Differences In Protein Abundance Research Articles

Predicting the efficacy of opioid sequestration by intravenous lipid emulsion using biologically relevant in vitro models of drug distribution

Intravenous lipid emulsions (ILE), among other uses, are utilized in the treatment of poisonings caused by lipophilic substances. The body of evidence regarding the benefits of this treatment is growing but information about opioids-ILE interaction is still very scarce. In this work, the impact of ILE on the distribution of buprenorphine, fentanyl and butorphanol used in various concentrations (100–500 ng/ml) was investigated. Two different in vitro models were used: disposition of the drugs in plasma after ultracentrifugation and distribution into the simulated biophase (cell monolayer of 3T3 fibroblasts or J774.E macrophages). We confirmed the ability of ILE to sequester the three drugs of interest which results in their decrease in the aqueous part of the plasma by 34.2–38.2%, 11.7–28.5% and 6.0–15.5% for buprenorphine, fentanyl and butorphanol, respectively. Moreover, ILE affected the drug distribution to the biophase in vitro, however, in this case the drug concentration in cells decreased by 97.3 ± 3.1%, 28.6 ± 5.4% and 13.0 ± 7.5% for buprenorphine, fentanyl and butorphanol, respectively. The two models revealed notable differences in ILE’s potential for drug sequestration, especially for buprenorphine. Similar, but not as pronounced tendencies were observed for the two other drugs. These discrepancies may result from the difference in protein abundance and resulting drug-protein binding in both systems. Nevertheless, the results obtained with both in vitro models correlated well with the partition coefficient (logP) values for these drugs.

The role of carbonic anhydrase-mediated tissue oxygen extraction in a marine teleost acclimated to hypoxia.

With the growing prevalence of hypoxia (O2 levels ≤2 mg l-1) in aquatic and marine ecosystems, there is increasing interest in the adaptive mechanisms fish may employ to better their performance in stressful environments. Here, we investigated the contribution of a proposed strategy for enhancing tissue O2 extraction - plasma-accessible carbonic anhydrase (CA-IV) - under hypoxia in a species of estuarine fish (red drum, Sciaenops ocellatus) that thrives in fluctuating habitats. We predicted that hypoxia-acclimated fish would increase the prevalence of CA-IV in aerobically demanding tissues to confer more efficient tissue O2 extraction. Furthermore, we predicted the phenotypic changes to tissue O2 extraction that occur with hypoxia acclimation may improve respiratory and swim performance under 100% O2 conditions (i.e. normoxia) when compared with performance in fish that have not been acclimated to hypoxia. Interestingly, there were no significant differences in relative CA-IV mRNA expression, protein abundance or enzyme activity between the two treatments, suggesting CA-IV function is maintained under hypoxia. Likewise, respiratory performance of hypoxia-acclimated fish was similar to that of control fish when tested in normoxia. Critical swim speed (Ucrit) was significantly higher in hypoxia-acclimated fish but translated to marginal ecological benefits with an increase of ∼0.3 body lengths per second. Instead, hypoxia-acclimated fish may have relied more heavily on anaerobic metabolism during their swim trials, utilizing burst swimming 1.5 times longer than control fish. While the maintenance of CA-IV may still be an important contributor for hypoxia tolerance, our evidence suggests hypoxia-acclimated red drum are using other mechanisms to cope in an O2-depleted environment.

Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting.

Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals.

Integrated changes in thermal stability and proteome abundance during altered nutrient states in Escherichia coli and human cells.

Altered thermal solubility measurement techniques are emerging as powerful tools to assess ligand binding, post-translational modification, protein-protein interactions, and many other cellular processes that affect protein state under various cellular conditions. Thermal solubility or stability profiling techniques are enabled on a global proteomic scale by employing isobaric tagging reagents that facilitate multiplexing capacity required to measure changes in the proteome across thermal gradients. Key among these is thermal proteomic profiling (TPP), which requires 8-10 isobaric tags per gradient and generation of multiple proteomic datasets to measure different replicates and conditions. Furthermore, using TPP to measure protein thermal stability state across different conditions may also require measurements of differential protein abundance. Here, we use the proteome integral stability alteration (PISA) assay, a higher throughput version of TPP, to measure global changes in protein thermal stability normalized to their protein abundance. We explore the use of this approach to determine changes in protein state between logarithmic and stationary phase Escherichia coli as well as glucose-starved human Hek293T cells. We observed protein intensity-corrected PISA changes in 290 and 350 proteins due to stationary phase transition in E. coli and glucose starvation, respectively. These data reveal several examples of proteins that were not previously associated with nutrient states by abundance alone. These include E. coli proteins such as putative acyl-CoA dehydrogenase (aidB) and chaperedoxin (cnoX) as well as human RAB vesicle trafficking proteins and many others which may indicate their involvement in metabolic diseases such as cancer.

Quantitative proteome profiling reveals molecular hallmarks of egg quality in Atlantic halibut: impairments of transcription and protein folding impede protein and energy homeostasis during early development

BackgroundTandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels.ResultsA total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs.ConclusionThe results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and β1 Integrins, E-Cadherin and HER-2.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance.

Pre-Activated Granulocytes from an Autoimmune Uveitis Model Show Divergent Pathway Activation Profiles upon IL8 Stimulation In Vitro.

In the pathophysiology of autoimmune-mediated uveitis, granulocytes have emerged as possible disease mediators and were shown to be pre-activated in equine recurrent uveitis (ERU), a spontaneous disease model. We therefore used granulocytes from ERU horses to identify early molecular mechanisms involved in this dysregulated innate immune response. Primary granulocytes from healthy and ERU horses were stimulated with IL8, and cellular response was analyzed with differential proteomics, which revealed significant differences in protein abundance of 170 proteins in ERU. Subsequent ingenuity pathway analysis identified three activated canonical pathways “PKA signaling”, “PTEN signaling” and “leukocyte extravasation”. Clustered to the leukocyte extravasation pathway, we found the membrane-type GPI-anchored protease MMP25, which was increased in IL8 stimulated ERU granulocytes. These findings point to MMP25 as a possible regulator of granulocyte extravasation in uveitis and a role of this molecule in the impaired integrity of the blood-retina-barrier. In conclusion, our analyses show a clearly divergent reaction profile of pre-activated granulocytes upon IL8 stimulation and provide basic information for further in-depth studies on early granulocyte activation in non-infectious ocular diseases. This may be of interest for the development of new approaches in uveitis diagnostics and therapy. Raw data are available via ProteomeXchange with identifier PXD013648.

Label-Free Quantitative Proteomics Reveal the Involvement of PRT6 in Arabidopsis thaliana Seed Responsiveness to Ethylene

In Arabidopsis thaliana, the breaking of seed dormancy in wild type (Col-0) by ethylene at 100 μL L−1 required at least 30 h application. A mutant of the proteolytic N-degron pathway, lacking the E3 ligase PROTEOLYSIS 6 (PRT6), was investigated for its role in ethylene-triggered changes in proteomes during seed germination. Label-free quantitative proteomics was carried out on dormant wild type Col-0 and prt6 seeds treated with (+) or without (−) ethylene. After 16 h, 1737 proteins were identified, but none was significantly different in protein levels in response to ethylene. After longer ethylene treatment (30 h), 2552 proteins were identified, and 619 Differentially Expressed Proteins (DEPs) had significant differences in protein abundances between ethylene treatments and genotypes. In Col, 587 DEPs were enriched for those involved in signal perception and transduction, reserve mobilization and new material generation, which potentially contributed to seed germination. DEPs up-regulated by ethylene in Col included S-adenosylmethionine synthase 1, methionine adenosyltransferase 3 and ACC oxidase involved in ethylene synthesis and of Pyrabactin Resistance1 acting as an ABA receptor, while DEPs down-regulated by ethylene in Col included aldehyde oxidase 4 involved in ABA synthesis. In contrast, in prt6 seeds, ethylene did not result in strong proteomic changes with only 30 DEPs. Taken together, the present work demonstrates that the proteolytic N-degron pathway is essential for ethylene-mediated reprogramming of seed proteomes during germination.

An insight into differential protein abundance throughout Leishmania donovani promastigote growth and differentiation

Leishmania donovani causes anthroponotic visceral leishmaniasis, responsible for about 50,000 annual deaths worldwide. Current therapies have considerable side effects. Drug resistance has been reported and no vaccine is available nowadays. The development of undifferentiated promastigotes in the sand fly vector’s gut leads to the promastigote form that is highly infective to the mammalian host. Fully differentiated promastigotes play a crucial role in the initial stages of mammalian host infection before internalization in the host phagocytic cell. Therefore, the study of protein levels in the promastigote stage is relevant for disease control, and proteomics analysis is an ideal source of vaccine candidate discovery. This study aims to get insight into the protein levels during the differentiation process of promastigotes by 2DE-MALDI-TOF/TOF. This partial proteome analysis has led to the identification of 75 proteins increased in at least one of the L. donovani promastigote differentiation and growth phases. This study has revealed the differential abundance of said proteins during growth and differentiation. According to previous studies, some are directly involved in parasite survival or are immunostimulatory. The parasite survival–related proteins are ascorbate peroxidase; cystathionine β synthase; an elongation factor 1β paralog; elongation factor 2; endoribonuclease L-PSP; an iron superoxide dismutase paralog; GDP-mannose pyrophosphorylase; several heat shock proteins—HSP70, HSP83-17, mHSP70-rel, HSP110; methylthioadenosine phosphorylase; two thiol-dependent reductase 1 paralogs; transitional endoplasmic reticulum ATPase; and the AhpC thioredoxin paralog. The confirmed immunostimulatory proteins are the heat shock proteins, enolase, and protein kinase C receptor analog. The potential immunostimulatory molecules according to findings in patogenic bacteria are fructose-1,6-diphophate aldolase, dihydrolipoamide acetyltransferase, isocitrate dehydrogenase, pyruvate dehydrogenase E1α and E1β subunits, and triosephosphate isomerase. These proteins may become disease control candidates through future intra-vector control methods or vaccines.

How in vitro maturation changes the proteome of ovine cumulus-oocyte complexes?

The present study evaluated the effects of in vitro maturation (IVM) on the proteome of cumulus-oocyte complexes (COCs) from ewes. Extracted COC proteins were analyzed by LC-MS/MS. Differences in protein abundances (p < 0.05) and functional enrichments in immature versusin vitro-matured COCs were evaluated using bioinformatics tools. There were 2550 proteins identified in the COCs, with 89 and 87 proteins exclusive to immature and mature COCs, respectively. IVM caused downregulation of 84 and upregulation of 34 proteins. Major upregulated proteins in mature COCs were dopey_N domain-containing protein, structural maintenance of chromosomes protein, ubiquitin-like modifier-activating enzyme 2. Main downregulated proteins in mature COCs were immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain 2, alpha-2-macroglobulin. Proteins exclusive to mature COCs and upregulated after IVM related to immune response, complement cascade, vesicle-mediated transport, cell cycle, and extracellular matrix organization. Proteins of immature COCs and downregulated after IVM were linked to metabolic processes, immune response, and complement cascade. KEGG pathways and miRNA-regulated genes attributed to downregulated and mature COC proteins related to complement and coagulation cascades, metabolism, humoral response, and B cell-mediated immunity. Thus, IVM influenced the ovine COC proteome. This knowledge supports the future development of efficient IVM protocols for Ovis aries.

The amniotic fluid proteome predicts imminent preterm delivery in asymptomatic women with a short cervix

Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix (< 25 mm) at 16–32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.

Signal Peptide Features Determining the Substrate Specificities of Targeting and Translocation Components in Human ER Protein Import.

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data.

Pilot Study Showing Feasibility of Phosphoproteomic Profiling of Pathway-Level Molecular Alterations in Barrett's Esophagus.

(1) Background: Barrett’s esophagus is a major risk factor for esophageal adenocarcinoma. In this pilot study, we employed precision mass spectrometry to map global (phospho)protein perturbations in Barrett’s esophagus lesions and adjacent normal tissue to glean insights into disease progression. (2) Methods: Biopsies were collected from two small but independent cohorts. Comparative analyses were performed between Barrett’s esophagus samples and adjacent matched (normal) tissues from patients with known pathology, while specimens from healthy patients served as additional controls. (3) Results: We identified and quantified 6810 proteins and 6395 phosphosites in the discovery cohort, revealing hundreds of statistically significant differences in protein abundances and phosphorylation states. We identified a robust proteomic signature that accurately classified the disease status of samples from the independent patient cohorts. Pathway-level analysis of the phosphoproteomic profiles revealed the dysregulation of specific cellular processes, including DNA repair, in Barrett’s esophagus relative to paired controls. Comparative analysis with previously published transcriptomic profiles provided independent evidence in support of these preliminary findings. (4) Conclusions: This pilot study establishes the feasibility of using unbiased quantitative phosphoproteomics to identify molecular perturbations associated with disease progression in Barrett’s esophagus to define potentially clinically actionable targets warranting further assessment.

1337-P: Sex-Specific Variation in Lipid Metabolism in Response to Nutritional Stress Is Associated with Differences in NASH Progression

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the US, encompassing a disease spectrum defined by steatosis, inflammation (NASH) , and fibrosis. Although the incidence of NASH is less in women compared with men, the mechanistic basis for this phenomenon is unclear. The aim of this study was to use high-resolution LC-MS/MS proteomics to determine sex-specific differences in liver protein abundance in healthy mice and in response to NASH that may contribute to protection in females. Male and female C57BL6J mice were fed regular chow (RC) or fast food (FF) diet (40% fat, 0.15% cholesterol, 42 g/L high-fructose corn syrup drinking water; n=18-20/g) to induce NASH. Liver weight and steatosis were significantly increased in male and female FF mice compared to RC counterparts, and the effects were more pronounced in males versus females. Plasma insulin and ALT followed similar patterns. Label-free differential mass spectrometry identified 2,917 proteins, where 314 and 445 proteins exhibited statistically significant changes in expression associated with sex and diet, and 2proteins showed a significant sex x diet interaction. Unsupervised hierarchical clustering of the 2proteins identified four groups of proteins defined by sex-specific differences in baseline abundance or response to FF-diet. Enrichment analysis of proteins within the four groups identified biological process related to 1) lipid catabolism and synthesis that were upregulated in FF males compared with females; 2) catabolism and oxidation-reduction reactions that were suppressed by FF diet in males but not females and 3) acyl-CoA hydrolase activity and fatty acid oxidation that were higher in RC females than males and induced by FF in males but not females. These data suggest that sex-specific differences in liver lipid metabolism at baseline and in response to nutritional stress may contribute to sexual dimorphism in NASH susceptibility. Disclosure M.Moon: None. A.Murali: None. F.Bello: None. J.Wang: None. M.J.Jurczak: None. Funding National Institutes of Health (DK114012)

Proteomic Profile of M. longissimus thoracis from Commercial Lambs Reared in Different Forage Systems

This study compared the protein composition of M. longissimus thoracis of lambs from six commercial forage production systems in New Zealand. A total of 286 proteins were identified based on liquid chromatography-tandem mass spectrometry. First, a binomial model showed that different production groups could be distinguished based on abundances of 16 proteins. Second, pair-wise comparisons were performed to search for protein abundance differences in meat due to animal sex (ewe vs. wether), diet (perennial ryegrass vs. chicory), and age (4 vs. 6–8 months old). Greater abundance of some myofibrillar and sarcoplasmic proteins were observed in lamb loins from ewes compared to wethers. Chicory diet and older age at slaughter were associated with meat with lower abundance of some myofibrillar proteins, possibly due to a greater proportion of muscle glycolytic fibres. The proteins that showed significant differences in their abundances due to production factors could be further investigated to understand their influence on meat quality.

Defects in the Proteome and Metabolome in Human Hypertrophic Cardiomyopathy.

Defects in energetics are thought to be central to the pathophysiology of hypertrophic cardiomyopathy (HCM); yet, the determinants of ATP availability are not known. The purpose of this study is to ascertain the nature and extent of metabolic reprogramming in human HCM, and its potential impact on contractile function. We conducted proteomic and targeted, quantitative metabolomic analyses on heart tissue from patients with HCM and from nonfailing control human hearts. In the proteomic analysis, the greatest differences observed in HCM samples compared with controls were increased abundances of extracellular matrix and intermediate filament proteins and decreased abundances of muscle creatine kinase and mitochondrial proteins involved in fatty acid oxidation. These differences in protein abundance were coupled with marked reductions in acyl carnitines, byproducts of fatty acid oxidation, in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, branched chain amino acids, and their breakdown products, were all significantly increased in HCM hearts. ATP content, phosphocreatine, nicotinamide adenine dinucleotide and its phosphate derivatives, NADP and NADPH, and acetyl CoA were also severely reduced in HCM compared with control hearts. Functional assays performed on human skinned myocardial fibers demonstrated that the magnitude of observed reduction in ATP content in the HCM samples would be expected to decrease the rate of cross-bridge detachment. Moreover, left atrial size, an indicator of diastolic compliance, was inversely correlated with ATP content in hearts from patients with HCM. HCM hearts display profound deficits in nucleotide availability with markedly reduced capacity for fatty acid oxidation and increases in ketone bodies and branched chain amino acids. These results have important therapeutic implications for the future design of metabolic modulators to treat HCM.

DCT‐specific COP9 Signalosome Deletion Activates the WNK4‐NCC Pathway and Mimics Familial Hyperkalemic Hypertension

With‐no‐lysine kinase 4 (WNK4) activates the NaCl cotransporter (NCC) which is the main pathway for sodium reabsorption in the distal convoluted tubule (DCT) of the kidney. The cullin‐RING ubiquitin ligase cullin 3 (CUL3) regulates WNK4 abundance via interaction through the substrate adaptor KLHL3. The monogenic disease familial hyperkalemic hypertension (FHHt) is caused by mutations in this pathway. Mutations in KLHL3 and WNK4 mainly disrupt formation of this complex, whereas, the CUL3 mutations impair binding to the COP9 signalosome (CSN), a deneddylase and upstream regulator of cullin‐RING ligases. Since FHHt is mostly a disease that affects the DCT, we proposed that DCT‐specific knockdown of the main CSN subunit, JAB1, would phenocopy FHHt. We deleted Jab1 in the DCT using the tamoxifen inducible NCC‐Cre‐ERT2 mouse model (DCT‐Jab1‐/‐). After five days of tamoxifen administration, proximal tubule, thick ascending limb, DCT, and connecting tubule/collecting duct segments were individually dissected and isolated to analyze protein abundance of each specific segment via Western blot. JAB1 and CUL3 protein abundance were substantially reduced in the DCT of DCT‐Jab1‐/‐ mice, whereas, WNK4 and phosphorylated NCC (pNCC) abundance were markedly elevated. Western blot analysis of cortical kidney tissue showed lower KLHL3 abundance and significantly higher abundance of phosphorylated SPAK (pSPAK), the intermediary kinase between WNK4 and NCC. Immunofluorescent staining of WNK4 and pSPAK showed increased expression and puncta formation in DCT1 and DCT2 tubules of DCT‐Jab1‐/‐ mice. This was not observed in other tubule segments that express WNK4 or pSPAK, such as the thick ascending limb and connecting tubule/collecting duct. Although there was obvious activation of the WNK4‐SPAK‐NCC pathway DCT‐Jab1‐/‐ mice, plasma analysis showed no significant differences in potassium, chloride or TCO2. However, challenging the DCT‐Jab1‐/‐ mice with a high potassium diet for seven days exaggerated the differences in pSPAK and pNCC protein abundance and led to a significant increase in plasma potassium. The results indicate that DCT‐specific disruption of JAB1 recapitulates many aspects of FHHt, including decreased KLHL3 and CUL3 abundance which leads to activation of the WNK4‐NCC pathway and a tendency toward hyperkalemia. These results implicate defective CSN binding as a key component of CUL3‐mediated familial hyperkalemic hypertension.

Serum Proteomics Identifies Immune Pathways and Candidate Biomarkers of Coronavirus Infection in Wild Vampire Bats

The apparent ability of bats to harbor many virulent viruses without showing disease is likely driven by distinct immune responses that coevolved with mammalian flight and the exceptional longevity of this order. Yet our understanding of the immune mechanisms of viral tolerance is restricted to a small number of bat–virus relationships and remains poor for coronaviruses (CoVs), despite their relevance to human health. Proteomics holds particular promise for illuminating the immune factors involved in bat responses to infection, because it can accommodate especially low sample volumes (e.g., sera) and thus can be applied to both large and small bat species as well as in longitudinal studies where lethal sampling is necessarily limited. Further, as the serum proteome includes proteins secreted from not only blood cells but also proximal organs, it provides a more general characterization of immune proteins. Here, we expand our recent work on the serum proteome of wild vampire bats (Desmodus rotundus) to better understand CoV pathogenesis. Across 19 bats sampled in 2019 in northern Belize with available sera, we detected CoVs in oral or rectal swabs from four individuals (21.1% positivity). Phylogenetic analyses identified all RdRp gene sequences in vampire bats as novel α-CoVs most closely related to known human CoVs. Across 586 identified serum proteins, we found no strong differences in protein composition nor abundance between uninfected and infected bats. However, receiver operating characteristic curve analyses identified seven to 32 candidate biomarkers of CoV infection, including AHSG, C4A, F12, GPI, DSG2, GSTO1, and RNH1. Enrichment analyses using these protein classifiers identified downregulation of complement, regulation of proteolysis, immune effector processes, and humoral immunity in CoV-infected bats alongside upregulation of neutrophil immunity, overall granulocyte activation, myeloid cell responses, and glutathione processes. Such results denote a mostly cellular immune response of vampire bats to CoV infection and identify putative biomarkers that could provide new insights into CoV pathogenesis in wild and experimental populations. More broadly, applying a similar proteomic approach across diverse bat species and to distinct life history stages in target species could improve our understanding of the immune mechanisms by which wild bats tolerate viruses.

Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation–reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

Proteome-Wide Identification and Functional Analysis of Lysine Crotonylation in Trichophyton rubrum Conidial and Mycelial Stages.

Lysine crotonylation is a newly discovered post-translational modification (PTM) with key roles in various important regulatory pathways. Despite its functional significance, there is limited knowledge about crotonylation in fungi. Trichophyton rubrum is the most common fungal pathogen in human infection and is considered a model organism of dermatophytes and human pathogenic filamentous fungi. In this study, we obtained a proteome-wide crotonylation profile of T. rubrum, leading to the identification of 14,019 crotonylated sites on 3144 proteins. The crotonylated proteins were significantly involved in translation and in various metabolic and biosynthetic processes. Some proteins related to fungal pathogenicity were also found to be targets of crotonylation. In addition, extensive crotonylation was found on histones, suggesting a role in epigenetic regulation. Furthermore, about half of the crotonylated proteins were specific to either the conidial or the mycelial stage, and functional enrichment analysis showed some differences between the two stages. The results suggest that the difference in crotonylation between the two stages is not due to differences in protein abundance. Crosstalk of crotonylation with acetylation, propionylation, and succinylation suggests distinct regulatory roles. This study is the first crotonylation analysis in dermatophytes and human pathogenic filamentous fungi. These results represent a solid foundation for further research on PTM regulatory mechanisms in fungi and should facilitate improved antifungal strategies against these medical important species.