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
BACKGROUND: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. RESULTS: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. CONCLUSIONS: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.
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DJ-1 is a small but relatively abundant protein of unknown function that may undergo stress-dependent cellular translocation and has been implicated in both neurodegenerative diseases and cancer. As such, DJ-1 may be an excellent study object to elucidate the relative influence of the cellular context on its interactome and for exploring whether acute exposure to oxidative stressors alters its molecular environment. Using quantitative mass spectrometry, we conducted comparative DJ-1 interactome analyses from in vivo cross-linked brains or livers and from hydrogen peroxide-treated or naïve embryonic stem cells. The analysis identified a subset of glycolytic enzymes, heat shock proteins 70 and 90, and peroxiredoxins as interactors of DJ-1. Consistent with a role of DJ-1 in Hsp90 chaperone biology, we document destabilization of Hsp90 clients in DJ-1 knockout cells. We further demonstrate the existence of a C106 sulfinic acid modification within DJ-1 and thereby establish that this previously inferred modification also exists in vivo. Our data suggest that caution has to be exerted in interpreting interactome data obtained from a single biological source material and identify a role of DJ-1 as an oxidative stress sensor and partner of a molecular machinery notorious for its involvement in cell fate decisions.
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Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo , Proteômica/métodos , Animais , Cisteína/química , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Espectrometria de Massas/métodos , Camundongos , Camundongos Knockout , Peroxirredoxinas/química , Proteína Desglicase DJ-1 , Proteoma , Ácidos Sulfínicos/químicaRESUMO
The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C)) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP(C) interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP(C) paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP(C) and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP(C) with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP(Sc). A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP(C) organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.
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Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligossacarídeos/metabolismo , Proteínas PrPC/metabolismo , Príons/metabolismo , Animais , Western Blotting , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Biologia Computacional/métodos , Proteínas Ligadas por GPI , Expressão Gênica , Ácido Láctico/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Isomerases de Dissulfetos de Proteínas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , TransfecçãoRESUMO
Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin-like protein (PUP). In this study, we report a proteome-wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine-mediated pupylation acceptor sites was obtained by collision-induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one-third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.
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Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Ubiquitinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Família Multigênica , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
Nucleolin is an essential cellular receptor to human respiratory syncytial virus (RSV). Pharmacological targeting of the nucleolin RNA binding domain RBD1,2 can inhibit RSV infections in vitro and in vivo; however, the site(s) on RBD1,2 which interact with RSV are not known. We undertook a series of experiments designed to: document RSV-nucleolin co-localization on the surface of polarized MDCK cells using immunogold electron microscopy, to identify domains on nucleolin that physically interact with RSV using biochemical methods and determine their biological effects on RSV infection in vitro, and to carry out structural analysis toward informing future RSV drug development. Results of immunogold transmission and scanning electron microscopy showed RSV-nucleolin co-localization on the cell surface, as would be expected for a viral receptor. RSV, through its fusion protein (RSV-F), physically interacts with RBD1,2 and these interactions can be competitively inhibited by treatment with Palivizumab or recombinant RBD1,2. Treatment with synthetic peptides derived from two 12-mer domains of RBD1,2 inhibited RSV infection in vitro, with structural analysis suggesting these domains are potentially feasible for targeting in drug development. In conclusion, the identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development.
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Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Receptores Virais/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Humanos , Imuno-Histoquímica , Células Madin Darby de Rim Canino , Microscopia Eletrônica , Palivizumab/farmacologia , NucleolinaRESUMO
Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.
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Benzodiazepinonas/química , Inibidores Enzimáticos/química , Peptidomiméticos/química , Presenilina-1/química , Proteínas Recombinantes de Fusão/química , Regulação Alostérica , Secretases da Proteína Precursora do Amiloide/química , Domínio Catalítico , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Simulação de Acoplamento Molecular , Presenilina-1/antagonistas & inibidores , Presenilina-1/genética , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-AtividadeRESUMO
The time-controlled transcardiac perfusion crosslinking (tcTPC) method differs from conventional perfusion fixation in that the crosslinking reagent is administered throughout the circulatory system for only a relatively short period of time, thereby allowing limited crosslinking to occur. Bait protein complexes are isolated by affinity capture (AC) under stringent conditions and are recovered from the AC matrix by acidic elution. Affinity-purified proteins are reduced, alkylated, and digested with a specific endoproteinase, such as trypsin. Subsequently, peptides are isotopically labeled, separated by reversed-phase chromatography and analyzed by quantitative tandem mass spectrometry (MS/MS). The proteins crosslinked to the bait protein during tcTPC are identified by database searches with conventional protein identification software. The tcTPC strategy offers unique advantages over alternative approaches for studying a subset of protein complexes which require a particular environment for their structural integrity, such as membrane protein complexes that are notorious for their tendency to dissociate upon detergent solubilization. The sensitivity and utility of this method are influenced by the spatial distribution of chemical groups within the bait protein complexes that can engage in productive crosslinks.