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Background: TNBCs have the highest mortality rate among all BC subtypes. There is thus an urgent and unmet need for effective targeted therapies in TNBC. Recently we, identified a novel agent ERX-41 that showed good efficacy in treating TNBC in preclinical mouse models, however, its molecular action remain unknown. In this study, we identified LIPA as novel molecular target of ERX-41. Methods: We have used CRISPR knockout pooled library and multiple TNBC models for identifying molecular target of ERX-41. Mechanistic studies were performed using LIPA mutants, RNA-seq, Turbo-ID mapping, Mass spectrometry, Immunoprecipitation, and Western blotting. The in vivo efficacy of ERX-41 was examined using four different patient-derived xenograft (PDX) models. We evaluated LIPA protein expression in TNBC using tissue microarray (TMA). Results: To identify the molecular target of ERX-41, we performed an unbiased CRISPR–Cas9 knockout (KO) screen in MDA-MB-231 cells and the results identified LIPA as a top hit. KO of LIPA alone (which encodes lysosomal acid lipase (LAL) abrogated cytotoxic response to ERX-41. Cellular thermal shift assays confirmed that ERX-41 binds to LAL. In silico modelling and mutational studies confirmed that ERX-41 interacts with LAL through residues in its LXXLL domain and that ERX-41 ability to induce ER stress and cell death in TNBC is independent of the lipase activity of LAL. Unbiased RNA-seq studies with and without ERX-41 in parental and LIPA KO SUM-159 cells revealed induction of genes involved in ER stress and UPR response by ERX-41 in parental SUM-159 cells but not in cells with LIPA KO. Ultrastructural studies using live-cell confocal microscopy show that LIPA KO abrogated ER morphological changes at 2 and 4 h after ERX-41 treatment. Further, subcellular localization studies showed LIPA localizes to endoplasmic reticulum (ER). Unbiased proteomic approaches (TurboID and DIA mass spec) identified a core set of proteins that were both LAL binders and affected by ERX-41 treatment. GO analyses of LAL binding proteins confirmed their involvement in protein folding. Tumor micro array (TMA) analyses confirmed that >80% of primary TNBC tumors had significant and detectable LAL protein expression in contrast, normal breast tissue had lower LAL expression. ERX-41 (10 mg/kg body weight) decreased growth of four distinct TNBC patient-derived xenografts (PDXs) in vivo. Conclusions: Our results identified a new molecular target (LAL) for ERX-41 and novel mechanism of action (disruption of protein folding and induction of ER stress) that may have utility in treating patients with TNBC. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Uday Pratap, Gangadhara R. Sareddy, Susan T. Weintraub, Ganesh V. Raj, Jung-Mo Ahn, Ratna K. Vadlamudi. Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-14.

International audience; The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) illustrates that protein expression has now been credibly detected (neXtProt PE1 level) for 18,407 (93.2%) of the 19,750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from datasets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.

Accumulating evidence indicates that extracellular vesicles (EVs) mediate endocrine functions and also pathogenic effects of neurodevelopmental perturbagens like ethanol. We performed mass-spectrometry on EVs secreted by fetal murine cerebral cortical neural stem cells (NSCs), cultured

Mass spectrometry has greatly improved the analysis of phosphorylation events in complex biological systems and on a large scale. Despite considerable progress, the correct identification of phosphorylated sites, their quantification, and their interpretation regarding physiological relevance remain challenging. The MS Resource Pillar of the Human Proteome Organization (HUPO) Human Proteome Project (HPP) initiated the Phosphopeptide Challenge as a resource to help the community evaluate methods, learn procedures and data analysis routines, and establish their own workflows by comparing results obtained from a standard set of 94 phosphopeptides (serine, threonine, tyrosine) and their nonphosphorylated counterparts mixed at different ratios in a neat sample and a yeast background. Participants analyzed both samples with their method(s) of choice to report the identification and site localization of these peptides, determine their relative abundances, and enrich for the phosphorylated peptides in the yeast background. We discuss the results from 22 laboratories that used a range of different methods, instruments, and analysis software. We reanalyzed submitted data with a single software pipeline and highlight the successes and challenges in correct phosphosite localization. All of the data from this collaborative endeavor are shared as a resource to encourage the development of even better methods and tools for diverse phosphoproteomic applications. All submitted data and search results were uploaded to MassIVE (littps://massive.ucsd.edu/) as data set MSV000085932 with ProteomeXchange identifier PXD020801.

The older adult population, estimated to double by 2050, is at increased risk of respiratory infections and other pulmonary diseases. Biochemical changes in the lung alveolar lining fluid (ALF) and in alveolar compartment cells can alter local immune responses as we age, generating opportunities for invading pathogens to establish successful infections. Indeed, the lung alveolar space of older adults is a pro-inflammatory, pro-oxidative, dysregulated environment that remains understudied. We performed an exploratory, quantitative proteomic profiling of the soluble proteins present in ALF, developing insight into molecular fingerprints, pathways, and regulatory networks that characterize the alveolar space in old age, comparing it to that of younger individuals. We identified 457 proteins that were significantly differentially expressed in older adult ALF, including increased production of matrix metalloproteinases, markers of cellular senescence, antimicrobials, and proteins of neutrophilic granule origin, among others, suggesting that neutrophils in the lungs of older adults could be potential contributors to the dysregulated alveolar environment with increasing age. Finally, we describe a hypothetical regulatory network mediated by the serum response factor that could explain the neutrophilic profile observed in the older adult population.

Diversity of phage propagation, physical properties, and assembly promotes the use of phages in ecological studies and biomedicine. However, observed phage diversity is incomplete. Bacillus thuringiensis siphophage, 0105phi-7-2, first described here, significantly expands known phage diversity, as seen via in-plaque propagation, electron microscopy, whole genome sequencing/annotation, protein mass spectrometry, and native gel electrophoresis (AGE). Average plaque diameter vs. plaque-supporting agarose gel concentration plots reveal unusually steep conversion to large plaques as agarose concentration decreases below 0.2%. These large plaques sometimes have small satellites and are made larger by orthovanadate, an ATPase inhibitor. Phage head–host-cell binding is observed by electron microscopy. We hypothesize that this binding causes plaque size-increase via biofilm evolved, ATP stimulated ride-hitching on motile host cells by temporarily inactive phages. Phage 0105phi7-2 does not propagate in liquid culture. Genomic sequencing/annotation reveals history as temperate phage and distant similarity, in a virion-assembly gene cluster, to prototypical siphophage SPP1 of Bacillus subtilis. Phage 0105phi7-2 is distinct in (1) absence of head-assembly scaffolding via either separate protein or classically sized, head protein-embedded peptide, (2) producing partially condensed, head-expelled DNA, and (3) having a surface relatively poor in AGE-detected net negative charges, which is possibly correlated with observed low murine blood persistence.

The PELP1 oncogene is commonly overexpressed in many cancers, including triple negative breast cancer (TNBC). However, the mechanisms by which PELP1 contributes to TNBC progression are not well understood. To elucidate these mechanisms, we generated CRISPR-Cas9 mediated PELP1 knockout TNBC cell lines, and alterations in the proteome were examined using global data-independent acquisition mass spectrometry (DIA-MS). Further mechanistic studies utilized shRNA knockdown, Western blotting, and RNA-seq approaches. TCGA data sets were utilized for determining the status of PELP1 in TNBC patient tumors and for examining its correlation with ribosomal proteins. Global DIA-MS studies revealed that 127 proteins are upregulated while 220 proteins are downregulated upon PELP1-KO. Bioinformatic analyses suggested that the oncogenic activities of PELP1 involve regulation of expression of ribosomal proteins and ribosomal complexes. RNA-seq studies further suggested PELP1 modulates the functions of transcription factor c-Myc in TNBC. TCGA data confirmed PELP1 has high expression in TNBC patient tumors, and this high expression pattern correlates with c-Myc, a regulator of ribosomal proteins. Collectively, our global approach studies suggest that PELP1 contributes to TNBC progression by modulation of cell cycle, apoptosis, and ribosome biogenesis pathways.

TRIM5α is an E3 ubiquitin ligase of the TRIM family that binds to the capsids of primate immunodeficiency viruses and blocks viral replication after cell entry. Here we investigate how synthesis of K63-linked polyubiquitin is upregulated by transient proximity of three RING domains in honeycomb-like assemblies formed by TRIM5α on the surface of the retroviral capsid. Proximity of three RINGs creates an asymmetric arrangement, in which two RINGs form a catalytic dimer that activates E2-ubiquitin conjugates and the disordered N-terminus of the third RING acts as the substrate for N-terminal autoubiquitylation. RING dimerization is required for activation of the E2s that contribute to the antiviral function of TRIM5α, UBE2W and heterodimeric UBE2N/V2, whereas the proximity of the third RING enhances the rate of each of the two distinct steps in the autoubiquitylation process: the initial N-terminal monoubiquitylation (priming) of TRIM5α by UBE2W and the subsequent extension of the K63-linked polyubiquitin chain by UBE2N/V2. The mechanism we describe explains how recognition of infection-associated epitope patterns by TRIM proteins initiates polyubiquitin-mediated downstream events in innate immunity.

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRLVHL)/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL. Using this approach to guide our development of a series of ABT263-derived and VHL-recruiting PROTACs, we generate a potent BCL-xL and BCL-2 (BCL-xL/2) dual degrader with significantly improved antitumor activity against BCL-xL/2-dependent leukemia cells. Our results provide experimental evidence that the accessibility of lysines on a target protein plays an important role in determining the selectivity and potency of a PROTAC in inducing protein degradation, which may serve as a conceptual framework to guide the future development of PROTACs., Simultaneous targeting of BCL-xL and BCL-2 is an attractive approach for cancer treatment. Based on information gained by computational structure modelling, the authors develop a PROTAC that induces degradation of both BCL-xL and BCL-2 and effectively targets BCL-xL/2-dependent leukaemia cells.

The 2021 Metrics of the HUPO Human Proteome Project (HPP) show that protein expression has now been credibly detected (neXtProt PE1 level) for 18 357 (92.8%) of the 19 778 predicted proteins coded in the human genome, a gain of 483 since 2020 from reports throughout the world reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 478 to 1421. This represents remarkable progress on the proteome parts list. The utilization of proteomics in a broad array of biological and clinical studies likewise continues to expand with many important findings and effective integration with other omics platforms. We present highlights from the Immunopeptidomics, Glycoproteomics, Infectious Disease, Cardiovascular, Musculo-Skeletal, Liver, and Cancers B/D-HPP teams and from the Knowledgebase, Mass Spectrometry, Antibody Profiling, and Pathology resource pillars, as well as ethical considerations important to the clinical utilization of proteomics and protein biomarkers.

The elderly population is at increased risk of acute and chronic respiratory infections and other pulmonary diseases, and it is estimated that this population will double in the next 30 years. Biochemical changes in the lung alveolar mucosa and lung cells alter local immune response as we age, creating opportunities for invading pathogens to establish successful infections. Indeed, the lungs of the elderly are a pro-inflammatory, pro-oxidative, dysregulated environment but this environment has remained understudied. We performed a comprehensive, quantitative proteomic profile of the lung mucosa in the elderly, developing insight into the molecular fingerprints, pathways, and regulatory networks that characterize the lung in old age. We identified neutrophils in the lungs of elderly individuals as possible contributors to dysregulated lung tissue environment. This study establishes a baseline for future investigations to develop strategies to mitigate susceptibility to respiratory infections in the elderly.

The placental villus syncytiotrophoblast, the nutrient-transporting and hormone-producing epithelium of the human placenta, is a critical regulator of fetal development and maternal physiology. However, the identities of the proteins synthesized and secreted by primary human trophoblast (PHT) cells remain unknown. Stable Isotope Labeling with Amino Acids in Cell Culture followed by mass spectrometry analysis of the conditioned media was used to identify secreted proteins and obtain information about their relative rates of synthesis in syncytialized multinucleated PHT cells isolated from normal term placental villus tissue (n = 4/independent placenta). A total of 1,344 proteins were identified, most of which have not previously been reported to be secreted by the human placenta or trophoblast. The majority of secreted proteins are involved in energy and carbon metabolism, glycolysis, biosynthesis of amino acids, purine metabolism, and fatty acid degradation. Histone family proteins and mitochondrial proteins were among proteins with the slowest synthesis rate whereas proteins associated with signaling and the plasma membrane were synthesized rapidly. There was a significant overlap between the PHT secretome and proteins known be secreted to the fetal circulation by the human placenta in vivo. The generated data will guide future experiments to determine the function of individual secreted proteins and will help us better understand how the placenta controls maternal and fetal physiology.

The Human Proteome Organization’s (HUPO) Human Proteome Project (HPP) developed Mass Spectrometry (MS) Data Interpretation Guidelines that have been applied since 2016. These guidelines have helped ensure that the emerging draft of the complete human proteome is highly accurate and with low numbers of false-positive protein identifications. Here, we describe an update to these guidelines based on consensus-reaching discussions with the wider HPP community over the past year. The revised 3.0 guidelines address several major and minor identified gaps. We have added guidelines for emerging data independent acquisition (DIA) MS workflows and for use of the new Universal Spectrum Identifier (USI) system being developed by the HUPO Proteomics Standards Initiative (PSI). In addition, we discuss updates to the standard HPP pipeline for collecting MS evidence for all proteins in the HPP, including refinements to minimum evidence. We present a new plan for incorporating MassIVE-KB into the HPP pipeline for the next (HPP 2020) cycle in order to obtain more comprehensive coverage of public MS data sets. The main checklist has been reorganized under headings and subitems, and related guidelines have been grouped. In sum, Version 2.1 of the HPP MS Data Interpretation Guidelines has served well, and this timely update to version 3.0 will aid the HPP as it approaches its goal of collecting and curating MS evidence of translation and expression for all predicted ∼20 000 human proteins encoded by the human genome., This work was funded in part by the National Institutes of Health grants R01GM087221 (EWD/RLM), R24GM127667 (EWD), U54EB020406 (EWD), R01HL133135 (RLM), U19AG02312 (RLM), U54ES017885 (GSO), U24CA210967-01 (GSO), R01LM013115 (NB) and P41GM103484 (NB); National Science Foundation grants ABI-1759980 (NB), DBI-1933311 (EWD), and IOS-1922871 (EWD); Canadian Institutes of Health Research 148408 (CMO); Korean Ministry of Health and Welfare HI13C2098 (YKP); French Ministry of Higher Education, Research and Innovation, ProFI project, ANR-10-INBS-08 (YV); also in part by the National Eye Institute (NEI), National Human Genome Research Institute (NHGRI), National Heart, Lung, and Blood Institute (NHLBI), National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of General Medical Sciences (NIGMS), and National Institute of Mental Health (NIMH) of the National Institutes of Health under Award Number U24HG007822 (SO) (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health).

International audience; Abstract The mechanisms of placental transfer of long chain polyunsaturated fatty acids are poorly understood, however fatty acid transporters in the syncytiotrophoblast microvillous (MVM) and basal plasma membrane (BM) are believed to be involved. Using LC-MS/MS and samples from normal term pregnant women, we found that the concentration of lysophosphatidylcholine-docosahexaenoyl (DHA-LPC) was 4-fold higher in umbilical vein plasma compared to maternal venous concentrations while conversely phosphatidylcholine (PC) containing DHA or arachidonic acid were higher in maternal circulation. Using primary human trophoblast cells incubated with DHA we show that DHA was highly incorporated into PC and LPC species in the placenta. Protein expression of Phosphatidylcholine Transfer Protein (PCTP) was 14-fold higher in MVM and the expression of MFSD2a, an LPC transporter, was 5-fold higher in BM than in MVM. Interestingly, BM MFSD2a expression was positively correlated with DHA-LPC in the umbilical vein. These findings suggest that syncytiotrophoblast takes up PC from the maternal circulation, as well as preferentially incorporating DHA into PC. Placental PC are converted to LPC forms, which are transported across the BM mediated by MFSD2a, with a strong selectivity for DHA.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. β-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of β(2)-adrenergic receptors (β(2)-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of β(2)-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective β(2)-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying β(2)-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition—and also functional aspects thereof—in livers of formoterol-treated animals. Our results suggest that β(2)-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete β-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion—all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic β(2)-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD. NEW & NOTEWORTHY Results of our study suggest that β(2)-adrenergic receptor (β(2)-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete β-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for β(2)-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.

SummaryThe androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR’s individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging AR’s domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by AR’s oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of AR’s cooperative interactions translate to consequences in development and disease.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumor's extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal noncanonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analogue into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor cells from those of non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers.

Interactions between macrophages and adipocytes are early molecular factors influencing adipose tissue (AT) dysfunction, resulting in high leptin, low adiponectin circulating levels and low-grade metaflammation, leading to insulin resistance (IR) with increased cardiovascular risk. We report the characterization of AT dysfunction through measurements of the adiponectin/leptin ratio (ALR), the adipo-insulin resistance index (Adipo-IRi), fasting/postprandial (F/P) immunometabolic phenotyping and direct F/P differential gene expression in AT biopsies obtained from symptom-free adults from the GEMM family study. AT dysfunction was evaluated through associations of the ALR with F/P insulin-glucose axis, lipid-lipoprotein metabolism, and inflammatory markers. A relevant pattern of negative associations between decreased ALR and markers of systemic low-grade metaflammation, HOMA, and postprandial cardiovascular risk hyperinsulinemic, triglyceride and GLP-1 curves was found. We also analysed their plasma non-coding microRNAs and shotgun lipidomics profiles finding trends that may reflect a pattern of adipose tissue dysfunction in the fed and fasted state. Direct gene differential expression data showed initial patterns of AT molecular signatures of key immunometabolic genes involved in AT expansion, angiogenic remodelling and immune cell migration. These data reinforce the central, early role of AT dysfunction at the molecular and systemic level in the pathogenesis of IR and immunometabolic disorders.

Obesity is a chronic condition associated with dyslipidemia and insulin resistance. Here, we show that the offspring of obese mothers are dyslipidemic and insulin resistant from the outset.Maternal and cord blood and placental tissues were collected following C-section at term. Patients were grouped as being normal weight (NW, BMI = 18–24.9) or obese (OB, BMI ≥ 30), and separated by fetal sex. We measured plasma lipids, insulin, and glucose in maternal and cord blood. Insulin resistance was quantified using the HOMA-IR. Placental markers of lipid and energy metabolism and relevant metabolites were measured by western blot and metabolomics, respectively.For OB women, total cholesterol was decreased in both maternal and cord blood, while HDL was decreased only in cord blood, independent of sex. In babies born to OB women, cord blood insulin and insulin resistance were increased. Placental protein expression of the energy and lipid metabolism regulators PGC1α, and SIRT3, ERRα, CPT1α, and CPT2 decreased with maternal obesity in a sex-dependent manner (P < 0.05). Metabolomics showed lower levels of acylcarnitines C16:0, C18:2, and C20:4 in OB women’s placentas, suggesting a decrease in β-oxidation. Glutamine, glutamate, alpha-ketoglutarate (αKG), and 2-hydroxyglutarate (2-HG) were increased, and the glutamine-to-glutamate ratio decreased (P < 0.05), in OB placentas, suggesting induction of glutamate into αKG conversion to maintain a normal metabolic flux.Newly-born offspring of obese mothers begin their lives dyslipidemic and insulin resistant. If not inherited genetically, such major metabolic perturbations might be explained by abnormal placental metabolism with potential long-term adverse consequences for the offspring’s health and wellbeing.

SUMMARY We report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants., Graphical Abstract, In Brief Ewing sarcoma is driven by the EWS-ETS fusion oncoprotein, but little is known about the extracellular signaling regulating this cancer. Zhou et al. report that the prodomain of GDF6 is a ligand for CD99, inhibiting Src through CSK and maintaining Ewing sarcoma growth in an autocrine fashion.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumor’s extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analog into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor-cells from non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers.

This report presents the results from the 2016 Association of Biomolecular Resource Facilities Proteome Informatics Research Group (iPRG) study on proteoform inference and false discovery rate (FDR) estimation from bottom-up proteomics data. For this study, 3 replicate Q Exactive Orbitrap liquid chromatography-tandom mass spectrometry datasets were generated from each of 4 Escherichia coli samples spiked with different equimolar mixtures of small recombinant proteins selected to mimic pairs of homologous proteins. Participants were given raw data and a sequence file and asked to identify the proteins and provide estimates on the FDR at the proteoform level. As part of this study, we tested a new submission system with a format validator running on a virtual private server (VPS) and allowed methods to be provided as executable R Markdown or IPython Notebooks. The task was perceived as difficult, and only eight unique submissions were received, although those who participated did well with no one method performing best on all samples. However, none of the submissions included a complete Markdown or Notebook, even though examples were provided. Future iPRG studies need to be more successful in promoting and encouraging participation. The VPS and submission validator easily scale to much larger numbers of participants in these types of studies. The unique "ground-truth" dataset for proteoform identification generated for this study is now available to the research community, as are the server-side scripts for validating and managing submissions.

Grafting of neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise for brain repair after injury or disease, but safety issues have hindered their clinical application. Employing nano-sized extracellular vesicles (EVs) derived from hiPSC-NSCs appears to be a safer alternative because they likely have similar neuroreparative properties as NSCs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. However, reliable methods for isolation, characterization and testing the biological properties of EVs are critically needed for translation. We investigated signatures of miRNAs and proteins and the biological activity of EVs, isolated from hiPSC-NSCs through a combination of anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC). AEC and SEC facilitated the isolation of EVs with intact ultrastructure and expressing CD9, CD63, CD81, ALIX and TSG 101. Small RNA sequencing, proteomic analysis, pathway analysis and validation of select miRNAs and proteins revealed that EVs were enriched with miRNAs and proteins involved in neuroprotective, anti-apoptotic, antioxidant, anti-inflammatory, blood-brain barrier repairing, neurogenic and Aβ reducing activities. Besides, EVs comprised miRNAs and/or proteins capable of promoting synaptogenesis, synaptic plasticity and better cognitive function. Investigations using an in vitro macrophage assay and a mouse model of status epilepticus confirmed the anti-inflammatory activity of EVs. Furthermore, the intranasal administration of EVs resulted in the incorporation of EVs by neurons, microglia and astrocytes in virtually all adult rat and mouse brain regions, and enhancement of hippocampal neurogenesis. Thus, biologically active EVs containing miRNAs and proteins relevant to brain repair could be isolated from hiPSC-NSC cultures, making them a suitable biologic for treating neurodegenerative disorders.

© The Author(s) 2020, The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP’s tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases., Parts of this work were supported by grants to ProteoRed PRB3-ISCIII, PT17/0019/0001 Comunidad de Madrid Grant B2017/BMD-3817 (F.J.C.); Korean Ministry of Health and Welfare HI13C2098 and HI16C0257 (Y.K.P.); NIH grants P30ES017885 and U24CA210967 (G.S.O.), 5U01HL-13104204, PADOM-SPO11347 and PARYB-SPO112285 (M.P.S.); NCI CPTAC U24CA210985 and NCI EDRN U24CA115102 (D.W.C.); NIH National Institute of General Medical Sciences R01GM087221 (E.W.D./R.L.M.) and R24GM127667 (E.W.D.); NIH National Institute on Aging U19AG023122 (R.L.M.); NSF DBI-1933311 (E.W.D.); CIHR COVID-19 Rapid Research Funding (F20-01013), CIHR Foundation Grant FDN:14840 and Canada Research Chair (C.M.O.); Investissement d’Avenir Infrastructures Nationales en Biologie et Santé ANR-10-INBS-08 (Proteomics French Infrastructure ProFI (Y.V.); Wellcome Trust WT101477MA and 208391/Z/17/Z (J.A.V.); Knut and Alice Wallenberg Foundation (M.U., C.L., J.M.S., E.L.); Brazilian CAPES 88887.130697, CNPq 440613/2016-7, FAPERJ E-26/210.173/2018 (G.B.D.) and FAPERJ E-26/202.650/2018 (F.C.S.N.), Australian Commonwealth NCRIS (M.S.B.); NHMRC 1010303 (M.S.B., E.C.N.); Cancer Council NSW RG19-04 (M.S.B., S.B.A., E.C.N.); Cancer Institute NSW Fellowship 15/ECF/1-38 (S.B.A.), Sydney Vital CINSW Translational Cancer Research Centre grant (M.S.B., S.B.A., S.A.), ‘Fight on the Beaches’ (M.S.B., S.B.A., E.C.N., S.A.) funding and an International Macquarie Research Excellence Scholarship (S.A.). M.S.B. thanks the Faculty of Medicine, Stanford University for a sabbatical visiting professorship.

Phage G has the largest capsid and genome of any known propagated phage. Many aspects of its structure, assembly, and replication have not been elucidated. Herein, we present the dsDNA-packed and empty phage G capsid at 6.1 Å and 9 Å resolution, respectively, using cryo-EM for structure determination and mass spectrometry for protein identification. The major capsid protein, gp27, is identified and found to share the HK97-fold universally conserved in all previously solved dsDNA phages. Trimers of the decoration protein, gp26, sit on the 3-fold axes and are thought to enhance the interactions of the hexameric capsomeres of gp27, for other phages encoding decoration proteins. Phage G’s decoration protein is longer than what has been reported in other phages, and we suspect the extra interaction surface area helps stabilize the capsid. We identified several additional capsid proteins, including a candidate for the prohead protease responsible for processing gp27. Furthermore, cryo-EM reveals a range of partially full, condensed DNA densities that appear to have no contact with capsid shell. Three analyses confirm that the phage G host is a Lysinibacillus, and not Bacillus megaterium: identity of host proteins in our mass spectrometry analyses, genome sequence of the phage G host and host range of phage G.

Background CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) are approved for the treatment of ER+ advanced breast cancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. We previously reported a novel therapeutic agent, ERX-11, that binds to the estrogen receptor (ER) and modulates ER-coregulator interactions. Here, we tested if the combination of ERX-11 with agents approved for ER+ BCa would be more potent. Methods We tested the effect of combination therapy using BCa cell line models, including those that have acquired resistance to tamoxifen, letrozole, or CDK4/6 inhibitors or have been engineered to express mutant forms of the ER. In vitro activity was tested using Cell Titer-Glo, MTT, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, RT-qPCR, and mass spectrometry approaches. Xenograft, patient-derived explants (PDEs), and xenograft-derived explants (XDE) were used for preclinical evaluation and toxicity. Results ERX-11 inhibited the proliferation of therapy-resistant BCa cells in a dose-dependent manner, including ribociclib resistance. The combination of ERX-11 and CDK4/6 inhibitor was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BCa cells, in vitro, in xenograft models in vivo, xenograft-derived explants ex vivo, and in primary patient-derived explants ex vivo. Importantly, the combination caused xenograft tumor regression in vivo. Unbiased global mass spectrometry studies demonstrated profound decreases in proliferation markers with combination therapy and indicated global proteomic changes in E2F1, ER, and ER coregulators. Mechanistically, the combination of ERX-11 and CDK4/6 inhibitor decreased the interaction between ER and its coregulators, as evidenced by immunoprecipitation followed by mass spectrometry studies. Biochemical studies confirmed that the combination therapy significantly altered the expression of proteins involved in E2F1 and ER signaling, and this is primarily driven by a transcriptional shift, as noted in gene expression studies. Conclusions Our results suggest that ERX-11 inhibited the proliferation of BCa cells resistant to both endocrine therapy and CDK4/6 inhibitors in a dose-dependent manner and that the combination of ERX-11 with a CDK4/6 inhibitor may represent a viable therapeutic approach.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF–exposed Mtb had reduced control and fewer phagosome–lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

SUMMARY BAF chromatin remodeling complexes play important roles in chromatin regulation and cancer. Here, we report that Ewing sarcoma cells are dependent on the autocrine signaling mediated by NELL2, a secreted glycoprotein that has been characterized as an axon guidance molecule. NELL2 uses Robo3 as the receptor to transmit critical growth signaling. NELL2 signaling inhibits cdc42 and upregulates BAF complexes and EWS-FLI1 transcriptional output. We demonstrate that cdc42 is a negative regulator of BAF complexes, inducing actin polymerization and complex disassembly. Furthermore, we identify NELL2highCD133highEWS-FLI1high and NELL2lowCD133lowEWS-FLI1low populations in Ewing sarcoma, which display phenotypes consistent with high and low NELL2 signaling, respectively. We show that NELL2, CD133, and EWS-FLI1 positively regulate each other and upregulate BAF complexes and cell proliferation in Ewing sarcoma. These results reveal a signaling pathway regulating critical chromatin remodeling complexes and cancer cell proliferation., Graphical Abstract, In brief BAF complexes play important roles in chromatin remodeling and cancers, but the signaling pathways regulating these complexes are poorly understood. Jayabal et al. report that NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth.

Rieske proteins play an essential role in electron transfer in the bc 1 complex. Rieske proteins contain a [2Fe-2S] cluster with one iron ligated by two histidines and the other iron ligated by two cysteines. All Rieske proteins have pH-dependent reduction potentials with the histidines ligating the cluster deprotonating in response to increases in pH. The addition of diethylpyrocarbonate (DEPC) modifies deprotonated histidines. The previous studies on the isolated Thermus thermophilus Rieske protein have used large excesses of DEPC, and this study examines what amino acids become modified under different molar equivalents of DEPC to protein. Increasing amounts of DEPC result in more modification, and higher pH values result in faster reaction. Upon modification, the protein also becomes reduced and ~6 equivalents of DEPC are needed for 50% of the reduction to occur. Which amino acids are modified first also points to the most reactive species on the protein. Mass spectrometry analysis shows that lysine 68 is the most reactive amino acid, followed by the ligating histidine 154 and two other surfaces lysines, 76 and 43. The modification of the ligating histidine at low numbers of DEPC equivalents and correlation with a similar number of equivalents needed to reduce the protein shows that this histidine can interact with neighboring groups, and these results can be extended to the protein within the bc 1 complex, where interaction with neighboring residues or molecules may allow reduction to occur. These results may shed light on how Rieske transfers electrons and protons in the bc 1 complex.

Aims: Protein S-glutathionylation, the formation of a mixed disulfide between glutathione and protein thiols, is an oxidative modification that has emerged as a new signaling paradigm, potentially linking oxidative stress to chronic inflammation associated with heart disease, diabetes, cancer, lung disease, and aging. Using a novel, highly sensitive, and selective proteomic approach to identify S-glutathionylated proteins, we tested the hypothesis that monocytes and macrophages sense changes in their microenvironment and respond to metabolic stress by altering their protein thiol S-glutathionylation status. Results: We identified over 130 S-glutathionylated proteins, which were associated with a variety of cellular functions, including metabolism, transcription and translation, protein folding, free radical scavenging, cell motility, and cell death. Over 90% of S-glutathionylated proteins identified in metabolically stressed THP-1 monocytes were also found in hydrogen peroxide (H2O2)-treated cells, suggesting that H2O2 mediates metabolic stress-induced protein S-glutathionylation in monocytes and macrophages. We validated our findings in mouse peritoneal macrophages isolated from both healthy and dyslipidemic atherosclerotic mice and found that 52% of the S-glutathionylated proteins found in THP-1 monocytes were also identified in vivo. Changes in macrophage protein S-glutathionylation induced by dyslipidemia were sexually dimorphic. Innovation: We provide a novel mechanistic link between metabolic (and thiol oxidative) stress, macrophage dysfunction, and chronic inflammatory diseases associated with metabolic disorders. Conclusion: Our data support the concept that changes in the extracellular metabolic microenvironment induce S-glutathionylation of proteins central to macrophage metabolism and a wide array of cellular signaling pathways and functions, which in turn initiate and promote functional and phenotypic changes in macrophages. Antioxid. Redox Signal. 25, 836–851.

Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241 , which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a “missing” β′ subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution. IMPORTANCE In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ϕKZ.

Lipid identification from data produced with high-throughput technologies is essential to the elucidation of the roles played by lipids in cellular function and disease. Software tools for identifying lipids from tandem mass (MS/MS) spectra have been developed, but they are often costly or lack the sophistication of their proteomics counterparts. We have developed Greazy, an open source tool for the automated identification of phospholipids from MS/MS spectra, that utilizes methods similar to those developed for proteomics. From user-supplied parameters, Greazy builds a phospholipid search space and associated theoretical MS/MS spectra. Experimental spectra are scored against search space lipids with similar precursor masses using a peak score based on the hypergeometric distribution and an intensity score utilizing the percentage of total ion intensity residing in matching peaks. The LipidLama component filters the results via mixture modeling and density estimation. We assess Greazy’s performance agains...

Introduction: Breast cancer (BC) is the most common cancer in women worldwide and leading cause of cancer death. More than 70% of cancers are estrogen receptor alpha (ERα) positive. Despite effective therapeutic strategies for treating hormone receptor-positive (HR+) breast cancer, resistance to endocrine therapy that is either de novo or acquired still occurs. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Studies have shown that estrogen receptor beta (ERβ) and its agonists inhibits the different types of cancers including BC. Herein, we investigated mechanism(s) for therapeutic efficacies of ERβ agonists in breast cancer. Methods: To investigate the mechanisms of ERβ agonists (S-Equol and LY500307), in the treatment of breast cancer, we used therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells and ERβ knock down cell lines, generated by CRISPR-Cas9 technology. The wild type and ERβ knock down cells with or without treatment of ERβ agonists were analyzed, for cell proliferation, protein (Western), Gene expression array analysis, RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses and RNA (RT-qPCR) analysis. Xenograft and patient derived BC explants (PDEX) were used to test the antiproliferative activity of ERβ agonists. Results: Our results demonstrated that treatment with ERβ agonists, S-Equol and LY500307 inhibited the growth of both endocrine therapy sensitive and resistant breast cancer cells. Gene expression array analysis of LY500307 treated breast cancer cells showed the modulation of signaling molecules involved in cell death and cell cycle pathways. RNA-seq analysis showed that treatment with S-equol modulated the ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis and Reactome pathway analysis revealed that S-equol treatment modulated the key proteins involved in DNA replication and cell cycle. Both ERβ agonists S-Equol and LY500307 inhibited the cell proliferation of parental cells whereas they could not elicits the inhibitory effect on the knock out cells. By using ERα positive PDX tumors, we have demonstrated that both the ERβ agonists inhibited the proliferation of human BC tumors. Our mechanistic studies confirmed that increased FOXO3 activation by S-equol in LTLT cells can reverse letrozole resistance. Our results showed that inhibiting FOXM1 by ERβ agonists significantly reduce the proliferation of letrozole-resistant cells. These changes were not observed in ERβ knockdown cells confirming the anti-proliferative role of ERβ in breast cancer progression. Conclusions: Collectively, our results suggest that ERβ agonists may offer a new targeted therapy for endocrine therapy resistant breast tumors. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294 and P30 CA-54174 NCI-Mays cancer center grant). Citation Format: Kumaraguruparan Ramasamy, Suryavathi Viswanadhapalli, M.A. Sureshkumar, Susan Weintraub, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4365.

Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between α2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Additional time-series experiments revealed the gephyrin regulating kinase ERK was inactivated by DZP at multiple time points. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.