Thiol-Mediated Enhancement of Nε-Acetyllysine Formation in Lens Proteins.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Quantifying Protein Acetylation in Diabetic Nephropathy from Formalin-Fixed Paraffin-Embedded Tissue.

PURPOSE: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end-stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant protein post-translational modifications. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism. EXPERIMENTAL DESIGN: A novel extraction and LC-MS/MS approach was adapted to quantify sites of lysine acetylation from formalin-fixed paraffin-embedded (FFPE) kidney tissue and from patients with DKD and non-diabetic donors (n = 5 and n = 7, respectively). RESULTS: Analysis of FFPE tissues identified 840 total proteins, with 225 of those significantly changing in patients with DKD. Acetylomic analysis quantified 289 acetylated peptides, with 69 of those significantly changing. Pathways impacted in DKD patients revealed numerous metabolic pathways, specifically mitochondrial function, oxidative phosphorylation, and sirtuin signaling. Differential protein acetylation in DKD patients impacted sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis. CONCLUSIONS AND CLINICAL RELEVANCE: A quantitative acetylomics platform was developed for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from DKD.

ACAD9 treatment with bezafibrate and nicotinamide riboside temporarily stabilizes cardiomyopathy and lactic acidosis.

Pathogenic ACAD9 variants cause complex I deficiency. Patients presenting in infancy unresponsive to riboflavin have high mortality. A six-month-old infant presented with riboflavin unresponsive lactic acidosis and life-threatening cardiomyopathy. Treatment with high dose bezafibrate and nicotinamide riboside resulted in marked clinical improvement including reduced lactate and NT-pro-brain type natriuretic peptide levels, with stabilized echocardiographic measures. After a long stable period, the child succumbed from cardiac failure with infection at 10.5 months. Therapy was well tolerated. Peak bezafibrate levels exceeded its EC50. The clinical improvement with this treatment illustrates its potential, but weak PPAR agonist activity of bezafibrate limited its efficacy.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Dexamethasone targets actin cytoskeleton signaling and inflammatory mediators to reverse sulfur mustard-induced toxicity in rabbit corneas.

PURPOSE: Sulfur mustard (SM), a bi-functional alkylating agent, was used during World War I and the Iran-Iraq war. SM toxicity is ten times higher in eyes than in other tissues. Cornea is exceptionally susceptible to SM-injuries due to its anterior positioning and mucous-aqueous interphase. Ocular SM exposure induces blepharitis, photosensitivity, dry eye, epithelial defects, limbal ischemia and stem cell deficiency, and mustard gas keratopathy leading to temporary or permanent vision impairments. We demonstrated that dexamethasone (Dex) is a potent therapeutic intervention against SM-induced corneal injuries; however, its mechanism of action is not well known. Investigations employing proteomic profiling (LC-MS/MS) to understand molecular mechanisms behind SM-induced corneal injury and Dex efficacy were performed in the rabbit cornea exposed to SM and then received Dex treatment. PEAKS studio was used to extract, search, and summarize peptide identity. Ingenuity Pathway Analysis was used for pathway identification. Validation was performed using immunofluorescence. One-Way ANOVA (FDR < 0.05; p < 0.005) and Student's t-test (p < 0.05) were utilized for analyzing proteomics and IF data, respectively. Proteomic analysis revealed that SM-exposure upregulated tissue repair pathways, particularly actin cytoskeleton signaling and inflammation. Prominently dysregulated proteins included lipocalin2, coronin1A, actin-related protein2, actin-related protein2/3 complex subunit2, actin-related protein2/3 complex subunit4, cell division cycle42, ezrin, bradykinin/kininogen1, moesin, and profilin. Upregulated actin cytoskeleton signaling increases F-actin formation, dysregulating cell shape and motility. Dex reversed SM-induced increases in the aforementioned proteins levels to near control expression profiles. Dex aids corneal wound healing and improves corneal integrity via actin cytoskeletal signaling and anti-inflammatory effects following SM-induced injuries.

Salmon Food-Specific Compounds and Their Metabolites Increase in Human Plasma and Are Associated with Cardiometabolic Health Indicators Following a Mediterranean-Style Diet Intervention.

BACKGROUND: Nutrimetabolomics allows for the comprehensive analysis of foods and human biospecimens to identify biomarkers of intake and begin to probe their associations with health. Salmon contains hundreds of compounds that may provide cardiometabolic benefits. OBJECTIVES: We used untargeted metabolomics to identify salmon food-specific compounds (FSCs) and their predicted metabolites that were found in plasma after a salmon-containing Mediterranean-style (MED) diet intervention. Associations between changes in salmon FSCs and changes in cardiometabolic health indicators (CHIs) were also explored. METHODS: For this secondary analysis of a randomized, crossover, controlled feeding trial, 41 participants consumed MED diets with 2 servings of salmon per week for 2 5-wk periods. CHIs were assessed, and fasting plasma was collected pre- and postintervention. Plasma, salmon, and 99 MED foods were analyzed using liquid chromatography-mass spectrometry-based metabolomics. Compounds were characterized as salmon FSCs if detected in all salmon replicates but none of the other foods. Metabolites of salmon FSCs were predicted using machine learning. For salmon FSCs and metabolites found in plasma, linear mixed-effect models were used to assess change from pre- to postintervention and associations with changes in CHIs. RESULTS: Relative to the other 99 MED foods, there were 508 salmon FSCs with 237 unique metabolites. A total of 143 salmon FSCs and 106 metabolites were detected in plasma. Forty-eight salmon FSCs and 30 metabolites increased after the intervention (false discovery rate <0.05). Increases in 2 annotated salmon FSCs and 2 metabolites were associated with improvements in CHIs, including total cholesterol, low-density lipoprotein cholesterol, triglycerides, and apolipoprotein B. CONCLUSIONS: A data-driven nutrimetabolomics strategy identified salmon FSCs and their predicted metabolites that were detectable in plasma and changed after consumption of a salmon-containing MED diet. Findings support this approach for the discovery of compounds in foods that may serve, upon further validation, as biomarkers or act as bioactive components influential to health. The trials supporting this work were registered at NCT02573129 (Mediterranean-style diet intervention) and NCT05500976 (ongoing clinical trial).

Hybrid insulin peptide isomers spontaneously form in pancreatic beta-cells from an aspartic anhydride intermediate.

Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain nongenomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes. A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here, we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets. Our research herein shows that these HIPs form spontaneously in beta-cells through a mechanism involving an aspartic anhydride intermediate. This mechanism leads to the formation of a regular HIP containing a standard peptide bond as well as a HIP-isomer containing an isopeptide bond by linkage to the carboxylic acid side chain of the aspartic acid residue. We used mass spectrometric analyses to confirm the presence of both HIP isomers in islets, thereby validating the occurrence of this novel reaction mechanism in beta-cells. The spontaneous formation of new peptide bonds within cells may lead to the development of neoepitopes that contribute to the pathogenesis of type 1 diabetes as well as other autoimmune diseases.

Click chemistry-based thiol redox proteomics reveals significant cysteine reduction induced by chronic ethanol consumption.

In the U.S., alcohol-associated liver disease (ALD) impacts millions of people and is a major healthcare burden. While the pathology of ALD is unmistakable, the molecular mechanisms underlying ethanol hepatotoxicity are not fully understood. Hepatic ethanol metabolism is intimately linked with alterations in extracellular and intracellular metabolic processes, specifically oxidation/reduction reactions. The xenobiotic detoxification of ethanol leads to significant disruptions in glycolysis, ß-oxidation, and the TCA cycle, as well as oxidative stress. Perturbation of these regulatory networks impacts the redox status of critical regulatory protein thiols throughout the cell. Integrating these key concepts, our goal was to apply a cutting-edge approach toward understanding mechanisms of ethanol metabolism in disrupting hepatic thiol redox signaling. Utilizing a chronic murine model of ALD, we applied a cysteine targeted click chemistry enrichment coupled with quantitative nano HPLC-MS/MS to assess the thiol redox proteome. Our strategy reveals that ethanol metabolism largely reduces the cysteine proteome, with 593 cysteine residues significantly reduced and 8 significantly oxidized cysteines. Ingenuity Pathway Analysis demonstrates that ethanol metabolism reduces specific cysteines throughout ethanol metabolism (Adh1, Cat, Aldh2), antioxidant pathways (Prx1, Mgst1, Gsr), as well as many other biochemical pathways. Interestingly, a sequence motif analysis of reduced cysteines showed a correlation for hydrophilic, charged amino acids lysine or glutamic acid nearby. Further research is needed to determine how a reduced cysteine proteome impacts individual protein activity across these protein targets and pathways. Additionally, understanding how a complex array of cysteine-targeted post-translational modifications (e.g., S-NO, S-GSH, S-OH) are integrated to regulate redox signaling and control throughout the cell is key to the development of redox-centric therapeutic agents targeted to ameliorate the progression of ALD.

Multi-Omics Analysis of Lung Tissue Demonstrates Changes to Lipid Metabolism during Allergic Sensitization in Mice.

Allergy and asthma pathogenesis are associated with the dysregulation of metabolic pathways. To understand the effects of allergen sensitization on metabolic pathways, we conducted a multi-omics study using BALB/cJ mice sensitized to house dust mite (HDM) extract or saline. Lung tissue was used to perform untargeted metabolomics and transcriptomics while both lung tissue and plasma were used for targeted lipidomics. Following statistical comparisons, an integrated pathway analysis was conducted. Histopathological changes demonstrated an allergic response in HDM-sensitized mice. Untargeted metabolomics showed 391 lung tissue compounds were significantly different between HDM and control mice (adjusted p < 0.05); with most compounds mapping to glycerophospholipid and sphingolipid pathways. Several lung oxylipins, including 14-HDHA, 8-HETE, 15-HETE, 6-keto-PGF1α, and PGE2 were significantly elevated in HDM-sensitized mice (p < 0.05). Global gene expression analysis showed upregulated calcium channel, G protein-signaling, and mTORC1 signaling pathways. Genes related to oxylipin metabolism such as Cox, Cyp450s, and cPla2 trended upwards. Joint analysis of metabolomics and transcriptomics supported a role for glycerophospholipid and sphingolipid metabolism following HDM sensitization. Collectively, our multi-omics results linked decreased glycerophospholipid and sphingolipid compounds and increased oxylipins with allergic sensitization; concurrent upregulation of associated gene pathways supports a role for bioactive lipids in the pathogenesis of allergy and asthma.

Neuronal SIRT3 Deletion Predisposes to Female-Specific Alterations in Cellular Metabolism, Memory, and Network Excitability.

Mitochondrial dysfunction is an early event in the pathogenesis of neurologic disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass spectrometry-based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability, and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.SIGNIFICANCE STATEMENT Mitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3-deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases, such as Alzheimer's disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia.

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65-75 years) were incubated with aggrelyte-2 (500 µM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%-30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC-MS/MS results showed Nε -acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.

The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease.

Inflammatory cholestatic liver diseases, including Primary Sclerosing Cholangitis (PSC), are characterized by periportal inflammation with progression to cirrhosis. The objective of this study was to examine interactions between oxidative stress and autophagy in cholestasis. Using hepatic tissue from male acute cholestatic (bile duct ligated) as well as chronic cholestatic (Mdr2KO) mice, localization of oxidative stress, the antioxidant response and induction of autophagy were analyzed and compared to human PSC liver. Concurrently, the ability of reactive aldehydes to post-translationally modify the autophagosome marker p62 was assessed in PSC liver tissue and in cell culture. Expression of autophagy markers was upregulated in human and mouse cholestatic liver. Whereas mRNA expression of Atg12, Lamp1, Sqstm1 and Map1lc3 was increased in acute cholestasis in mice, it was either suppressed or not significantly changed in chronic cholestasis. In human and murine cholestasis, periportal hepatocytes showed increased IHC staining of ubiquitin, 4-HNE, p62, and selected antioxidant proteins. Increased p62 staining colocalized with accumulation of 4-HNE-modified proteins in periportal parenchymal cells as well as with periportal macrophages in both human and mouse liver. Mechanistically, p62 was identified as a direct target of lipid aldehyde adduction in PSC hepatic tissue and in vitro cell culture. In vitro LS-MS/MS analysis of 4-HNE treated recombinant p62 identified carbonylation of His123, Cys128, His174, His181, Lys238, Cys290, His340, Lys341 and His385. These data indicate that dysregulation of autophagy and oxidative stress/protein damage are present in the same periportal hepatocyte compartment of both human and murine cholestasis. Thus, our results suggest that both increased expression as well as ineffective autophagic degradation of oxidatively-modified proteins contributes to injury in periportal parenchymal cells and that direct modification of p62 by reactive aldehydes may contribute to autophagic dysfunction.

Redox state and the sirtuin deacetylases are major factors that regulate the acetylation status of the stress protein NQO1.

The stress induced protein NQO1 can participate in a wide range of biological pathways which are dependent upon the interaction of NQO1 with protein targets. Many of the protein-protein interactions involving NQO1 have been shown to be regulated by the pyridine nucleotide redox balance. NQO1 can modify its conformation as a result of redox changes in pyridine nucleotides and sites on the C-terminal and helix seven regions of NQO1 have been identified as potential areas that may be involved in redox-dependent protein-protein interactions. Since post-translational modifications can modify the functionality of proteins, we examined whether redox-dependent conformational changes induced in NQO1 would alter lysine acetylation. Recombinant NQO1 was incubated with and without NADH then acetylated non-enzymatically by acetic anhydride or S-acetylglutathione (Ac-GSH). NQO1 acetylation was determined by immunoblot and site-specific lysine acetylation was quantified by mass spectrometry (MS). NQO1 was readily acetylated by acetic anhydride and Ac-GSH. Interestingly, despite a large number of lysine residues (9%) in NQO1 only a small subset of lysines were acetylated and the majority of these were located in or near the functional C-terminal or helix seven regions. Reduction of NQO1 by NADH prior to acetylation resulted in almost complete protection of NQO1 from lysine acetylation as confirmed by immunoblot analysis and MS. Lysines located within the redox-active C-terminus and helix seven regions were readily acetylated when NQO1 was in an oxidized conformation but were protected from acetylation when NQO1 was in the reduced conformation. To investigate regulatory mechanisms of enzymatic deacetylation, NQO1 was acetylated by Ac-GSH then exposed to purified sirtuins (SIRT 1-3) or histone deacetylase 6 (HDAC6). NQO1 could be deacetylated by all sirtuin isoforms and quantitative MS analysis performed using SIRT2 revealed very robust deacetylation of NQO1, specifically at K262 and K271 in the C-terminal region. No deacetylation of NQO1 by HDAC6 was detected. These data demonstrate that the same subset of key lysine residues in the C-terminal and helix seven regions of NQO1 undergo redox dependent acetylation and are regulated by sirtuin-mediated deacetylation.

Proteomic analysis of alcohol-associated hepatitis reveals glycoprotein NMB (GPNMB) as a novel hepatic and serum biomarker.

Alcohol consumption remains a leading cause of liver disease worldwide, resulting in a complex array of hepatic pathologies, including steatosis, steatohepatitis, and cirrhosis. Individuals who progress to a rarer form of alcohol-associated liver disease (ALD), alcohol-associated hepatitis (AH), require immediate life-saving intervention in the form of liver transplantation. Rapid onset of AH is poorly understood and the metabolic mechanisms contributing to the progression to liver failure remain undetermined. While multiple mechanisms have been identified that contribute to ALD, no cures exist and mortality from AH remains high. To identify novel pathways associated with AH, our group utilized proteomics to investigate AH-specific biomarkers in liver explant tissues. The goal of the present study was to determine changes in the proteome as well as epigenetic changes occurring in AH. Protein abundance and acetylomic analyses were performed utilizing nHPLC-MS/MS, revealing significant changes to proteins associated with metabolic and inflammatory fibrosis pathways. Here, we describe a novel hepatic and serum biomarker of AH, glycoprotein NMB (GPNMB). The anti-inflammatory protein GPNMB was significantly increased in AH explant liver and serum compared to healthy donors by 50-fold and 6.5-fold, respectively. Further, bioinformatics analyses identified an AH-dependent decrease in protein abundance across fatty acid degradation, biosynthesis of amino acids, and carbon metabolism. The greatest increases in protein abundance were observed in pathways for focal adhesion, lysosome, phagosome, and actin cytoskeleton. In contrast with the hyperacetylation observed in murine models of ALD, protein acetylation was decreased in AH compared to normal liver across fatty acid degradation, biosynthesis of amino acids, and carbon metabolism. Interestingly, immunoblot analysis found epigenetic marks were significantly increased in AH explants, including Histone H3K9 and H2BK5 acetylation. The increased acetylation of histones likely plays a role in the altered proteomic profile observed, including increases in GPNMB. Indeed, our results reveal that the AH proteome is dramatically impacted through unanticipated and unknown mechanisms. Understanding the origin and consequences of these changes will yield new mechanistic insight for ALD as well as identify novel hepatic and serum biomarkers, such as GPNMB.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin.

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

NQO1 protects obese mice through improvements in glucose and lipid metabolism.

Chronic nutrient excess leads to metabolic disorders and insulin resistance. Activation of stress-responsive pathways via Nrf2 activation contributes to energy metabolism regulation. Here, inducible activation of Nrf2 in mice and transgenesis of the Nrf2 target, NQO1, conferred protection from diet-induced metabolic defects through preservation of glucose homeostasis, insulin sensitivity, and lipid handling with improved physiological outcomes. NQO1-RNA interaction mediated the association with and inhibition of the translational machinery in skeletal muscle of NQO1 transgenic mice. NQO1-Tg mice on high-fat diet had lower adipose tissue macrophages and enhanced expression of lipogenic enzymes coincident with reduction in circulating and hepatic lipids. Metabolomics data revealed a systemic metabolic signature of improved glucose handling, cellular redox, and NAD+ metabolism while label-free quantitative mass spectrometry in skeletal muscle uncovered a distinct diet- and genotype-dependent acetylation pattern of SIRT3 targets across the core of intermediary metabolism. Thus, under nutritional excess, NQO1 transgenesis preserves healthful benefits.

Single-Cell and Population Transcriptomics Reveal Pan-epithelial Remodeling in Type 2-High Asthma.

The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses.

Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek® ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream® ). High-performance liquid chromatography followed by nano-flow liquid chromatography-mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5-fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre-analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano-flow liquid chromatography-mass spectrometry.

Defining decreased protein succinylation of failing human cardiac myofibrils in ischemic cardiomyopathy.

Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.

Lysine malonylation and propionylation are prevalent in human lens proteins.

Acylated lysine residues represent major chemical modifications in proteins. We investigated the malonylation and propionylation of lysine residues (MalK, PropK) in the proteins of aging human lenses. Western blot results showed that the two modifications are present in human lens proteins. Liquid chromatography-mass spectrometry (LC-MS/MS) results showed 4-18 and 4-32 pmol/mg protein of MalK and PropK, respectively, in human lens proteins with no apparent changes related to aging. Mass spectrometry results revealed that MalK- and PropK-modified lysine residues are present in all major crystallins, other cytosolic proteins, and membrane and cytoskeletal proteins of the lens. Several mitochondrial and cytosolic proteins in cultured human lens epithelial cells showed MalK and PropK modifications. Sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) were present in human lens epithelial and fiber cells. Moreover, lens epithelial cell lysate deacylated propionylated and malonylated lysozyme. The absence of SIRT3 and SIRT5 led to higher PropK and MalK levels in mouse lenses. Together, these data suggest that MalK and PropK are widespread modifications in lens and SIRT3 and SIRT5 could regulate their levels in lens epithelial cells.