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Background & Aims The human gut microbiota can regulate production of serotonin (5-hydroxytryptamine [5-HT]) from enterochromaffin cells. However, the mechanisms underlying microbial-induced serotonin signaling are not well understood. Methods Adult germ-free mice were treated with sterile media, live Bifidobacterium dentium, heat-killed B dentium, or live Bacteroides ovatus. Mouse and human enteroids were used to assess the effects of B dentium metabolites on 5-HT release from enterochromaffin cells. In vitro and in vivo short-chain fatty acids and 5-HT levels were assessed by mass spectrometry. Expression of tryptophan hydroxylase, short-chain fatty acid receptor free fatty acid receptor 2, 5-HT receptors, and the 5-HT re-uptake transporter (serotonin transporter) were assessed by quantitative polymerase chain reaction and immunostaining. RNA in situ hybridization assessed 5-HT–receptor expression in the brain, and 5-HT–receptor–dependent behavior was evaluated using the marble burying test. Results B dentium mono-associated mice showed increased fecal acetate. This finding corresponded with increased intestinal 5-HT concentrations and increased expression of 5-HT receptors 2a, 4, and serotonin transporter. These effects were absent in B ovatus-treated mice. Application of acetate and B dentium–secreted products stimulated 5-HT release in mouse and human enteroids. In situ hybridization of brain tissue also showed significantly increased hippocampal expression of 5-HT–receptor 2a in B dentium–treated mice relative to germ-free controls. Functionally, B dentium colonization normalized species-typical repetitive and anxiety-like behaviors previously shown to be linked to 5-HT–receptor 2a. Conclusions These data suggest that B dentium, and the bacterial metabolite acetate, are capable of regulating key components of the serotonergic system in multiple host tissues, and are associated with a functional change in adult behavior., Graphical abstract

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism., Graphical abstract Fully-humanized, bioengineered miR-328-3p agent reduces glycolysis through the suppression of protein levels of targeted transporters GLUT1 and LAT1 in human osteosarcoma cells, leading to synergistic antiproliferative effects when combined with chemotherapeutic drugs.Image 1

Well-orchestrated maternal-fetal crosstalk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium, and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal-fetal crosstalk. Herein, focusing on ligand–receptor complexes and coupled pathways at the maternal-fetal interface in sheep, we provide the first comprehensive proteomic map of ligand-receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand-receptor pathway cascades at the maternal-fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus-endometrium crosstalk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception.

Highlights • Fluorimetric quantification of entrapped FITC-BSA is possible. • Two separate methods allow its quantification in the nanogram range. • Dequenching of FITC-BSA by enzymatic degradation or alkaline unfolding. • Calcium phosphate increases FITC-BSA loading of PLGA nanoparticles by 2.7-fold. • Nanoparticle characteristics were optimized for inner ear drug delivery., Although therapeutically active proteins are highly efficacious, their content in protective nanoparticles is often too low to elicit adequate plasma levels. A strategy to increase protein loading is the in-situ generation of calcium phosphate as a protein adsorbent. To verify this approach, a highly sensitive and reliable fluorimetric method for quantification of incorporated fluorescein-labelled bovine serum albumin (FITC-BSA) as a model protein drug was developed. Dequenching the fluorescein label by pronase E, which digests the protein backbone, and dissolving the nanoparticle matrix in acetonitrile enabled FITC-BSA quantification in the nanogram per milliliter range. This test was confirmed by a second assay involving alkaline hydrolysis of FITC-BSA and the matrix. Nanoparticles prepared with calcium phosphate contained 40 µg FITC-BSA/mg and nanoparticles without calcium phosphate only 15 µg FITC-BSA/mg, representing a 2.7-fold increase in model protein loading. In this work the nanoparticle preparation procedure was optimized in terms of size for administration in the inner ear, but the range of applications is not limited.

Influenza A virus (IAV) infection casts a significant burden on society. It has particularly high morbidity and mortality rates in patients suffering from metabolic disorders. The aim of this study was to relate metabolic changes with IAV susceptibility using well-characterized inbred mouse models. We compared the highly susceptible DBA/2J (D2) mouse strain for which IAV infection is lethal with the C57BL/6J (B6) strain, which exhibits a moderate course of disease and survives IAV infection. Previous studies showed that D2 has higher insulin and glucose levels and is predisposed to develop diet-induced type 2 diabetes. Using high-resolution liquid chromatography–coupled MS, the plasma metabolomes of individual animals were repeatedly measured up to 30 days postinfection. The biggest metabolic difference between these strains in healthy and infected states was in the levels of malonylcarnitine, which was consistently increased 5-fold in D2. Other interstrain and intrastrain differences in healthy and infected animals were observed for acylcarnitines, glucose, branched-chain amino acids, and oxidized fatty acids. By mapping metabolic changes to canonical pathways, we found that mitochondrial beta-oxidation is likely disturbed in D2 animals. In noninfected D2 mice, this leads to increased glycerolipid production and reduced acylcarnitine production, whereas in infected D2 animals, peroxisomal beta-oxidation becomes strongly increased. From these studies, we conclude that metabolic changes caused by a distortion of mitochondrial and peroxisomal metabolism might impact the innate immune response in D2, leading to high viral titers and mortality.

Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell–based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag–based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration–associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease., Graphical Abstract, Highlights • Proteomics data were integrated with prior transcriptomic (RNA-Seq) data on RPE-EMT. • Dysregulated RPE-EMT proteome shares commonality with malignancy-associated EMT. • Altered RPE-EMT proteome signatures correlated with known AMD-associated risk factors. • Protein kinases and phosphatases crosstalk modulate RPE-EMT., In Brief EMT can play a role in retinal diseases. Here, we present a comprehensive proteomic analysis aimed at defining the temporal protein expression changes associated with EMT of stem cell–derived retinal pigment epithelial cells. Tandem mass tag and direct data-independent acquisition MS approaches were performed after inducing RPE-EMT by enzymatic dissociation. We present integration of our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into the RPE-EMT progression.

The integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy.

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR–Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR–Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16., Graphical Abstract, Highlights • TSR1 of ADAMTS16 can be C-mannosylated. • Deletion of DPY19L1 or DPY19L3 in hiPSCs caused reduced secretion of ADAMTS16. • Targeting of dpy19l3 in medaka occasionally led to coloboma., In Brief We identified ADAMTS16 as a target protein for C-mannosylation and showed that this modification is needed for proper secretion of ADAMTS16. Targeting a distinct C-mannosyltransferase by CRISPR–Cas9 in medaka fish embryos caused defects in eye development. We conclude that these developmental defects are caused by reduced secretion of ADAMTS16 when C-mannosylation is missing.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell–cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS., Graphical abstract, Highlights • Semiautomated peptide immunoprecipitation on reusable antibody cartridges. • Application of TOMAHAQ for MHC-I detection and quantitation. • Routine analysis of >4000 unique MHC-I peptides from 250 million cells via automation. • Quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS., In Brief This manuscript highlights a new semiautomated MHC-I peptide immunoprecipitation method used in conjunction with the multiplexed quantitative technique TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification) for the detection and quantitation of MHC-I peptides. This combination of techniques allows the routine analysis of >4000 unique MHC-I peptides from 250 million cells and in with targeted analysis, quantitative sensitivity down to the low amol/μl level.

Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom–up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples., Graphical abstract, Highlights • Microwave-assisted labeling of histones using TMA for bottom–up proteomics. • TMA enables discrimination of isobaric peptides by improved chromatographic behavior. • TMA facilitates histone quantification from MS1-level spectral information. • TMA provides excellent performance for monitoring hPTM pattern in the tissues., In Brief The discrimination of isobaric peptides represents a common challenge in histone characterization. This study reports TMA as a novel derivatization reagent for bottom–up proteomics of histone proteoforms. TMA substantially improves separation of positional isomers, which is a prerequisite for identification and quantification of post-translationally modified histone forms.

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation–enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count–based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture–based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested., Graphical Abstract, Highlights • EDEM2 affinity proteomics and proteome-wide analysis of sucrose density fractionation. • New EDEM2 partners involved in ER quality control and ERAD. • Soluble tyrosinase degradation is EDEM2 dependent in melanoma cells. • Glycoproteomics and SILAC-based proteomics reveal novel EDEM2 potential substrates., In Brief Various pathologies including neurodegenerative diseases and cancers result from disruptions to or stress of ER homeostasis. One key protein proposed to act in quality control processes maintaining ER homeostasis is EDEM2. Using affinity proteomics and sucrose-density sedimentation, we identified several new EDEM2 partners involved in quality control. Moreover, we defined novel endogenous candidates for EDEM2-dependent degradation by combining glycoproteomics and SILAC-based proteomics. Our data suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously thought.

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A2 inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A2, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments., Graphical abstract This study investigated the small molecule inhibitors varespladib, marimastat and dimercaprol in neutralising coagulopathic Crotalinae (pit vipers) snake venom toxins by using nanofractionation analytics which combines bioassays, liquid chromatography and mass spectrometry in parallel. Results obtained highlight their potential as future snakebite treatments.Image 1

The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumor microenvironment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell-, and tumor grade–specific N- and O-glycosylation in surgically removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade–specific alterations of the oligomannosidic-, paucimannosidic-, and branched sialylated complex-type N-glycans, and dynamic remodeling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ∼7400 unique N-glycopeptides from 500 N-glycoproteins and ∼500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell–derived glycoproteins. Furthermore, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumor microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Furthermore, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms., Graphical Abstract, Highlights • Glycomics and glycoproteomics of PCa tissues during disease progression. • Cell-specific dynamics of pauci- and oligomannosylation during PCa progression. • Increased N-glycan branching and core 2-type O-glycosylation in ECM glycoproteins. • Increased N-site occupancy and oligosaccharyltransferase expression in PCa., In Brief Integrated glycomics and glycoproteomics with reference to the established Tissue Atlas were used to uncover the cell-, protein-, site-, and structure-specific N- and O-glycosylation in prostate cancer tissues spanning five disease grades relatively to benign prostatic hyperplasia control tissues. Dynamic cell-specific changes in the paucimannosylation and oligomannosylation of known bone marrow– and prostate-derived glycoproteins, respectively, and increased N-glycan branching and core 2-type O-glycosylation of known extracellular matrix glycoproteins were found to be key changes associated with prostate cancer progression.

Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.

Protein ubiquitylation is an important posttranslational modification that governs most cellular processes. Signaling functions of ubiquitylation are very diverse and involve proteolytic as well as nonproteolytic events, such as localization, regulation of protein interactions, and control of protein activity. The intricacy of ubiquitin signaling is further complicated by several different polyubiquitin chain types that are likely recognized and interpreted by different protein readers. For example, K48-linked ubiquitin chains represent the most abundant chain topology and are the canonical degradation signals, but have been implicated in degradation-independent functions as well, likely requiring a variety of protein readers. Ubiquitin binding domains that interact with polyubiquitin chains are likely at the center of ubiquitin signal recognition and transmission, but their structure and selectivity are largely unexplored. Here we report identification and characterization of the ubiquitin interacting motif-like (UIML) domain of the yeast transcription factor Met4 as a strictly K48-polyubiquitin specific binding unit using methods such as biolayer interferometry (BLI), pull-down assays, and mass spectrometry. We further used the selective binding property to develop an affinity probe for purification of proteins modified with K48-linked polyubiquitin chains. The affinity probe has a Kd = 100 nM for K48 tetra-ubiquitin and shows no detectable interaction with either monoubiquitin or any other polyubiquitin chain configuration. Our results define a short strictly K48-linkage-dependent binding motif and present a new affinity reagent for the K48-polyubiquitin-modified proteome. Our findings benefit the ubiquitin field in analyses of the role of K48-linked polyubiquitylation and increase our understanding of chain topology selective ubiquitin chain recognition., Graphical Abstract, Highlights • The UIML domain of Met4 is a K48-selective polyubiquitin binding domain. • Development of a K48 polyubiquitin chain-specific probe using the UIML domain. • Binding constants to K48 tetra-ubiquitin were determined by biolayer interferometry. • Tandem arrangement of UIML increases affinity and maintains K48-chain specificity. • The UIMLx2 probe can enrich K48 ubiquitylated proteins from yeast and HeLa cells., In Brief Ubiquitylation is a highly complex protein modification. The diversity of the ubiquitin signal is due to the variety of different ubiquitin chains that are assembled in cells. Villamil and colleagues identified a ubiquitin binding domain with high selectivity for only one specific chain type, K48-linked polyubiquitin. They used this UIML domain to generate a K48-ubiquitin chain selective affinity reagent for proteome-wide studies of proteins modified by this specific ubiquitin chain.

Polyethylene glycols (PEGs) in general use are polydisperse molecules with molecular weight (MW) distributed around an average value applied in their designation e.g., PEG 4000. Previous research has shown that PEGs can act as P-glycoprotein (P-gp) inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs. However, questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed. In this study, we examined the mechanism of uptake of PEGs by MDCK-mock cells, in particular, the effect of MW and interaction with P-gp by MDCK-hMDR1 and A549 cells. The results show that: (a) the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors; (b) PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil; and (c) uptake of PEGs of low MW (5000 Da) occurs by a combination of passive diffusion and caveolae-mediated endocytosis. These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems., Graphical abstract PEGs are taken up by cells and act as P-gp substrates. Low molecular weight (MW) PEGs cross cell membranes by passive diffusion, whereas those high MW PEGs enter cells by a combination of passive diffusion and caveolae-mediated endocytosis.Image 1

Autophagy is a major cellular quality control system responsible for the degradation of proteins and organelles in response to stress and damage to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase ubiquitin-specific protease 11 (USP11) restricts autophagy and that KO of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human amyloid-β peptide 42 aggregation-induced paralysis. USP11 coprecipitated with autophagy-specific class III phosphatidylinositol 3-kinase complex I and limited its interaction with nuclear receptor-binding factor 2, thus decreasing lipid kinase activity of class III phosphatidylinositol 3-kinase complex I and subsequent recruitment of effectors such as WD-repeat domain phosphoinositide-interacting proteins to the autophagosomal membrane. Accordingly, more WD-repeat domain phosphoinositide-interacting protein 2 puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mechanistic target of rapamycin, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

Mitochondrial DNA is located in organelle that house essential metabolic reactions and contains high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes, and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1, and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol γ, while increasing NAD+ levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.

Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. Here, we profiled the aging proteome and metabolome of the Drosophila eye and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Of note, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. Our integration of the proteomic, metabolomic, and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. We propose that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases. Data are available via ProteomeXchange with identifier PXD027090., Graphical Abstract, Highlights • Tissue-specific changes in protein abundance occur in the aging Drosophila eye. • Increase in mitochondrial metabolism enzyme abundance in the aging eye. • Decrease in corneal lens protein abundance and calcium buffering in the aging eye. • Dysregulated metabolism impacts vitamin B and methionine metabolism in the aging eye., In Brief Hall et al. profiled the proteome, transcriptome, and metabolome of the aging Drosophila eye. The integrated analysis revealed changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of immune response.

Immune checkpoint inhibitors and adoptive lymphocyte transfer–based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)–driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale MS-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar numbers of peptides were identified from both tumor types. Cell line and patient-specific databases (DBs) were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA class I immunopeptidome MS data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germ line (CG) antigen DB with 285 antigens. This allowed us to identify 40 class I-presented CG antigen–derived peptides. The class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long noncoding RNA (lncRNA) DB, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by class I. We validated tandem MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA class I-binding assays to demonstrate binding to class I proteins. In summary, we provide direct evidence of HLA class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR-mutant lung cancers., Graphical Abstract, Highlights • Proteogenomics identified ∼35,000 class I-presented peptides. • CG antigen and PTM peptides identified in melanoma and lung cancer. • De novo search identified variant and lncRNA-derived peptides. • A new strategy to identify class I-presented lncRNA-derived peptides developed., In Brief Cancer immunotherapy is ineffective in low TMB EGFR-mutant lung adenocarcinoma. Qi et al. performed a comprehensive proteogenomic profiling of HLA class I-presented immunopeptides in high TMB melanoma and low TMB EGFR-mutant lung cancer. Similar numbers of immunopeptides were identified from both. Variant, CG antigen, PTM, and lncRNA-derived peptides were identified. A novel strategy to identify lncRNA-derived peptides was developed. The direct identification of class I-presented immunopeptides will potentially accelerate precision immunotherapy for low TMB tumors.

Extracellular vesicle (EV) proteins from acute myeloid leukemia (AML) cell lines were analyzed using mass spectrometry. The analyses identified 2450 proteins, including 461 differentially expressed proteins (290 upregulated and 171 downregulated). CD53 and CD47 were upregulated and were selected as candidate biomarkers. The association between survival of patients with AML and the expression levels of CD53 and CD47 at diagnosis was analyzed using mRNA expression data from The Cancer Genome Atlas database. Patients with higher expression levels showed significantly inferior survival than those with lower expression levels. ELISA results of the expression levels of CD53 and CD47 from EVs in the bone marrow of patients with AML at diagnosis and at the time of complete remission with induction chemotherapy revealed that patients with downregulated CD53 and CD47 expression appeared to relapse less frequently. Network model analysis of EV proteins revealed several upregulated kinases, including LYN, CSNK2A1, SYK, CSK, and PTK2B. The potential cytotoxicity of several clinically applicable drugs that inhibit these kinases was tested in AML cell lines. The drugs lowered the viability of AML cells. The collective data suggest that AML cell–derived EVs could reflect essential leukemia biology., Graphical Abstract, Highlights • AML-derived EVs are key cargo proteins carrying parental AML cell information. • Proteomics of AML-derived EVs identify biomarkers predicting therapeutic responses. • EVs' CD53 and CD47 expression levels may be related to the prognosis of AML patients., In Brief We analyzed proteomics of AML cell–derived extracellular vesicles (EVs) that carry key information of parental cells. CD53 and CD47 were selected as predictive EV markers for AML, which showed good correlation with relapse-free survival of patients. In addition, several tyrosine kinases were enriched in AML cell–derived EVs, which may serve as potential therapeutic targets for AML.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are important biological markers and cardiac function regulators. Natriuretic peptide receptor A (NPRA) binds to an ANP or BNP ligand and induces transmembrane signal transduction by elevating the intracellular cyclic guanosine monophosphate (cGMP) levels. However, the metabolic phenotype and related mechanisms induced by NPRA deletion remain ambiguous. Here, we constructed myocardial-specific NPRA deletion mice and detected the heart functional and morphological characteristics by histological analysis and explored the altered metabolic pattern and the expression patterns of proteins by liquid chromatography–mass spectrometry (LC-MS)-based omics technology. NPRA deficiency unexpectedly did not result in significant cardiac remodeling or dysfunction. However, compared with the matched littermates, NPRA-deficient mice had significant metabolic differences. Metabolomic analysis showed that the metabolite levels varied in cardiac tissues and plasma. In total, 33 metabolites were identified in cardiac tissues and 54 were identified in plasma. Compared with control mice, NPRA-deficient mice had 20 upregulated and six downregulated metabolites in cardiac tissues and 25 upregulated and 23 downregulated metabolites in plasma. Together, NPRA deficiency resulted in increased nucleotide biosynthesis and histidine metabolism only in heart tissues and decreased creatine metabolism only in plasma. Further proteomic analysis identified 136 differentially abundant proteins in cardiac tissues, including 54 proteins with higher abundance and 82 proteins with lower abundance. Among them, cytochrome c oxidase subunit 7c and 7b (Cox7c, Cox7b), ATP synthase, H+ transporting, mitochondrial Fo complex subunit F2 (ATP5J2), ubiquinol-cytochrome c reductase, complex III subunit X (Uqcr10), and myosin heavy chain 7 (Myh7) were mainly involved in related metabolic pathways. These results revealed the essential role of NPRA in metabolic profiles and may elucidate new underlying pathophysiological mechanisms of NPRA in cardiovascular diseases., Graphical Abstract, Highlights • Metabolomic analysis of cardiac tissues from NPRA-deficient mice. • Metabolomic analysis of plasma from NPRA-deficient mice. • TMT-based proteomic analysis of cardiac tissues in NPRA-deficient mice. • Mechanistic insights into NPRA in mice from metabolomes and proteomes., In Brief Metabolomic analysis in mice revealed that natriuretic peptide receptor A (NPRA) is mainly involved in nucleotide biosynthesis and histidine metabolism in cardiac tissues, and in creatine metabolism, TCA cycle and pentose phosphate pathway in the plasma. Furthermore, proteomics revealed that Cox7c, Cox7b, ATP5J2, Uqcr10, and Myh7 play a vital role in the regulation of metabolic pathway. Together, deterioration of NPRA results in metabolic dysfunction involved with a protein and metabolite-interacting pathway.

Many small ORFs embedded in long noncoding RNA (lncRNA) transcripts have been shown to encode biologically functional polypeptides (small ORF-encoded polypeptides [SEPs]) in different organisms. Despite some novel SEPs have been found, the identification is still hampered by their poor predictability, diminutive size, and low relative abundance. Here, we take advantage of NONCODE, a repository containing the most complete collection and annotation of lncRNA transcripts from different species, to build a novel database that attempts to maximize a collection of SEPs from human and mouse lncRNA transcripts. In order to further improve SEP discovery, we implemented two effective and complementary polypeptide enrichment strategies using 30-kDa molecular weight cutoff filter and C8 solid-phase extraction column. These combined strategies enabled us to discover 353 SEPs from eight human cell lines and 409 SEPs from three mouse cell lines and eight mouse tissues. Importantly, 19 of them were then verified through in vitro expression, immunoblotting, parallel reaction monitoring, and synthetic peptides. Subsequent bioinformatics analysis revealed that some of the physical and chemical properties of these novel SEPs, including amino acid composition and codon usage, are different from those commonly found in canonical proteins. Intriguingly, nearly 65% of the identified SEPs were found to be initiated with non-AUG start codons. The 762 novel SEPs probably represent the largest number of SEPs detected by MS reported to date. These novel SEPs might not only provide new clues for the annotation of noncoding elements in the genome but also serve as a valuable resource for functional study., Graphical abstract, Highlights • Complementary enrichment strategies combined with membrane filtration and C8 SPE. • A combined database with the comprehensive putative SEPs and canonical proteins used. • Seven hundred sixty-two novel SEPs identified from human cell lines, mouse cell lines, and mouse tissues. • Nineteen SEPs have been validated by fusion expression or synthetic peptides., In Brief This study proposed a new and effective strategy for the improved discovery and identification of novel SEPs, including the construction of databases maximally collecting all putative small ORFs from human and mouse lncRNA transcripts in NONCODE and the effective enrichment of polypeptides based on 30-kDa molecular weight cutoff (MWCO) membrane and C8 solid-phase extraction column. This effort led to the discovery of 762 novel lncRNA-encoded SEPs from multiple cell lines and tissues.

Seneca Valley virus (SVV) or commonly known as senecavirus A, is one of the picornavirus that is associated with vesicular disease and neonatal mortality in swine herds. Our previous study found that SVV replicates extremely faster in porcine Instituto Biologico-Rim Suino-2 (IBRS-2) cells than that in porcine kidney-15 (PK-15) cells. However, the underlying mechanism remains unknown. In this study, we comprehensively compared the expression features between IBRS-2 cells and PK-15 cells in response to SVV infection by an unbiased high-throughput quantitative proteomic analysis. We found that the innate immune response–related pathways were efficiently activated in PK-15 cells but not in IBRS-2 cells during SVV infection. A large amount of interferon (IFN)-stimulated genes were induced in PK-15 cells. In contrast, no IFN-stimulated genes were induced in IBRS-2 cells. Besides, we determined similar results in the two cell lines infected by another porcine picornavirus foot-and-mouth disease virus. Further study demonstrated that the Janus kinase signal transducer and activator of transcription signaling pathway was functioning properly in both IBRS-2 and PK-15 cells. A systematic screening study revealed that the aberrant signal transduction from TANK-binding kinase 1 to IFN regulatory factor 3 in the retinoic acid–inducible gene I–like receptor signaling pathway in IBRS-2 cells was the fundamental cause of the different innate immune response manifestation and different viral replication rate in the two cell lines. Together, our findings determined the different features of IBRS-2 and PK-15 cell lines, which will help for clarification of the pathogenesis of SVV. Besides, identification of the underlying mechanisms will provide new targets and an insight for decreasing the viral clearance rate and probably improve the oncolytic effect by SVV in cancer cells., Graphical Abstract, Highlights • Divergent innate immune responses were triggered by SVV in IBRS-2 and PK-15 cells. • SVV induced higher levels of type I IFN in PK-15 cells than in IBRS-2 cells. • IBRS-2 cell line has an aberrant RLR pathway but an intact type I IFN pathway. • TBK1-mediated antiviral signal transduction was dysfunctional in IBRS-2 cells., In Brief Both IBRS-2 and PK-15 cells have been widely used for porcine picornavirus research. However, the virus replicates faster and causes severer CPE in IBRS-2 cells than in PK-15 cells, and the underlying mechanism remains unknown. Proteomic analyses suggested that the RLR pathway was in a dysfunctional state in IBRS-2 cells. We finally determined that the disabled signal transduction from TBK1 to IRF3 in IBRS-2 cells was the fundamental cause of dysfunction of the RLR pathway during porcine picornavirus infection.

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover, and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse life spans including the longest-lived mammal, the bowhead whale. We show that organismal life span is negatively correlated with turnover rates of highly abundant proteins. In comparison with mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production, and reduced reactive oxygen species levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of a rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins., Graphical abstract, Highlights • Cross-species proteomic analyses show that turnover is correlated with life span. • Correlation between life span and turnover is evident for abundant proteins. • Long-lived naked mole rats also have lower ATP production and ROS levels. • Despite having slow turnover, naked mole rats tolerate protein misfolding stress., In Brief A proteomic approach was used to measure protein turnover rates within fibroblasts isolated from species with diverse life spans. We found that life span is negatively correlated with turnover rates of highly abundant proteins. Despite having slower rates of protein turnover, long-lived naked mole rat cells tolerate protein misfolding stress more effectively than short-lived mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys., Graphical abstract fx1, Highlights • We investigated oxidative stress in muscle from the GRMD dog model of DMD. • Excess neutrophils were associated with hypochlorous acid generation in GRMD muscle. • GRMD muscle exhibited oxidative damage to proteins, and protein thiol oxidation. • These data imply a role of immune cell generated oxidative stress in GRMD pathology.

The sample preparation of samples containing bovine serum albumin (BSA), e.g., as used in transdermal Franz diffusion cell (FDC) solutions, was evaluated using an analytical quality-by-design (QbD) approach. Traditional precipitation of BSA by adding an equal volume of organic solvent, often successfully used with conventional HPLC-PDA, was found insufficiently robust when novel fused-core HPLC and/or UPLC-MS methods were used. In this study, three factors (acetonitrile (%), formic acid (%) and boiling time (min)) were included in the experimental design to determine an optimal and more suitable sample treatment of BSA-containing FDC solutions. Using a QbD and Derringer desirability (D) approach, combining BSA loss, dilution factor and variability, we constructed an optimal working space with the edge of failure defined as D

Human age-related diseases, including obesity and type 2 diabetes (T2DM), have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS) subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly) pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM., Graphical abstract fx1, Highlights • First high-throughput redox proteomic analysis of human adipocyte mitochondria. • Thiol protein oxidation is inversely correlated to protein levels in adipocyte mitochondria. • OXPHOS Mitochondrial- vs. nuclear-encoded protein modules are altered in T2DM. • Redox proteomics highlights defects in protein import through the MIA pathway in aging and T2DM. • Complex IV emerges as a common target of oxidative modifications connecting aging and T2DM.

Dihomo-γ-linolenic acid (DGLA) and its downstream fatty acid arachidonic acid (AA) are both nutritionally important ω–6 polyunsaturated fatty acids (ω–6s). Evidence shows that, via COX-mediated peroxidation, DGLA and its metabolites (1-series prostaglandins) are associated with anti-tumor activity, while AA and its metabolites (2-series prostaglandins) could be tightly implicated in various cancer diseases. However, it still remains a mystery why DGLA and AA possess contrasting bioactivities. Our previous studies showed that DGLA could go through an exclusive C-8 oxygenation pathway during COX-catalyzed lipid peroxidation in addition to a C-15 oxygenation pathway shared by both DGLA and AA, and that the exclusive C-8 oxygenation could lead to the production of distinct DGLA׳s free radical derivatives that may be correlated with DGLA׳s anti-proliferation activity. In the present work, we further investigate the anti-cancer effect of DGLA׳s free radical derivatives and their associated molecular mechanisms. Our study shows that the exclusive DGLA׳s free radical derivatives from C-8 oxygenation lead to cell growth inhibition, cell cycle arrest and apoptosis in the human colon cancer cell line HCA-7 colony 29, probably by up-regulating the cancer suppressor p53 and the cell cycle inhibitor p27. In addition, these exclusive radical derivatives were also able to enhance the efficacy of 5-Fluorouracil (5-FU), a widely used chemo-drug for colon cancer. For the first time, we show how DGLA׳s radical pathway and metabolites are associated with DGLA׳s anti-cancer activities and able to sensitize colon cancer cells to chemo-drugs such as 5-FU. Our findings could be used to guide future development of a combined chemotherapy and dietary care strategy for colon cancer treatment., Graphical abstract, Highlight • The free radical metabolites from DGLA peroxidation may possess anticancer activity. • The DGLA׳s radical derivatives could induce cell cycle arrest in colon cancer cell. • The DGLA׳s radical derivatives could induce apoptosis in colon cancer cell. • The DGLA׳s radical derivatives could sensitize colon cancer cell to 5-fluororacil.

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity.

While the long-term physiological adaptation of the neuromuscular system to changed functional demands is usually reflected by unilateral skeletal muscle transitions, the progressive degeneration of distinct motor neuron populations is often associated with more complex changes in the abundance and/or isoform expression pattern of contractile proteins and metabolic enzymes. In order to evaluate these intricate effects of primary motor neuronopathy on the skeletal muscle proteome, label-free MS was employed to study global alterations in the WR (wobbler) mouse model of progressive neurodegeneration. In motor neuron disease, fibre-type specification and the metabolic weighting of bioenergetic pathways appear to be strongly influenced by both a differing degree of a subtype-specific vulnerability of neuromuscular synapses and compensatory mechanisms of fibre-type shifting. Proteomic profiling confirmed this pathobiochemical complexity of disease-induced changes and showed distinct alterations in 72 protein species, including a variety of fibre-type-specific isoforms of contractile proteins, metabolic enzymes, metabolite transporters and ion-regulatory proteins, as well as changes in molecular chaperones and various structural proteins. Increases in slow myosin light chains and the troponin complex and a decrease in fast MBP (myosin-binding protein) probably reflect the initial preferential loss of the fast type of neuromuscular synapses in motor neuron disease., The systematic biochemical analysis of muscle from the wobbler mouse model of motor neuron disease suggests that the loss of neuromuscular synapses causes complex changes in the protein profile of contractile tissues, affecting especially the contractile apparatus and energy metabolism.

OPC (oligodendrocyte progenitor cell) death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H–I (hypoxia–ischemia) in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL–Bax association in white matter following H–I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax–cofilin association was disrupted during glutamate-induced OPC apoptosis.

Mass spectrometry, in the past five years, has increased in speed, accuracy and use. With the ability of the mass spectrometers to identify increasing numbers of proteins the identification of undesirable peptides (those not from the protein sample) has also increased. Most undesirable contaminants originate in the laboratory and come from either the user (e.g. keratin from hair and skin), or from reagents (e.g. trypsin), that are required to prepare samples for analysis. We found that a significant amount of MS instrument time was spent sequencing peptides from abundant contaminant proteins. While completely eliminating non-specific protein contamination is not feasible, it is possible to reduce the sequencing of these contaminants. For example, exclusion lists can provide a list of masses that can be used to instruct the mass spectrometer to ‘ignore’ the undesired contaminant peptides in the list. We empirically generated be-spoke exclusion lists for several model organisms (Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae and Xenopus laevis), utilising information from over 500 mass spectrometry runs and cumulative analysis of these data. Here we show that by employing these empirically generated lists, it was possible to reduce the time spent analysing contaminating peptides in a given sample thereby facilitating more efficient data acquisition and analysis. Biological significance Given the current efficacy of the Mass Spectrometry instrumentation, the utilisation of data from ~500 mass spec runs to generate be-spoke exclusion lists and optimise data acquisition is the significance of this manuscript. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012]., Graphical abstract, Highlights • We looked at the contribution of ‘contaminating’ peptides to MS acquisition time. • We found that 30–50% of time was wasted on contaminant peptide sequencing. • We tested whether the application of an exclusion list would solve this problem. • Exclusion lists generated for specific species from > 500 mass spectrometry runs. • Exclusion lists enabled more efficient analysis with coverage and isoform data.