Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Protein cysteine thiols undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates protein-membrane and protein-protein interactions critical to an array of biological processes, from homeostatic lung surfactant function to cellular transformation. The most widely used S-acylation assays, including acyl-biotin exchange (ABE) and acyl resin-assisted capture (Acyl-RAC), utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. ABE and Acyl-RAC have enabled both the proteome-wide identification of S-palmitoylation sites and basic biochemical studies. Yet, these assays generally utilize hundreds of micrograms to milligrams of input material and require numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome this, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment from 20-50 micrograms of input protein. The method is compatible with protein-level detection of Sacylated proteins as well as S-acyl site-based identification and quantification using on-quartz isobaric (tandem mass tag) labeling and LC-MS/MS. Also described are conditions for long-term hydroxylamine storage, which further expedites the assay and minimizes waste. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional intact protein detection and chemoproteomic workflows.

Nontargeted Plasma Proteomic Analysis of Renal Disease and Pulmonary Hypertension in Patients with Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by red blood cell sickling, vaso-occlusion, hemolytic anemia, damage to multiple organ systems, and, as a result, shortened life expectancy. Sickle cell disease nephropathy (SCDN) and pulmonary hypertension (pHTN) are common and frequently co-occurring complications of SCD; both are associated with markedly accelerated mortality. To identify candidate circulating biomarkers of SCDN and pHTN, we used mass spectrometry to quantify the relative abundance of >1000 proteins in plasma samples from 189 adults with SCD from the Outcome Modifying Genes in SCD (OMG-SCD) cohort (ProteomeXchange identifier PXD048716). Forty-four proteins were differentially abundant in SCDN, most significantly cystatin-C and collagen α-1(XVIII) chain (COIA1), and 55 proteins were dysregulated in patients with SCDN and pHTN, most significantly insulin-like growth factor-binding protein 6 (IBP6). Network analysis identified a module of 133 coregulated proteins significantly associated with SCDN, that was enriched for extracellular matrix proteins, insulin-like growth factor binding proteins, cell adhesion proteins, EGF-like calcium binding proteins, and several cadherin family members. Collectively, these data provide a comprehensive understanding of plasma protein changes in SCDN and pHTN which validate numerous studies of chronic kidney disease and suggest shared profiles of protein disruption in kidney dysfunction and pHTN among SCD patients.

Differences in the Circulating Proteome in Individuals with versus without Sickle Cell Trait.

BACKGROUND: Sickle cell trait affects approximately 8% of Black individuals in the United States, along with many other individuals with ancestry from malaria-endemic regions worldwide. While traditionally considered a benign condition, recent evidence suggests that sickle cell trait is associated with lower eGFR and higher risk of kidney diseases, including kidney failure. The mechanisms underlying these associations remain poorly understood. We used proteomic profiling to gain insight into the pathobiology of sickle cell trait. METHODS: We measured proteomics ( N =1285 proteins assayed by Olink Explore) using baseline plasma samples from 592 Black participants with sickle cell trait and 1:1 age-matched Black participants without sickle cell trait from the prospective Women's Health Initiative cohort. Age-adjusted linear regression was used to assess the association between protein levels and sickle cell trait. RESULTS: In age-adjusted models, 35 proteins were significantly associated with sickle cell trait after correction for multiple testing. Several of the sickle cell trait-protein associations were replicated in Black participants from two independent cohorts (Atherosclerosis Risk in Communities study and Jackson Heart Study) assayed using an orthogonal aptamer-based proteomic platform (SomaScan). Many of the validated sickle cell trait-associated proteins are known biomarkers of kidney function or injury ( e.g. , hepatitis A virus cellular receptor 1 [HAVCR1]/kidney injury molecule-1 [KIM-1], uromodulin [UMOD], ephrins), related to red cell physiology or hemolysis (erythropoietin [EPO], heme oxygenase 1 [HMOX1], and α -hemoglobin stabilizing protein) and/or inflammation (fractalkine, C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 [MCP-1], and urokinase plasminogen activator surface receptor [PLAUR]). A protein risk score constructed from the top sickle cell trait-associated biomarkers was associated with incident kidney failure among those with sickle cell trait during Women's Health Initiative follow-up (odds ratio, 1.32; 95% confidence interval, 1.10 to 1.58). CONCLUSIONS: We identified and replicated the association of sickle cell trait with a number of plasma proteins related to hemolysis, kidney injury, and inflammation.

The urinary proteome infers dysregulation of mitochondrial, lysosomal, and protein reabsorption processes in chronic kidney disease of unknown etiology (CKDu).

Chronic kidney disease (CKD) of uncertain etiology (CKDu) is a global health concern affecting tropical farming communities. CKDu is not associated with typical risk factors (e.g., diabetes) and strongly correlates with environmental drivers. To gain potential insights into disease etiology and diagnosis, here we report the first urinary proteome comparing patients with CKDu and non-CKDu controls from Sri Lanka. We found 944 differentially abundant proteins. In silico analyses identified 636 proteins of likely kidney and urogenital origin. As expected, renal tubular injury in patients with CKDu was evinced by increases in albumin, cystatin C, and ß2-microglobulin. However, several proteins typically elevated under CKD, including osteopontin and α-N-acetylglucosaminidase, were decreased in patients with CKDu. Furthermore, urinary excretion of aquaporins found higher in CKD was lower in CKDu. Comparisons with previous CKD urinary proteome datasets revealed a unique proteome for CKDu. Notably, the CKDu urinary proteome was relatively similar to that of patients with mitochondrial diseases. Furthermore, we report a decrease in endocytic receptor proteins responsible for protein reabsorption (megalin and cubilin) that correlated with an increase in abundance of 15 of their cognate ligands. Functional pathway analyses identified kidney-specific differentially abundant proteins in patients with CKDu denoted significant changes in the complement cascade and coagulation systems, cell death, lysosomal function, and metabolic pathways. Overall, our findings provide potential early detection markers to diagnose and distinguish CKDu and warrant further analyses on the role of lysosomal, mitochondrial, and protein reabsorption processes and their link to the complement system and lipid metabolism in CKDu onset and progression.NEW & NOTEWORTHY CKDu is a global health concern debilitating a number of tropical rural farming communities. In the absence of typical risk factors like diabetes and hypertension and the lack of molecular markers, it is crucial to identify potential early disease markers. Here, we detail the first urinary proteome profile to distinguish CKDu from CKD. Our data and in silico pathway analyses infer the roles of mitochondrial, lysosomal, and protein reabsorption processes in disease onset and progression.

Octreotide may improve pharyngocutaneous fistula healing through downregulation of cystatins: A pilot study.

Background: Pharyngocutaneous fistula (PCF) and salivary leaks are well known complications of head and neck surgery. The medical management of PCF has included the use of octreotide without a well-defined understanding of its therapeutic mechanism. We hypothesized that octreotide induces alterations in the saliva proteome and that these alterations may provide insight into the mechanism of action underlying improved PCF healing. We undertook an exploratory pilot study in healthy controls that involved collecting saliva before and after a subcutaneous injection of octreotide and performing proteomic analysis to determine the effects of octreotide. Methods: Four healthy adult participants provided saliva samples before and after subcutaneous injection of octreotide. A mass-spectrometry based workflow optimized for the quantitative proteomic analysis of biofluids was then employed to analyze changes in salivary protein abundance after octreotide administration. Results: There were 3076 human, 332 Streptococcus mitis, 102 G. haemolyans, and 42 Granulicatella adiacens protein groups quantified in saliva samples. A paired statistical analysis was performed using the generalized linear model (glm) function in edgeR. There were and ~300 proteins that had a p < .05 between the pre- and post-octreotide groups ~50 proteins with an FDR-corrected p < .05 between pre- and post-groups. These results were visualized using a volcano plot after filtering on proteins quantified by 2 more or unique precursors. Both human and bacterial proteins were among the proteins altered by octreotide treatment. Notably, four isoforms of the human cystatins, belonging to a family of cysteine proteases, that had significantly lower abundance after treatment. Conclusion: This pilot study demonstrated octreotide-induced downregulation of cystatins. By downregulation of cystatins in the saliva, there is decreased inhibition of cysteine proteases such as Cathepsin S. This results in increased cysteine protease activity that has been linked to enhanced angiogenic response, cell proliferation and migration that have resulted in improved wound healing. These insights provide first steps at furthering our understanding of octreotide's effects on saliva and reports of improved PCF healing.

A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart.

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.

Characterization of Small-Molecule-Induced Changes in Parkinson's-Related Trafficking via the Nedd4 Ubiquitin Signaling Cascade.

The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson's disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KDapp = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its co-localization. Ubiquitin enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking-associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism and revealing trafficking scaffold protein TFG as a Nedd4 substrate.

The effect of pathogen inactivation on cryoprecipitate: a functional and quantitative evaluation.

BACKGROUND: As a pooled donor blood product, cryoprecipitate (cryo) carries risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on haemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics and in vitro haemostatic capacity of PI-cryo. MATERIALS AND METHODS: Whole blood (WB)- and apheresis (APH)-derived plasma was subject to PI with INTERCEPT® Blood System (Cerus Corporation, Concord, CA, USA) and cryo was prepared from treated plasma. Protein levels in PI-cryo and paired controls were quantified using liquid chromatography-tandem mass spectrometry. Functional haemostatic properties of PI-cryo were assessed using a microparticle (MP) prothrombinase assay, thrombin generation assay, and an in vitro coagulopathy model subjected to thromboelastometry. RESULTS: Over 300 proteins were quantified across paired PI-cryo and controls. PI did not alter the expression of coagulation factors, but levels of platelet-derived proteins and platelet-derived MPs were markedly lower in the WB PI-cryo group. Compared to controls, WB (but not APH) cryo samples demonstrated significantly lower MP prothrombinase activity, prolonged clotting time, and lower clot firmness on thromboelastometry after PI. However, PI did not affect overall thrombin generation variables in either group. DISCUSSION: Data from this study suggest that PI via INTERCEPT® Blood System does not significantly impact the coagulation factor content or function of cryo but reduces the higher MP content in WB-derived cryo. PI-cryo products may confer benefits in reducing pathogen transmission without affecting haemostatic function, but further in vivo assessment is warranted.

Interleukin-13 disrupts type 2 pneumocyte stem cell activity.

The T helper 2 (Th2) inflammatory cytokine interleukin-13 (IL-13) has been associated with both obstructive and fibrotic lung diseases; however, its specific effect on the epithelial stem cells in the gas exchange compartment of the lung (alveolar space) has not been explored. Here, we used in vivo lung models of homeostasis and repair, ex vivo organoid platforms, and potentially novel quantitative proteomic techniques to show that IL-13 disrupts the self-renewal and differentiation of both murine and human type 2 alveolar epithelial cells (AEC2s). Significantly, we find that IL-13 promotes ectopic expression of markers typically associated with bronchiolar airway cells and commonly seen in the alveolar region of lung tissue from patients with idiopathic pulmonary fibrosis. Furthermore, we identify a number of proteins that are differentially secreted by AEC2s in response to IL-13 and may provide biomarkers to identify subsets of patients with pulmonary disease driven by "Th2-high" biology.

The Salmonella Secreted Effector SarA/SteE Mimics Cytokine Receptor Signaling to Activate STAT3.

Bacteria masterfully co-opt and subvert host signal transduction. As a paradigmatic example, Salmonella uses two type-3 secretion systems to inject effector proteins that facilitate Salmonella entry, establishment of an intracellular niche, and modulation of immune responses. We previously demonstrated that the Salmonella anti-inflammatory response activator SarA (Stm2585, GogC, PagJ, SteE) activates the host transcription factor STAT3 to drive expression of immunomodulatory STAT3-targets. Here, we demonstrate-by sequence, function, and biochemical measurement-that SarA mimics the cytoplasmic domain of glycoprotein 130 (gp130, IL6ST). SarA is phosphorylated at a YxxQ motif, facilitating binding to STAT3 with greater affinity than gp130. Departing from canonical gp130 signaling, SarA function is JAK-independent but requires GSK-3, a key regulator of metabolism and development. Our results reveal that SarA undergoes host phosphorylation to recruit a STAT3-activating complex, circumventing cytokine receptor activation. Effector mimicry of gp130 suggests GSK-3 can regulate normal cytokine signaling, potentially enabling metabolic and immune crosstalk.

ERK-dependent proteasome degradation of Txnip regulates thioredoxin oxidoreductase activity.

Dynamic control of thioredoxin (Trx) oxidoreductase activity is essential for balancing the need of cells to rapidly respond to oxidative/nitrosative stress and to temporally regulate thiol-based redox signaling. We have previously shown that cytokine stimulation of the respiratory epithelium induces a precipitous decline in cell S-nitrosothiol, which depends upon enhanced Trx activity and proteasome-mediated degradation of Txnip (thioredoxin-interacting protein). We now show that tumor necrosis factor-α-induced Txnip degradation in A549 respiratory epithelial cells is regulated by the extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase pathway and that ERK inhibition augments both intracellular reactive oxygen species and S-nitrosothiol. ERK-dependent Txnip ubiquitination and proteasome degradation depended upon phosphorylation of a PXTP motif threonine (Thr349) located within the C-terminal α-arrestin domain and proximal to a previously characterized E3 ubiquitin ligase-binding site. Collectively, these findings demonstrate the ERK mitogen-activated protein kinase pathway to be integrally involved in regulating Trx oxidoreductase activity and that the regulation of Txnip lifetime via ERK-dependent phosphorylation is an important mediator of this effect.

Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.

Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.

A comparative analysis of egg provisioning using mass spectrometry during rapid life history evolution in sea urchins.

A dramatic life history switch that has evolved numerous times in marine invertebrates is the transition from planktotrophic (feeding) to lecithotrophic (nonfeeding) larval development-an evolutionary tradeoff with many important developmental and ecological consequences. To attain a more comprehensive understanding of the molecular basis for this switch, we performed untargeted lipidomic and proteomic liquid chromatography-tandem mass spectrometry on eggs and larvae from three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and the distantly related planktotroph Lytechinus variegatus. We identify numerous molecular-level changes possibly associated with the evolution of lecithotrophy in H. erythrogramma. We find the massive lipid stores of H. erythrogramma eggs are largely composed of low-density, diacylglycerol ether lipids that, contrary to expectations, appear to support postmetamorphic development and survivorship. Rapid premetamorphic development in this species may instead be powered by upregulated carbohydrate metabolism or triacylglycerol metabolism. We also find proteins involved in oxidative stress regulation are upregulated in H. erythrogramma eggs, and apoB-like lipid transfer proteins may be important for echinoid oogenic nutrient provisioning. These results demonstrate how mass spectrometry can enrich our understanding of life history evolution and organismal diversity by identifying specific molecules associated with distinct life history strategies and prompt new hypotheses about how and why these adaptations evolve.

Identification of a Novel Inhibitor of Human Rhinovirus Replication and Inflammation in Airway Epithelial Cells.

Human rhinovirus (RV), the major cause of the common cold, triggers the majority of acute airway exacerbations in patients with asthma and chronic obstructive pulmonary disease. Nitric oxide, and the related metabolite S-nitrosoglutathione, are produced in the airway epithelium via nitric oxide synthase (NOS) 2 and have been shown to function in host defense against RV infection. We hypothesized that inhibitors of the S-nitrosoglutathione-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), might potentiate the antiviral properties of airway-derived NOS2. Using in vitro models of RV-A serotype 16 (RV-A16) and mNeonGreen-H1N1pr8 infection of human airway epithelial cells, we found that treatment with a previously characterized GSNOR inhibitor (4-[[2-[[(3-cyanophenyl)methyl]thio]-4-oxothieno-[3,2-d]pyrimidin-3(4H)-yl]methyl]-benzoic acid; referred to as C3m) decreased RV-A16 replication and expression of downstream proinflammatory and antiviral mediators (e.g., RANTES [regulated upon activation, normal T cell expressed and secreted], CXCL10, and Mx1), and increased Nrf2 (nuclear factor erythroid 2-related factor 2)-dependent genes (e.g., SQSTM1 and TrxR1). In contrast, C3m had no effect on influenza virus H1N1pr8 replication. Moreover, a structurally dissimilar GSNOR inhibitor (N6022) did not alter RV replication, suggesting that the properties of C3m may be specific to rhinovirus owing to an off-target effect. Consistent with this, C3m antiviral effects were not blocked by either NOS inhibition or GSNOR knockdown but appeared to be mediated by reduced intercellular adhesion molecule 1 transcription and increased shedding of soluble intercellular adhesion molecule 1 protein. Collectively these data show that C3m has novel antirhinoviral properties that may synergize with, but are unrelated to, its GSNOR inhibitor activity.

Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, transcription factors (TFs) regulate the periodic expression of many genes during the cell cycle, including gene products required for progression through cell-cycle events. Experimental evidence coupled with quantitative models suggests that a network of interconnected TFs is capable of regulating periodic genes over the cell cycle. Importantly, these dynamical models were built on transcriptomics data and assumed that TF protein levels and activity are directly correlated with mRNA abundance. To ask whether TF transcripts match protein expression levels as cells progress through the cell cycle, we applied a multiplexed targeted mass spectrometry approach (parallel reaction monitoring) to synchronized populations of cells. We found that protein expression of many TFs and cell-cycle regulators closely followed their respective mRNA transcript dynamics in cycling wild-type cells. Discordant mRNA/protein expression dynamics was also observed for a subset of cell-cycle TFs and for proteins targeted for degradation by E3 ubiquitin ligase complexes such as SCF (Skp1/Cul1/F-box) and APC/C (anaphase-promoting complex/cyclosome). We further profiled mutant cells lacking B-type cyclin/CDK activity ( clb1-6) where oscillations in ubiquitin ligase activity, cyclin/CDKs, and cell-cycle progression are halted. We found that a number of proteins were no longer periodically degraded in clb1-6 mutants compared with wild type, highlighting the importance of posttranscriptional regulation. Finally, the TF complexes responsible for activating G1/S transcription (SBF and MBF) were more constitutively expressed at the protein level than at periodic mRNA expression levels in both wild-type and mutant cells. This comprehensive investigation of cell-cycle regulators reveals that multiple layers of regulation (transcription, protein stability, and proteasome targeting) affect protein expression dynamics during the cell cycle.

UBE2N Promotes Melanoma Growth via MEK/FRA1/SOX10 Signaling.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry-based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma.Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462-72. ©2018 AACR.

New Strategies and Challenges in Lung Proteomics and Metabolomics. An Official American Thoracic Society Workshop Report.

This document presents the proceedings from the workshop entitled, "New Strategies and Challenges in Lung Proteomics and Metabolomics" held February 4th-5th, 2016, in Denver, Colorado. It was sponsored by the National Heart Lung Blood Institute, the American Thoracic Society, the Colorado Biological Mass Spectrometry Society, and National Jewish Health. The goal of this workshop was to convene, for the first time, relevant experts in lung proteomics and metabolomics to discuss and overcome specific challenges in these fields that are unique to the lung. The main objectives of this workshop were to identify, review, and/or understand: (1) emerging technologies in metabolomics and proteomics as applied to the study of the lung; (2) the unique composition and challenges of lung-specific biological specimens for metabolomic and proteomic analysis; (3) the diverse informatics approaches and databases unique to metabolomics and proteomics, with special emphasis on the lung; (4) integrative platforms across genetic and genomic databases that can be applied to lung-related metabolomic and proteomic studies; and (5) the clinical applications of proteomics and metabolomics. The major findings and conclusions of this workshop are summarized at the end of the report, and outline the progress and challenges that face these rapidly advancing fields.

The Diacetyl-Exposed Human Airway Epithelial Secretome: New Insights into Flavoring-Induced Airways Disease.

Bronchiolitis obliterans (BO) is an increasingly important lung disease characterized by fibroproliferative airway lesions and decrements in lung function. Occupational exposure to the artificial food flavoring ingredient diacetyl, commonly used to impart a buttery flavor to microwave popcorn, has been associated with BO development. In the occupational setting, diacetyl vapor is first encountered by the airway epithelium. To better understand the effects of diacetyl vapor on the airway epithelium, we used an unbiased proteomic approach to characterize both the apical and basolateral secretomes of air-liquid interface cultures of primary human airway epithelial cells from four unique donors after exposure to an occupationally relevant concentration (∼1,100 ppm) of diacetyl vapor or phosphate-buffered saline as a control on alternating days. Basolateral and apical supernatants collected 48 h after the third exposure were analyzed using one-dimensional liquid chromatography tandem mass spectrometry. Paired t tests adjusted for multiple comparisons were used to assess differential expression between diacetyl and phosphate-buffered saline exposure. Of the significantly differentially expressed proteins identified, 61 were unique to the apical secretome, 81 were unique to the basolateral secretome, and 11 were present in both. Pathway enrichment analysis using publicly available databases revealed that proteins associated with matrix remodeling, including degradation, assembly, and new matrix organization, were overrepresented in the data sets. Similarly, protein modifiers of epidermal growth factor receptor signaling were significantly altered. The ordered changes in protein expression suggest that the airway epithelial response to diacetyl may contribute to BO pathogenesis.