Label-free Quantitative Mass Spectrometry Research Articles

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth.

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto–maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/- placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.

Laser Capture Microdissection-Assisted Protein Biomarker Discovery from Coccidioides-Infected Lung Tissue.

Laser capture microdissection (LCM) coupled to label-free quantitative mass spectrometry is a viable strategy to identify biomarkers from infected tissues. In this study, LCM was employed to take a “snapshot” of proteins produced in vivo during Coccidiodies spp. infection in human lungs. Proteomic analysis of LCM lung sections revealed hundreds of hosts and Coccidioidal proteins. Twenty-seven highly abundant Coccidioides spp. proteins were identified which do not share significant sequence orthology with human proteins. Three of the 27 Coccidioidal proteins are also potential Coccidoides-specific biomarkers, as they also do not share sequence homology to any other pathogenic fungus or microbe. Gene ontology analysis of the 27 biomarker candidate proteins revealed enriched hydrolase activity and increased purine and carbohydrate metabolism functions. Finally, we provide proteomic evidence that all 27 biomarker candidates are produced by the fungus when grown in vitro in a media- and growth-phase dependent manner.

Mass spectrometry-based abundance atlas of ABC transporters in human liver, gut, kidney, brain and skin.

ABC transporters (ATP‐binding cassette transporter) traffic drugs and their metabolites across membranes, making ABC transporter expression levels a key factor regulating local drug concentrations in different tissues and individuals. Yet, quantification of ABC transporters remains challenging because they are large and low‐abundance transmembrane proteins. Here, we analysed 200 samples of crude and membrane‐enriched fractions from human liver, kidney, intestine, brain microvessels and skin, by label‐free quantitative mass spectrometry. We identified 32 (out of 48) ABC transporters: ABCD3 was the most abundant in liver, whereas ABCA8, ABCB2/TAP1 and ABCE1 were detected in all tissues. Interestingly, this atlas unveiled that ABCB2/TAP1 may have TAP2‐independent functions in the brain and that biliary atresia (BA) and control livers have quite different ABC transporter profiles. We propose that meaningful biological information can be derived from a direct comparison of these data sets.

Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

ObjectiveThe adamalysin metalloproteinase 15 (ADAM15) has been shown to protect against development of osteoarthritis in mice. Here, we have investigated factors that control ADAM15 levels in cartilage. DesignSecretomes from wild-type and Adam15−/− chondrocytes were compared by label-free quantitative mass spectrometry. mRNA was isolated from murine knee joints, either with or without surgical induction of osteoarthritis on male C57BL/6 mice, and the expression of Adam15 and other related genes quantified by RT-qPCR. ADAM15 in human normal and osteoarthritic cartilage was investigated similarly and by fluorescent immunohistochemistry. Cultured HTB94 chondrosarcoma cells were treated with various anabolic and catabolic stimuli, and ADAM15 mRNA and protein levels evaluated. ResultsThere were no significant differences in the secretomes of chondrocytes from WT and Adam15−/− cartilage. Expression of ADAM15 was not altered in either human or murine osteoarthritic cartilage relative to disease-free controls. However, expression of ADAM15 was markedly reduced upon aging in both species, to the extent that expression in joints of 18-month-old mice was 45-fold lower than in that 4.5-month-old animals. IL-13 increased expression of ADAM15 in HTB94 ​cells by 2.5-fold, while modulators of senescence and autophagy pathways had no effect. Expression of Il13 in the joint was reduced with aging, suggesting this cytokine may control ADAM15 levels in the joint. ConclusionExpression of the chondroprotective metalloproteinase ADAM15 is reduced in aging human and murine joints, possibly due to a concomitant reduction in IL-13 expression. We thus propose IL-13 as a novel factor contributing to increased osteoarthritis risk upon aging.

NQO1 protects obese mice through improvements in glucose and lipid metabolism

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

Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes.

Photocaged cell-permeable ubiquitin probe holds promise in profiling the activity of cellular deubiquitinating enzymes (DUBs) with the much needed temporal control. Here we report a new photocaged cell-permeable ubiquitin probe that undergoes photoactivation upon 365 nm UV treatment and enables intracellular deubiquitinating enzyme profiling. We used a semisynthetic approach to generate modular ubiquitin-based probe containing a tetrazole-derived warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine cell-penetrating peptide (cR10) conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live cells. Upon 365 nm UV irradiation, the tetrazole group is converted to a nitrilimine intermediate in situ, which reacts with nearby nucleophilic cysteine residue from the DUB active site. The new photocaged cell-permeable probe showed good reactivity toward purified DUBs, including USP2, UCHL1, and UCHL3, upon photoirradiation. The Ub-tetrazole probe was also assessed in HeLa cell lysate and showed robust labeling only upon photoactivation. We further carried out protein profiling in intact HeLa cells using the new photocaged cell-permeable ubiquitin probe and identified DUBs captured by the probe using label-free quantitative (LFQ) mass spectrometry. Importantly, the photocaged cell-permeable ubiquitin probe captured DUBs specifically in respective G1/S and G2/M phases in synchronized HeLa cells. Moreover, using this probe DUBs were profiled at different time points following the release of HeLa cells from G1/S phase. Our results showed that photocaged cell-permeable probe represents a valuable new tool for achieving a better understanding of the cellular functions of DUBs.

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.

Dynamic changes in the brain protein interaction network correlates with progression of A\u03b242 pathology in Drosophila

Alzheimer’s disease (AD), the most prevalent form of dementia, is a progressive and devastating neurodegenerative condition for which there are no effective treatments. Understanding the molecular pathology of AD during disease progression may identify new ways to reduce neuronal damage. Here, we present a longitudinal study tracking dynamic proteomic alterations in the brains of an inducible Drosophila melanogaster model of AD expressing the Arctic mutant Aβ42 gene. We identified 3093 proteins from flies that were induced to express Aβ42 and age-matched healthy controls using label-free quantitative ion-mobility data independent analysis mass spectrometry. Of these, 228 proteins were significantly altered by Aβ42 accumulation and were enriched for AD-associated processes. Network analyses further revealed that these proteins have distinct hub and bottleneck properties in the brain protein interaction network, suggesting that several may have significant effects on brain function. Our unbiased analysis provides useful insights into the key processes governing the progression of amyloid toxicity and forms a basis for further functional analyses in model organisms and translation to mammalian systems.

Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes.

BioID, a proximity biotinylation technique, offers a valuable approach to examine the interactions occurring within protein complexes that complements traditional protein biochemical methods. BioID has various advantages that are beneficial to the study of complexes, including an ability to detect insoluble and transient proteins. We have applied BioID to the study of integrin adhesion complexes (IACs), which are located at the junction between the plasma membrane and actin cytoskeleton. The use of multiple BioID baits enables a complex-wide, spatial annotation of IACs, which in turn facilitates the detection of novel proximal interactors and provides insights into IAC architecture. This article describes the labeling and affinity purification of IAC-proximal proteins and their analysis by label-free quantitative mass spectrometry. The article also outlines steps to identify high-confidence proximity interactors, and to interrogate the topology and functional relevance of proximity interaction networks through bioinformatic analyses. © 2020 The Authors. Basic Protocol 1: Proximity biotinylation of integrin adhesion complex components Basic Protocol 2: Mass spectrometry data processing by MaxQuant and detection of high-confidence proximal interactors Basic Protocol 3: Bioinformatic analysis and data visualization.

Abstract LB-095: Synergistic cancer immunotherapy using tumor tissue derived exosomes and artificially produced bacterial outer membrane vesicles

Abstract Checkpoint inhibitors work only in cancers that host inflammatory cells, and “cold” tumors normally do not respond. Therefore, making “cold” tumors “hot” is required to increase the response rate to immunooncology therapies in general. Bacteria and bacterial products have been utilized for cancer immunotherapy for more than 100 years, but currently no such treatment is available because of the severe side effects that are observed. In this study, we produced artificial outer membrane vesicles (aOMVs) from Escherichia coli outer membrane, and injected them together with cancer tissue-derived exosomes to booster an immune response to the malignancy. Firstly, outer membranes were obtained from E. coli by chemical means, followed by ionic stress and applied mild energy to generate aOMVs. The yield and purity of aOMVs were analyzed by nanoparticle tracking analysis and transmission electron microscopy. The protein and RNA contents were examined by label-free quantitative mass spectrometry and bioanalyzer. Inflammation was evaluated in macrophage cell line (RAW 264.7) and mice in vivo, and bone marrow-derived dendritic cells were used to assess the immunomodulatory functions of the aOMVs. For the study of antitumor activity, mice were subcutaneously inoculated with B16F10 cells and then subcutaneously immunized with aOMVs and melanoma exosomes five times at 3-day intervals. Also, anti-mouse PD-1 antibody was intraperitoneally injected into mice 1 day prior to immunization to investigate the effects of combination therapy. To elucidate the immunogenic mechanism, blood and spleen were obtained for antibody titer and splenocyte function study. As a result, bacterial aOMVs presented nanosized spherical shape with closed membranes and exhibited high yield and purity with very few cytosolic components. These aOMVs do not cause pro-inflammatory cytokine responses in RAW 264.7 cells and mice in vivo, despite high exposure levels. The aOMVs could be taken up by dendritic cells to stimulate cytokine and maturation marker expression. Co-immunization with aOMVs and melanoma tissue-derived exosomes elicited tumor regression in melanoma-bearing mice through Th-1 type T cell immunity and anti-tumor exosome IgG antibody production. Also, the immunotherapeutic effect of aOMVs was synergistically enhanced by anti-PD-1 inhibitor. Collectively, bacterial aOMVs can be produced in a large quantities with high purity, but are “detoxified" compared to naturally released OMVs. The non-toxic aOMVs are powerful adjuvants for eliciting specific anti-tumor response, suggesting that aOMVs may be novel bacterial vesicle-mimetics clinically applicable as cancer treatment. Citation Format: Kyong-su Park, Kristina Svennerholm, Rossella Crescitelli, Cecilia Lässer, Inta Gribonika, Jan Lötvall. Synergistic cancer immunotherapy using tumor tissue derived exosomes and artificially produced bacterial outer membrane vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-095.

Abstract 3947: Mass-spectrometry analysis of metastatic matrisome from MC38 experimental liver metastasis

Abstract Prognosis of patients with colorectal cancer liver metastasis is generally dismal due to unavailability of effective therapeutic modalities. Thus, more basic research is required to extend our knowledge about mechanisms underlying development of hepatic metastasis. The extracellular matrix (ECM) represents a backbone scaffold supporting tissue integrity and establishing cell-cell communications; tumor stroma is highly enriched in certain ECM components which have been suggested as drivers of metastatic dissemination. We performed a label-free quantitative mass-spectrometry (MS) analysis of the ECM isolated from experimental liver metastasis generated by intrasplenic injection of MC38 colon cancer cells. As a group of comparison, we used unaffected mouse liver tissue. Extraction of the ECM was performed utilizing an original method of detergent decellularization, fractionation, followed by biochemical enrichment (i.e. depletion of polysaccharides and nucleic acids) developed in our lab. Label-free MS analysis of resultant ECM samples revealed 2328 proteins, of which 140 were classified as murine matrisome components. Inter-sample variability between ECM molecules was insignificant. Among 140 identified ECM molecules, 13 matrix proteins were dramatically upregulated in metastasis tissues in comparison with tumor-free liver ECM. A combination of these 13 ECM proteins significantly predicted overall survival from colorectal adenocarcinoma based on assessment of 486 patients (P<0.01). One of the most prominent ECM candidates was Annexin A1 (ANXA1), an ECM-affiliated protein consistently overrepresented in the murine metastatic tissue by 7 fold with the highest significance score (P<0.0001). We further validated ANXA1 overexpression in human hepatic metastasis tissues. Higher concentrations of tumor ANXA1 tended to predict colorectal cancer prognosis with borderline significance (P=0.06). Using immunohistochemistry, we identified major sources of ANXA1 in the tumor microenvironment, which were the cancer cells, myeloid cells and T cells. ANXA1 has been previously linked to chemoresistance and altered TGF-beta signaling in cancer cells. To explore the functional role of ANXA1, we performed a stable knockdown of this molecule using shRNA transfection. In vitro, ANXA1-deficient cancer cells displayed slower growth rates but elevated migratory capabilities. In vivo, ANXA1 knockdown cells grew significantly slower as compared to vector control cells when injected subcutaneously, suggesting its role in tumor outgrowth. To conclude, here were report a comprehensive proteomics-aided ECM characterization of MC38 murine colorectal liver metastasis and suggest ANXA1 as a possible target for this disease. Citation Format: Arseniy E. Yuzhalin, Dihua Yu, Ruth J. Muschel. Mass-spectrometry analysis of metastatic matrisome from MC38 experimental liver metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3947.

Proteome-Wide Analyses Provide New Insights into the Compatible Interaction of Rice with the Root-Knot Nematode Meloidogyne graminicola.

The root-knot nematode Meloidogyne graminicola is an important pathogen in rice, causing huge yield losses annually worldwide. Details of the interaction between rice and M. graminicola and the resistance genes in rice still remain unclear. In this study, proteome-wide analyses of the compatible interaction of the japonica rice cultivar “Nipponbare” (NPB) with M. graminicola were performed. In total, 6072 proteins were identified in NPB roots with and without infection of M. graminicola by label-free quantitative mass spectrometry. Of these, 513 specifically or significantly differentially expressed proteins were identified to be uniquely caused by nematode infection. Among these unique proteins, 99 proteins were enriched on seven Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. By comparison of protein expression and gene transcription, LOC_Os01g06600 (ACX, a glutaryl-CoA dehydrogenase), LOC_Os09g23560 (CAD, a cinnamyl-alcohol dehydrogenase), LOC_Os03g39850 (GST, a glutathione S-transferase) and LOC_Os11g11960 (RPM1, a disease resistance protein) on the alpha-linolenic acid metabolism, phenylpropanoid biosynthesis, glutathione metabolism and plant–pathogen interaction pathways, respectively, were all associated with disease defense and identified to be significantly down-regulated in the compatible interaction of NPB with nematodes, while the corresponding genes were remarkably up-regulated in the roots of a resistant rice accession “Khao Pahk Maw” with infection of nematodes. These four genes likely played important roles in the compatible interaction of rice with M. graminicola. Conversely, these disease defense-related genes were hypothesized to be likely involved in the resistance of resistant rice lines to this nematode. The proteome-wide analyses provided many new insights into the interaction of rice with M. graminicola.

Label-free quantitative mass spectrometry from formalin-fixed paraffin-embedded samples of nasopharyngeal carcinoma: Preliminary results from a non-endemic European cohort of patients.

Report our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis. Nasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis. FFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients' received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan-Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated. 28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein-Barr (EBV) and Herpes simplex (HSV) viruses' related proteins significantly present in early-stage primary NPC (T1 and T2, p<0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients (p<0.01). Median survival from this M1 group was <1 year (p<0.001). FFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis.

Idiopathic subglottic stenosis (iSGS) is a chronic inflammatory condition that causes dyspnea and affects middle-aged women of White race and non-Latino or Hispanic ethnicity. To better characterize its phenotype and pathogenesis, we assessed the proteomic and genomic methylation signatures of subglottic tissue collected from iSGS patients compared to controls. Molecular analysis of clinical biospecimens. We collected subglottic tissue biopsies from 12 patients during direct laryngoscopy, immediately prior to surgical treatment of iSGS; as well as from 4 age-, sex-, and race/ethnicity-matched control patients undergoing other direct laryngoscopic procedures. We isolated protein and genomic DNA, acquired proteomic data using label-free quantitative mass spectrometry techniques, and acquired genome-wide methylation data using bisulfite conversion and a microarray platform. We compared molecular profiles across the iSGS and control groups, and with respect to clinical course in the iSGS group. Eight of the 12 iSGS patients underwent subsequent blood collection and plasma isolation for further assessment. Proteomic analysis revealed 42 differentially abundant proteins in the iSGS biopsies compared to controls, inferring enrichment of biological pathways associated with early wound healing, innate immunity, matrix remodeling, and metabolism. Proteome-based hierarchical clustering organized patients into two iSGS and one control subgroups. Methylation analysis revealed five hypermethylated genes in the iSGS biopsies compared to controls, including the biotin recycling enzyme biotinidase (BTD). Follow-up analysis showed elevated plasma BTD activity in iSGS patients compared to both controls and published normative data. iSGS exhibits distinct proteomic and genomic methylation signatures. These signatures expand current understanding of the iSGS phenotype, support the possibility of disease subgroups, and should inform the direction of future experimental studies. Not applicable Laryngoscope, 131:E540-E546, 2021.

Robust Summarization and Inference in Proteome-wide Label-free Quantification

Label-Free Quantitative mass spectrometry based workflows for differential expression (DE) analysis of proteins impose important challenges on the data analysis because of peptide-specific effects and context dependent missingness of peptide intensities. Peptide-based workflows, like MSqRob, test for DE directly from peptide intensities and outperform summarization methods which first aggregate MS1 peptide intensities to protein intensities before DE analysis. However, these methods are computationally expensive, often hard to understand for the non-specialized end-user, and do not provide protein summaries, which are important for visualization or downstream processing. In this work, we therefore evaluate state-of-the-art summarization strategies using a benchmark spike-in dataset and discuss why and when these fail compared with the state-of-the-art peptide based model, MSqRob. Based on this evaluation, we propose a novel summarization strategy, MSqRobSum, which estimates MSqRob's model parameters in a two-stage procedure circumventing the drawbacks of peptide-based workflows. MSqRobSum maintains MSqRob's superior performance, while providing useful protein expression summaries for plotting and downstream analysis. Summarizing peptide to protein intensities considerably reduces the computational complexity, the memory footprint and the model complexity, and makes it easier to disseminate DE inferred on protein summaries. Moreover, MSqRobSum provides a highly modular analysis framework, which provides researchers with full flexibility to develop data analysis workflows tailored toward their specific applications.

Bioenergetic shift and actin cytoskeleton remodelling as acute vascular adaptive mechanisms to angiotensin II in murine retina and ophthalmic artery

Ocular vascular dysfunction is a major contributing factor to the pathogenesis of glaucoma. In recent years, there has been a renewed interest in the role of angiotensin II (Ang II) in mediating the disease progression. Despite its (patho)physiological importance, the molecular mechanisms underlying Ang II-mediated oxidative stress remain largely unexplored in the ocular vasculature. Here, we provide the first direct evidence of the alterations of proteome and signalling pathways underlying Ang II-elicited oxidative insult independent of arterial pressure changes in the ophthalmic artery (OA) and retina (R) employing an in vitro experimental model. Both R and OA were isolated from male C57Bl/6J mice (n = 15/group; n = 5/biological replicate) and incubated overnight in medium containing either vehicle or Ang II (0.1 μM) at physiological conditions. Label-free quantitative mass spectrometry (MS)-based proteomics analysis identified a differential expression of 107 and 34 proteins in the R and OA, respectively. Statistical and bioinformatics analyses revealed that protein clusters involved in actin cytoskeleton and integrin-linked kinase signalling were significantly activated in the OA. Conversely, a large majority of differentially expressed retinal proteins were involved in dysregulation of numerous energy-producing and metabolic signalling pathways, hinting to a possible shift in retinal cell bioenergetics. Particularly, Ang II-mediated downregulation of septin-7 (Sept7; p < 0.01) and superoxide dismutase [Cu-Zn] (Sod1; p < 0.05), and upregulation of troponin T, fast skeletal muscle (Tnnt3; p < 0.05) and tropomyosin alpha-3 chain (Tpm3; p < 0.01) in the OA, and significant decreased expressions of two crystallin proteins (Cryab; p < 0.05 and Crybb2; p < 0.0001) in the R were verified at the mRNA level, corroborating our proteomics findings. In summary, these results demonstrated that exogenous application of Ang II over an acute time period caused impairment of retinal bioenergetics and cellular demise, and actin cytoskeleton-mediated vascular remodelling in the OA.

Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics.

5-Lipoxygenase (5-LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5-LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter-interacting proteins, obtained by DNA pulldown and label-free quantitative mass spectrometry. Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel-like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G-quadruplex structures, which may play a functional role in ALOX5 gene regulation.

Changes in the proteome of sea urchin Paracentrotus lividus coelomocytes in response to LPS injection into the body cavity.

BackgroundThe immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated.Principal findingsIn the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis.ConclusionsOur data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways. Data are available via ProteomeXchange with identifier PXD008439.

Comparative proteomic analysis of round and elongated spermatids during spermiogenesis in mice.

Spermiogenesis in mammals is an exclusive process during which haploid round spermatids mature into spermatozoa in the testis. Any abnormality in the process of spermiogenesis may result in male infertility. The aim of the present study was to characterize the differentially expressed proteins between round and elongated spermatids in mice using label-free quantitative mass spectrometry. Of the 2411 proteins identified in this study, 333 were differentially expressed with a ≥10-fold change, including 208 upregulated proteins and 125 downregulated proteins in round spermatids relative to elongated spermatids. Gene Ontology analysis showed that these differentially expressed proteins were categorized into 10 types of subcellular localizations, 9 molecular functions, and were involved in 9 biological processes. All the identified proteins participated in 268 different pathways. In addition, ubiquitin-mediated proteolysis and the proteasome pathway, autophagy, lysosome, and apoptosis pathways were involved in the mechanism of spermiogenesis. Our data may provide valuable information for a better understanding of spermiogenesis and help improve the diagnosis and treatment of male factor infertility.

Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome.

Protein kinases are important mediators of signal transduction in cellular pathways, and calcium-dependent protein kinases (CDPKs) compose a unique class of calcium-dependent kinases present in plants and apicomplexans, including Plasmodium parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite Plasmodium falciparum grows inside red blood cells, and P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage P. falciparum phosphoproteome by performing large-scale phosphoproteomic profiling on tightly synchronized parasites just prior to egress, identifying 2,704 phosphorylation sites on 919 proteins. Using a conditional knockdown of PfCDPK5, we identify 58 phosphorylation sites on 50 proteins with significant reduction in levels of PfCDPK5-deficient parasites. Furthermore, gene ontology analysis of the identified proteins reveals enrichment in transmembrane- and membrane-associated proteins and in proteins associated with transport activity. Among the identified proteins is PfNPT1, a member of the apicomplexan-specific novel putative transporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, P. falciparum egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics.IMPORTANCE The malaria parasite Plasmodium falciparum is a major cause of morbidity and mortality globally. The P. falciparum parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is essential for egress; parasites deficient in PfCDPK5 remain trapped inside their host cells. We have used a label-free quantitative mass spectrometry approach to identify the phosphoproteome of schizont-stage parasites just prior to egress and identify 50 proteins that display a significant reduction in phosphorylation in PfCDPK5-deficient parasites. We show that a member of the Apicomplexan-specific transport protein family, PfNPT1 is a potential substrate of PfCDPK5 and is localized to the parasite plasma membrane. P. falciparum egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.