bims-prodis 2018-05-13 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2018–05–13
nine papers selected by
Nancy Gough, Bioserendipity

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.
Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3.
Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.
Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids.
Quantitative proteomics analysis of differentially expressed proteins in ruptured and unruptured cerebral aneurysms by iTRAQ.
The Chinese herbal formula Zhibai Dihuang Granule treat Yin-deficiency-heat syndrome rats by regulating the immune responses.
HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.
Proteome analysis of the potential serum biomarkers for chronic benzene poisoning.
Quantitative proteomics and biochemical analyses reveal the role of endoplasmin in the regulation of the expression and secretion of A Disintegrin And Metalloproteinase 12.

Reprod Biomed Online. 2018 Apr 21. pii: S1472-6483(18)30220-7. [Epub ahead of print]

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

Fatemehsadat Amjadi, Mehdi Mehdizadeh, Mahnaz Ashrafi, Davood Nasrabadi, Sara Taleahmad, Mehdi Mirzaei, Vivek Gupta, Ghasem Hosseini Salekdeh, Reza Aflatoonian.

   RESEARCH QUESTION: What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)?
DESIGN: In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources.
RESULTS: Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton.
CONCLUSIONS: The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.

Keywords:  Endometrium; Luteal phase; PCOS; Polycystic ovary syndrome; Proliferative phase; Proteomics

DOI:  https://doi.org/10.1016/j.rbmo.2018.04.043
BMC Biol. 2018 May 07. 16(1): 47

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3.

Angelika Schmidt, Francesco Marabita, Narsis A Kiani, Catharina C Gross, Henrik J Johansson, Szabolcs Éliás, Sini Rautio, Matilda Eriksson, Sunjay Jude Fernandes, Gilad Silberberg, Ubaid Ullah, Urvashi Bhatia, Harri Lähdesmäki, Janne Lehtiö, David Gomez-Cabrero, Heinz Wiendl, Riitta Lahesmaa, Jesper Tegnér.

   BACKGROUND: Regulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.
RESULTS: To gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.
CONCLUSION: The data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.

Keywords:  Data integration; FOXP3; Proteomics; RNA sequencing (RNA-Seq); Regulatory T cells; T cell differentiation; TGF-β; Treg; iTreg

DOI:  https://doi.org/10.1186/s12915-018-0518-3
Kidney Int. 2018 May 04. pii: S0085-2538(18)30181-9. [Epub ahead of print]

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.

Nino Kvirkvelia, Malgorzata McMenamin, Marie Warren, Ravirajsinh N Jadeja, Sai Karthik Kodeboyina, Ashok Sharma, Wenbo Zhi, Paul M O'Connor, Raghavan Raju, Rudolf Lucas, Michael P Madaio.

  To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

Keywords:  PKC-α inhibition; glomerular endothelial cells; mitochondrial dysfunction; nephritis; proteomics; targeted delivery

DOI:  https://doi.org/10.1016/j.kint.2018.01.032
Reprod Sci. 2018 Jan 01. 1933719118773420

Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids.

M Fairuz B Jamaluddin, Prathima B Nagendra, Pravin Nahar, Christopher Oldmeadow, Pradeep S Tanwar.

  Uterine leiomyomas (fibroids) are the most common gynecological tumors, which are enriched in the extracellular matrix (ECM). Fibroids are leading cause of abnormal uterine bleeding and hysterectomy. One of the major questions yet to be answered is the overproduction of specific ECM components in human uterine fibroids, particularly in relation to mutations in the driver gene mediator complex subunit 12 ( MED12). Surgical specimens from 14 patients with uterine leiomyoma having fibroids and corresponding adjacent normal myometrium (ANM) were utilized to analyze genetic and proteomic expression patterns in the tissue samples. MED12 mutations in the fibroids were screened by Sanger sequencing. iTRAQ was used to label the peptides in small-, medium-, and large-sized fibroid samples of annotated MED12 mutation from the same patient. The mixtures of the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the differential expression proteins. Using isobaric tagged-based quantitative mass spectrometry on 3 selected patients, ECM-related protein tenascin-C (TNC) was observed significantly upregulated (>1.5-fold) with a confidence corresponding to false discovery rate (FDR) <1% in small-, medium-, and large-sized fibroid samples regardless of MED12 mutation status. The TNC was validated on additional patient samples using Western blotting (WB) and immunohistochemistry (IHC) and confirmed significant overexpression of this protein in fibroids compared to matched ANM. Proteomic analyses have identified the increased ECM protein expression, TNC, as a hallmark of uterine fibroids regardless of MED12 mutations. Further functional studies focusing on the upregulated ECM proteins in leiomyogenesis will lead to the identification of novel ECM drug targets for fibroid treatment.

Keywords:  ECM; fibroids; infertility; tumor; uterus

DOI:  https://doi.org/10.1177/1933719118773420
J Proteomics. 2018 May 03. pii: S1874-3919(18)30195-7. [Epub ahead of print]

Quantitative proteomics analysis of differentially expressed proteins in ruptured and unruptured cerebral aneurysms by iTRAQ.

Pengjun Jiang, Jun Wu, Xin Chen, Bo Ning, Qingyuan Liu, Zhengsong Li, Maogui Li, Fan Yang, Yong Cao, Rong Wang, Shuo Wang.

  The underlying pathophysiological mechanisms involved in cerebral aneurysms rupture remain unclear. This study was performed to investigate the differentially expressed proteins between ruptured and unruptured aneurysms using quantitative proteomics. The aneurysmal walls of six ruptured aneurysms and six unruptured aneurysms were collected during the surgical operation. The isobaric tags for relative and absolute quantification (iTRAQ) were used to identify the differentially expressed proteins and western blotting was performed to validate the expression of the proteins of interest. Bioinformatics analysis of the differentially expressed proteins was also performed using the KEGG database and GO database. Between ruptured and unruptured aneurysms, 169 proteins were found differently expressed, including 74 up-regulated proteins and 95 down-regulated proteins with a fold change ≥ 2 and p value ≤ .05. KEGG pathway analysis revealed that phagosome, focal adhesion and ECM-receptor interaction were the most common pathways involved in aneurysm rupture. In addition, the differential expressions of ITGB3, CRABP1 and S100A9 were validated by western blotting. Through the iTRAQ method, we found that inflammatory responses and cell-matrix interactions may play a significant role in the rupture of cerebral aneurysms. These findings provide a basis for better understanding of pathophysiological mechanisms associated with aneurysm rupture.
BIOLOGICAL SIGNIFICANCE: Intracranial aneurysm is the leading cause of life-threating subarachnoid hemorrhage which can cause 45% patients die within 30 days and severe morbidity in long-term survivors. With a high prevalence ranging from 1% to 5% in general population, cerebral aneurysm has become a widespread health hazard over past decades. Though great advances have been achieved in the diagnosis and treatment of this disease, the underlying pathophysiological mechanisms of aneurysm rupture remains undetermined and a lot of uncertainty still exists surrounding the treatment of unruptured cerebral aneurysms. Clarifying the mechanism associated with aneurysm rupture is important for estimating the rupture risk, as well as the development of new treatment strategy. Some previous studies have analyzed the molecular differences between ruptured and unruptured IAs at gene and mRNA levels, but further comprehensive proteomic studies are relatively rare. Here we performed a comparative proteomics study to investigate the differentially expressed proteins between ruptured IAs (RIAs) and unruptured IAs (UIAs). Results of our present study will provide more insights into the pathogenesis of aneurysm rupture at protein level. With a better understanding of pathophysiological mechanisms associated with aneurysm rupture, some noninvasive treatment strategies may be developed in the future.

Keywords:  Intracranial aneurysm; Proteomics analysis; Rupture; iTRAQ

DOI:  https://doi.org/10.1016/j.jprot.2018.05.001
J Ethnopharmacol. 2018 May 02. pii: S0378-8741(17)32634-X. [Epub ahead of print]

The Chinese herbal formula Zhibai Dihuang Granule treat Yin-deficiency-heat syndrome rats by regulating the immune responses.

Chang-Ming Liu, Jing Chen, Su Yang, Lian-Gen Mao, Ting-Ting Jiang, Hui-Hui Tu, Zhong-Liang Chen, Yu-Ting Hu, Lin Gan, Zhong-Jie Li, Ji-Cheng Li.

   ETHNOPHARMACOLOGICAL RELEVANCE: Zhibai Dihuang Granule (ZDG), a traditional Chinese medicine (TCM) made from eight Chinese herbs, has been classically used to treat Yin-deficiency-heat (YDH) syndrome. ZDG is well known with the therapeutic efficacy of nourishing Yin and decreasing internal heat in clinic, but the mechanism of ZDG's therapeutic effect is still not clear.
MATERIALS AND METHODS: High doses of triiodothyronine (T3) were given intraperitoneally to induce Hyperthyroid YDH syndrome in SD rats. The animals were then treated with ZDG for one week. The iTRAQ-coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique was used to screen the differentially expressed serum proteins between ZDG treated rats and YDH syndrome rats. The differentially expressed proteins were analyzed by bioinformatics method and were verified by enzyme-linked immunosorbent assay (ELISA).
RESULTS: A total of 55 differentially expressed proteins were identified, including 23 up-regulated proteins (>1.25 fold, p<0.05) and 32 down-regulated proteins (<0.80 fold, p<0.05). Among the differentially expressed proteins, 26 proteins returned to normal after ZDG treatment. Bioinformatics analysis showed that these proteins were mainly involved in immune response, including regulation of immune system process, complement activation, and humoral immune response mediated by circulating immunoglobulin. ELISA revealed significantly increased levels of Zinc-alpha-2-glycoprotein (Azgp1), L-selectin, C-reactive protein (Crp), Plasminogen (Plg), Kininogen 1 (Kng1), and significantly decreased levels of Mannose binding lectin 2 (Mbl2) and Complement C1qb chain (C1qb) in ZDG treated rats compared with YDH syndrome rats. Bioinformatics analyses indicated that Azgp1 participated in antigen processing and presentation, Crp, C1qb, and Mbl2 were involved in complement activation, while L-selectin, Plg, and Kng1 were involved in regulating the inflammatory response.
CONCLUSIONS: Our study provides experimental evidence to understand the therapeutic mechanism of ZDG in YDH syndrome. The results suggested that ZDG may regulate the complement activation and inflammatory response, and promote the ability to recognize antigens to alleviate YDH syndrome.

Keywords:  Traditional Chinese Medicine; Yin-deficiency-heat syndrome; Zhibai Dihuang Granule; immune response; proteomics

DOI:  https://doi.org/10.1016/j.jep.2018.05.001
Aging Cell. 2018 May 05. e12710

HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

Michael K Lin, Jin Yang, Chun Wei Hsu, Anuradha Gore, Alexander G Bassuk, Lewis M Brown, Ryan Colligan, Jesse D Sengillo, Vinit B Mahajan, Stephen H Tsang.

  High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.

Keywords:  Neurodegenerative diseases; age related macular degeneration; genetics; mass spectrometry

DOI:  https://doi.org/10.1111/acel.12710
Environ Toxicol Pharmacol. 2018 Apr 27. pii: S1382-6689(18)30081-4. [Epub ahead of print]60 157-164

Proteome analysis of the potential serum biomarkers for chronic benzene poisoning.

Zhimin Zhang, Peimao Li, Dafeng Lin, Dianpeng Wang, Yanfang Zhang.

  The aim of our study is to seek novel specific biomarkers which could provide clues to the mechanism of chronic benzene poisoning (CBP) and might also be used as specific markers for early detection and diagnosis. In this study, a comparative serological proteome analysis between normal controls and CBP patients at three different levels of poisoning were performed via a 2D-DIGE and MALDI-TOF-MS. As the result a total of 10 proteins were found significantly altered between the normal and the mild, moderate and severe poisoning. The identified differentially expressed proteins were classified according to their molecular functions, biological processes, and protein classes, and three important serum proteins among them, apolipoproteinA-1, alpha-1-antitrypsin and complement C3, were further confirmed by immune turbidimetric analysis for their significant up-regulation in the CBP patients. Our findings suggest that these differential proteins may help elucidate the mechanism of CBP and provide potential biomarkers for diagnosis.

Keywords:  Benzene; Biomarkers; Chronic poisoning; Hematotoxicity; Serum proteins

DOI:  https://doi.org/10.1016/j.etap.2018.04.017
J Proteomics. 2018 May 02. pii: S1874-3919(18)30194-5. [Epub ahead of print]

Quantitative proteomics and biochemical analyses reveal the role of endoplasmin in the regulation of the expression and secretion of A Disintegrin And Metalloproteinase 12.

Lipeng Xiong, Wenwen Yan, Emmanuel Zubia, Yanqing Zhou, Yang Zhang, Qianqian Duan, Mahesh Narayan, Guoqiang Xu.

  A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in multiple cancers such as breast and cervical cancers and its high expression reduces the overall patient survival rate. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently in cells are not clear. Here, we use affinity purification and mass spectrometry to identify the ADAM12S-interacting proteins. Spectral counting and MaxQuant label-free quantification reveal that ADAM12S but not ADAM12L specifically interacts with a subset of endoplasmic reticulum proteins, such as endoplasmin (GRP94), 78 kDa glucose-regulated protein (GRP78), and UDP-glucose:glycoprotein glucosyltransferase I (UGGT1), that regulate the folding and processing of secreted proteins. Further biochemical experiments validate the interaction between ADAM12S and several of its interacting proteins. Computational docking analysis demonstrates that GRP94 preferentially interacts with ADAM12S over ADAM12L. The data also suggest that both the protein expression level and the secretion of ADAM12S are regulated by GRP94 expression and knockdown. Our results reveal a link between these two proteins that are highly expressed in cancer cells. Furthermore, our studies define a new ADAM12S-specific regulator that may contribute to the cancer development.
SIGNIFICANCE: A Disintegrin And Metalloproteinase 12 (ADAM12) is highly expressed in many cancers such as lung, breast, and cervical cancers. ADAM12 has two major splicing variants, the long membrane-anchored form ADAM12L and the short secreted form ADAM12S. However, how they are regulated and whether they are modulated similarly or differently are not completely understood. We use affinity purification and label-free quantitative proteomics to identify the ADAM12S-interacting proteins. Our results reveal that ADAM12S specifically interacts with a subset of endoplasmic reticulum proteins, including endoplasmin (GRP94), UDP-glucose:glycoprotein glucosyltransferase I (UGGT1), and neutral α-glucosidase AB (GANAB). Computer modeling reveals that ADAM12S interacts with the surface amino acids of GRP94 more strongly than ADAM12L. Biochemical experiments further reveal that GRP94 regulates both the protein level and the secretion of ADAM12S. Database mining finds that both GRP94 and ADAM12 are highly expressed in multiple cancers and their high expression is correlated with poor patient survival rate. Taken together, our work discovers a new upstream regulator for ADAM12S, which may contribute to its distinct functions in the regulation of the migration and invasion of cancer cells.

Keywords:  ADAM12S; Docking; Endoplasmin (GRP94); Label-free quantification; Secretion; Spectral counting

DOI:  https://doi.org/10.1016/j.jprot.2018.04.033