bims-prodis 2018-11-25 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2018–11–25
34 papers selected by
Nancy Gough, Bioserendipity

Carcinogenic Helicobacter pylori strains selectively dysregulate the in vivo gastric proteome, which may be associated with stomach cancer progression.
The future of protein biomarker research in type 2 diabetes mellitus.
Proteome Investigation of Rat Lungs subjected to Ex Vivo Perfusion (EVLP).
Obesity shows preserved plasma proteome in large independent clinical cohorts.
MALDI-Imaging for Classification of Epithelial Ovarian Cancer Histotypes from a Tissue Microarray using Machine Learning Methods.
The use of proteomics to understand antiviral immunity.
Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma.
Tumour-derived extracellular vesicles as a novel source of protein biomarkers for cancer diagnosis and monitoring.
From Analysis of Ischemic Mouse Brain Proteome to Identification of Human Serum Clusterin as a Potential Biomarker for Severity of Acute Ischemic Stroke.
Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives.
Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples.
Differences in plasma fibrin clot composition in patients with thrombotic antiphospholipid syndrome compared with venous thromboembolism.
Constitutional absence of epithelial integrin α3 impacts the composition of the cellular microenvironment of ILNEB keratinocytes.
Pazopanib-induced changes in protein expression signatures of extracellular vesicles in synovial sarcoma.
Quantitative Proteomics Reveals Antibiotics Resistance Function of Outer Membrane Proteins in Aeromonas hydrophila.
Diverse Protein Profiles in CNS Myeloid Cells and CNS Tissue From Lipopolysaccharide- and Vehicle-Injected APPSWE/PS1ΔE9 Transgenic Mice Implicate Cathepsin Z in Alzheimer's Disease.
A Proteomic Approach for Understanding the Mechanisms of Delayed Corneal Wound Healing in Diabetic Keratopathy Using Diabetic Model Rat.
Development and Validation of Apolipoprotein AI-Associated Lipoprotein Proteome Panel for the Prediction of Cholesterol Efflux Capacity and Coronary Artery Disease.
Dataset on the comparative proteomic profiling of mouse saliva and serum from wild type versus the dystrophic mdx-4cv mouse model of dystrophinopathy.
ASSESSING AUTOMATED SAMPLE PREPARATION TECHNOLOGIES FOR HIGH-THROUGHPUT PROTEOMICS OF FROZEN WELL CHARACTERIZED TISSUES FROM SWEDISH BIOBANKS.
mTORC1 is a key mediator of RON-dependent breast cancer metastasis with therapeutic potential.
Integration of transcriptome and proteome profiles in glioblastoma: looking for the missing link.
SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.
Extracellular Vesicles from Neurosurgical Aspirates Identifies Chaperonin Containing TCP1 Subunit 6A (CCT6A) as a Potential Glioblastoma Biomarker with Prognostic Significance.
TSG101 promotes the proliferation, migration and invasion of hepatocellular carcinoma cells by regulating the PEG10.
The use of proteomic technologies to study molecular mechanisms of multidrug resistance in cancer.
Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity.
Compound kushen injection suppresses human acute myeloid leukaemia by regulating the Prdxs/ROS/Trx1 signalling pathway.
Cluster of Differentiation 46 is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28).
Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth.
Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways.
Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.
Proteomic Identification of Allergenic Proteins of Morus alba L. Pollenexacerbation.
ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay.

Mol Cell Proteomics. 2018 Nov 19. pii: mcp.RA118.001181. [Epub ahead of print]

Carcinogenic Helicobacter pylori strains selectively dysregulate the in vivo gastric proteome, which may be associated with stomach cancer progression.

Jennifer M Noto, Kristie Rose, Amanda J Hachey, Alberto G Delgado, Judith Romero-Gallo, Lydia E Wroblewski, Barbara G Schneider, Shailja C Shah, Timothy L Cover, Keith T Wilson, Dawn A Israel, Juan Carlos Roa, Kevin L Schey, Yana Zavros, M Blanca Piazuelo, Richard M Peek.

  Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+ H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly upregulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared to gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.

Keywords:  Cancer biomarker(s); G3BP2; Gastrointestinal disease; Helicobacter pylori; Immunohistochemistry; Infectious disease; Inflammatory response; Microbiology; Molecular biology*; Pathway Analysis; RABEP2; Tandem Mass Spectrometry; iTRAQ

DOI:  https://doi.org/10.1074/mcp.RA118.001181
Expert Rev Proteomics. 2018 Nov 23.

The future of protein biomarker research in type 2 diabetes mellitus.

Roel Tans, Lars Verschuren, Hans J C T Wessels, Stephan J L Bakker, Cees J Tack, Jolein Gloerich, Alain J van Gool.

   INTRODUCTION: The onset of Type 2 Diabetes Mellitus (T2DM) is strongly associated with obesity and subsequent perturbations in immuno-metabolic responses. To understand the complexity of these systemic changes and better monitor the health status of people at risk, validated clinical biomarkers are needed. Omics technologies are increasingly applied to measure the interplay of genes, proteins and metabolites in biological systems, which is imperative in understanding molecular mechanisms of disease and selecting the best possible molecular biomarkers for clinical use. Areas covered: This review describes the complex onset of T2DM, the contribution of obesity and adipose tissue inflammation to the T2DM disease mechanism, and the output of current biomarker strategies. A new biomarker approach is described that combines published and new self-generated data to merge multiple -omes (i.e. genome, proteome, metabolome etc.) towards understanding of mechanism of disease on the individual level and design multi-parameter biomarker panels that drive significant impacts on personalized healthcare. Expert commentary: We here propose an approach to use cross-omics analyses to contextualize published biomarker data and better understand molecular mechanisms of health and disease. This will improve the current and future innovation gaps in translation of discovered putative biomarkers to clinically applicable biomarker tests.

Keywords:  Type 2 Diabetes Mellitus; X-omics; adipose tissue inflammation; innovation gaps; protein biomarkers; translational medicine

DOI:  https://doi.org/10.1080/14789450.2018.1551134
Molecules. 2018 Nov 22. pii: E3061. [Epub ahead of print]23(12):

Proteome Investigation of Rat Lungs subjected to Ex Vivo Perfusion (EVLP).

Valentina Roffia, Antonella De Palma, Caterina Lonati, Dario Di Silvestre, Rossana Rossi, Marco Mantero, Stefano Gatti, Daniele Dondossola, Franco Valenza, Pierluigi Mauri, Francesco Blasi.

  Ex vivo lung perfusion (EVLP) is an emerging procedure that allows organ preservation, assessment and reconditioning, increasing the number of marginal donor lungs for transplantation. However, physiological and airflow measurements are unable to unveil the molecular mechanisms responsible of EVLP beneficial effects on lung graft and monitor the proper course of the treatment. Thus, it is urgent to find specific biomarkers that possess these requirements but also accurate and reliable techniques that identify them. The purpose of this study is to give an overview on the potentiality of shotgun proteomic platforms in characterizing the status and the evolution of metabolic pathways during EVLP in order to find new potential EVLP-related biomarkers. A nanoLC-MS/MS system was applied to the proteome analysis of lung tissues from an optimized rat model in three experimental groups: native, pre- and post-EVLP. Technical and biological repeatability were evaluated and, together with clustering analysis, underlined the good quality of data produced. In-house software and bioinformatics tools allowed the label-free extraction of differentially expressed proteins among the three examined conditions and the network visualization of the pathways mainly involved. These promising findings encourage further proteomic investigations of the molecular mechanisms behind EVLP procedure.

Keywords:  Ex Vivo Lung Perfusion (EVLP); nanoLC-MS/MS; proteomics; transplantation

DOI:  https://doi.org/10.3390/molecules23123061
Sci Rep. 2018 Nov 19. 8(1): 16981

Obesity shows preserved plasma proteome in large independent clinical cohorts.

Ornella Cominetti, Antonio Núñez Galindo, John Corthésy, Armand Valsesia, Irina Irincheeva, Martin Kussmann, Wim H M Saris, Arne Astrup, Ruth McPherson, Mary-Ellen Harper, Robert Dent, Jörg Hager, Loïc Dayon.

Holistic human proteome maps are expected to complement comprehensive profile assessment of health and disease phenotypes. However, methodologies to analyze proteomes in human tissue or body fluid samples at relevant scale and performance are still limited in clinical research. Their deployment and demonstration in large enough human populations are even sparser. In the present study, we have characterized and compared the plasma proteomes of two large independent cohorts of obese and overweight individuals using shotgun mass spectrometry (MS)-based proteomics. Herein, we showed, in both populations from different continents of about 500 individuals each, the concordance of plasma protein MS measurements in terms of variability, gender-specificity, and age-relationship. Additionally, we replicated several known and new associations between proteins, clinical and molecular variables, such as insulin and glucose concentrations. In conclusion, our MS-based analyses of plasma samples from independent human cohorts proved the practical feasibility and efficiency of a large and unified discovery/replication approach in proteomics, which was also recently coined "rectangular" design.

DOI: https://doi.org/10.1038/s41598-018-35321-7
Proteomics Clin Appl. 2018 Nov 24. e1700181

MALDI-Imaging for Classification of Epithelial Ovarian Cancer Histotypes from a Tissue Microarray using Machine Learning Methods.

Oliver Klein, Frederic Kanter, Hagen Kulbe, Paul Jank, Carsten Denkert, Grit Nebrich, Wolfgang D Schmitt, Zhiyang Wu, Catarina A Kunze, Jalid Sehouli, Silvia Darb-Esfahani, Ioana Braicu, Jan Lellmann, Herbert Thiele, Eliane T Taube.

   PURPOSE: Precise histological classification of epithelial ovarian cancer (EOC) has immanent diagnostic and therapeutic consequences, but remains challenging in histological routine. The aim of this pilot study was to examine the potential of MALDI-Imaging mass spectrometry in combination with machine learning methods to classify EOC histological subtypes from tissue microarray.
EXPERIMENTAL DESIGN: Formalin-fixed-paraffin-embedded tissue of 20 patients with ovarian clear-cell, 14 low-grade serous, 19 high-grade serous ovarian carcinomas and 14 serous borderline tumors were analysed using MALDI-Imaging. Classifications were computed by linear discriminant analysis (LDA), support vector machines with linear (SVM-lin) and radial basis function kernels (SVM-rbf), a neural network (NN), and a convolutional neural network (CNN).
RESULTS: MALDI-Imaging and machine learning methods results in classification of EOC histotypes with mean accuracy of 80% for LDA, 80% SVM-lin, 74% SVM-rbf, 83% NN and 85% CNN. Based on sensitivity (69%-100%) and specificity (90-99%), CCN and NN were most suited to EOC classification.
CONCLUSION AND CLINICAL RELEVANCE: The pilot study demonstrates the potential of MALDI-Imaging derived proteomic classifiers in combination with machine learning algorithms to discriminate EOC histotypes. Applications might support the development of new prognostic parameters in the assessment of EOC. This article is protected by copyright. All rights reserved.

Keywords:  histotype classification; imaging mass spectrometry; machine learning; ovarian cancer

DOI:  https://doi.org/10.1002/prca.201700181
Semin Cell Dev Biol. 2018 Dec;pii: S1084-9521(16)30451-7. [Epub ahead of print]84 22-29

The use of proteomics to understand antiviral immunity.

Amanda P Woon, Anthony W Purcell.

  Viruses are intracellular pathogens that cause a vast array of diseases, which are often severe and typified by high morbidity and mortality rates. Viral infections continue to be a global health burden and effective vaccines and therapeutics are constantly sought to prevent and treat these infections. The development of such treatments generally relies on understanding the mechanisms that underpin efficient host antiviral immune responses. This review summarises recent developments in our understanding of antiviral adaptive immunity and in particular, highlights the use of mass spectrometry to elucidate viral antigens and their processing and presentation to T cells and other immune effectors. These processed peptides serve as potential vaccine candidates or may facilitate clinical monitoring, diagnosis and immunotherapy of infectious diseases.

Keywords:  Antigen presentation; Antigen processing; Immunopeptidomics; Mass spectrometry; Proteomics; Virus

DOI:  https://doi.org/10.1016/j.semcdb.2017.12.002
Sci Signal. 2018 Nov 20. pii: eaap9752. [Epub ahead of print]11(557):

Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma.

Kristina B Emdal, Anna-Kathrine Pedersen, Dorte B Bekker-Jensen, Alicia Lundby, Shana Claeys, Katleen De Preter, Frank Speleman, Chiara Francavilla, Jesper V Olsen.

Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis.

DOI: https://doi.org/10.1126/scisignal.aap9752
Proteomics. 2018 Nov 23. e1800155

Tumour-derived extracellular vesicles as a novel source of protein biomarkers for cancer diagnosis and monitoring.

Olivia Ruhen, Katie Meehan.

  "Liquid biopsies" have received attention as a complementary tool for traditional tissue biopsies that may enhance the spectrum of analysis for tumour-derived factors. One such factor gaining prominence in the liquid biopsy field are extracellular vesicles (EVs), membrane-bound nanovesicles which are secreted by cells into biofluids such as blood, urine and saliva. EVs are released in both physiological and pathological conditions and can transport a variety of molecules, including proteins, metabolites, RNA, microRNAs and DNA, to distant sites throughout the body. As such, they are emerging as a promising source of tumour biomarkers for the non-invasive diagnosis, prognosis and monitoring of cancer patients. In particular, the wealth of tumour-related information that can be gleaned from the EV proteomic cargo has become apparent through mass spectrometric analysis, which has provided new benchmarks for clinically-focused biomarker research. In this review, we have explored the current achievements in the use of mass spectrometry for identifying potential EV-derived protein biomarkers of cancer, and summarised the techniques and challenges involved in this pursuit. This article is protected by copyright. All rights reserved.

Keywords:  cancer-associated extracellular vesicles; clinically-derived biofluids; comprehensive proteomic analyses

DOI:  https://doi.org/10.1002/pmic.201800155
Transl Stroke Res. 2018 Nov 21.

From Analysis of Ischemic Mouse Brain Proteome to Identification of Human Serum Clusterin as a Potential Biomarker for Severity of Acute Ischemic Stroke.

Hailong Song, Hui Zhou, Zhe Qu, Jie Hou, Weilong Chen, Weiwu Cai, Qiong Cheng, Dennis Y Chuang, Shanyan Chen, Shuwei Li, Jilong Li, Jianlin Cheng, C Michael Greenlief, Yuan Lu, Agnes Simonyi, Grace Y Sun, Chenghan Wu, Jiankun Cui, Zezong Gu.

  Ischemic stroke is a devastating neurological disease that can cause permanent brain damage, but to date, few biomarkers are available to reliably assess the severity of injury during acute onset. In this study, quantitative proteomic analysis of ischemic mouse brain detected the increase in expression levels of clusterin (CLU) and cystatin C (CST3). Since CLU is a secretary protein, serum samples (n = 70) were obtained from acute ischemic stroke (AIS) patients within 24 h of stroke onset and together with 70 matched health controls. Analysis of CLU levels indicated significantly higher levels in AIS patients than healthy controls (14.91 ± 4.03 vs. 12.79 ± 2.22 ng/L; P = 0.0004). Analysis of serum CST3 also showed significant increase in AIS patients as compared with healthy controls (0.90 ± 0.19 vs. 0.84 ± 0.12 ng/L; P = 0.0064). The serum values of CLU were also positively correlated with the NIH Stroke Scale (NIHSS) scores, the time interval after stroke onset, as well as major stroke risk factors associated with lipid profile. These data demonstrate that elevated levels of serum CLU and CST3 are independently associated with AIS and may serve as peripheral biomarkers to aid clinical assessment of AIS and its severity. This pilot study thus contributes to progress toward preclinical proteomic screening by using animal models and allows translation of results from bench to bedside.

Keywords:  Biomarkers; Clusterin; Cystatin C; Ischemic stroke; Proteomics

DOI:  https://doi.org/10.1007/s12975-018-0675-2
Proteomics. 2018 Nov 23. e1800247

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives.

Maria N Barrachina, Beatriz Calderón-Cruz, Lucía Fernandez-Rocca, Ángel García.

  Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids and nucleic acids. In this review, we provide some guidelines for plasma-derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease studies. For EVs analysis, blood samples should be collected using a 21-gauge needle, preferably in citrate tubes, and plasma stored for up to one year at -80°, using a single freeze-thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization can be done by transmission electron microscopy, particle enumeration techniques (nanoparticle-tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label-free gel-free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular-related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of cardiovascular disease. This article is protected by copyright. All rights reserved.

Keywords:  biomarkers; cardiometabolic disease; cardiovascular disease; circulating microparticles; exosomes; extracellular vesicles; microvesicles; proteomics

DOI:  https://doi.org/10.1002/pmic.201800247
Proteomics Clin Appl. 2018 Nov 24. e1700166

Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples.

Camilla Restellini, Alessandro Cuomo, Michela Lupia, Marco Giordano, Tiziana Bonaldi, Roberta Noberini.

   PURPOSE: Profiling histone post-translational modifications (PTMs) in clinical samples holds great potential for the identification of epigenetic biomarkers and the discovery of novel epigenetic targets. Mass spectrometry (MS)-based approaches to analyze histone PTMs in clinical samples usually rely on SDS-PAGE separation following histone enrichment in order to eliminate detergents and further isolate histones. However, this limits the digestions options and hence the modification coverage.
EXPERIMENTAL DESIGN AND RESULTS: The aim of this study was the implementation of a procedure involving acetone protein precipitation followed by histone enrichment through a C18 StageTip column to obtain histone preparations suitable for various in-solution digestion protocols. Among them, we tested the Arg-C digestion, which allows profiling histone H4 modifications, and the Prop-PIC method, which improves the detection of short and hydrophilic peptides. We verified the feasibility of this approach for the analysis of different types of samples, including formalin-fixed paraffin-embedded pathology tissues, and employed it to profile histone H4 modifications in cancer samples and normal tissues, identifying previously reported differences, as well as novel ones.
CONCLUSIONS AND CLINICAL RELEVANCE: This protocol widens the number of applications available in the toolbox of clinical epigenomics, allowing the investigation of a larger spectrum of marks in patient samples. This article is protected by copyright. All rights reserved.

Keywords:  epigenetic mark; histone post-translational modifications; mass spectrometry; proteomics; reversed-phase chromatography

DOI:  https://doi.org/10.1002/prca.201700166
Sci Rep. 2018 Nov 23. 8(1): 17301

Differences in plasma fibrin clot composition in patients with thrombotic antiphospholipid syndrome compared with venous thromboembolism.

Aneta Stachowicz, Michal Zabczyk, Joanna Natorska, Maciej Suski, Rafał Olszanecki, Ryszard Korbut, Jacek R Wiśniewski, Anetta Undas.

The prothrombotic fibrin clot phenotype has been reported in patients with thrombotic antiphospholipid syndrome (APS) and venous thromboembolism (VTE). Protein composition of plasma fibrin clots in APS has not been studied. We evaluated 23 patients with thrombotic APS, 19 with VTE alone, and 20 well-matched controls. A proteomic analysis of fibrin clots generated from citrated plasma was based on liquid chromatography-mass spectrometry. Plasma levels of thrombospondin-1 (TSP1), apolipoprotein(a), A-I, and B-100, complement components (C)3a, C5b-C9, histidine-rich glycoprotein (HRG), and prothrombin were evaluated using immunoenzymatic tests. In plasma fibrin clots of APS patients, compared with VTE subjects and controls, we identified decreased amounts of (pro)thrombin, antithrombin-III, apolipoprotein A-I, and HRG with no differences in plasma levels of antithrombin, prothrombin, along with lower plasma HRG and apolipoprotein A-I. In APS patients, plasma HRG positively correlated with amounts of clot-bound HRG, while apolipoprotein A-I was inversely associated with clot-bound levels of this protein. The most predominant proteins within the clots of APS patients were bone marrow proteoglycan, C5-C9, immunoglobulins, apolipoprotein B-100, platelet-derived proteins, and TSP1. Our study is the first to demonstrate differences in the protein composition of fibrin clots generated from plasma of thrombotic APS patients versus those with VTE alone.

DOI: https://doi.org/10.1038/s41598-018-35034-x
Matrix Biol. 2018 Dec;pii: S0945-053X(18)30281-6. [Epub ahead of print]74 62-76

Constitutional absence of epithelial integrin α3 impacts the composition of the cellular microenvironment of ILNEB keratinocytes.

Yinghong He, Kerstin Thriene, Melanie Boerries, Ingrid Hausser, Claus-Werner Franzke, Hauke Busch, Joern Dengjel, Cristina Has.

  Integrin α3β1, a major epidermal adhesion receptor is critical for organization of the basement membrane during development and wound healing. Integrin α3 deficiency leads to interstitial lung disease, nephrotic syndrome and epidermolysis bullosa (ILNEB), an autosomal recessive multiorgan disease characterized by basement membrane abnormalities in skin, lung and kidney. The pathogenetic chains from ITGA3 mutation to tissue abnormalities are still unclear. Although integrin α3 was reported to regulate multiple extracellular proteins, the composition of the extracellular compartment of integrin α3-negative keratinocytes has not been resolved so far. In a comprehensive approach, quantitative proteomics of deposited extracellular matrix, conditioned cultured media as well as of the intracellular compartment of keratinocytes isolated from an ILNEB patient and from normal skin were performed. By mass spectrometry-based proteomics, 167 proteins corresponding to the GO terms "extracellular" and "cell adhesion", or included in the "human matrisome" were identified in the deposited extracellular matrix, and 217 in the conditioned media of normal human keratinocytes. In the absence of integrin α3, 33% and 26% respectively were dysregulated. Dysregulated proteins were functionally related to integrin α3 or were known interaction partners. The results show that in the absence of integrin α3 ILNEB keratinocytes produce a fibronectin-rich microenvironment and make use of fibronectin-binding integrin subunits αv and α5. The most important results were validated in monolayer and organotypic coculture models. Finally, the in vivo relevance of the most dysregulated components was demonstrated by immunostainings of skin, kidney and lung samples of three ILNEB patients.

Keywords:  Basement membrane; Epidermolysis bullosa; Extracellular matrix; Fibronectin; Genodermatosis; Integrin α3; Keratinocyte; Proteomics

DOI:  https://doi.org/10.1016/j.matbio.2018.07.001
Biochem Biophys Res Commun. 2018 Nov 30. pii: S0006-291X(18)32148-X. [Epub ahead of print]506(3): 723-730

Pazopanib-induced changes in protein expression signatures of extracellular vesicles in synovial sarcoma.

Kumiko Shiozawa, Yusuke Yoshioka, Zhiwei Qiao, Ji Shuting, Takahiro Ochiya, Tadashi Kondo.

  Secreted proteins enclosed in extracellular vesicles can act as intercellular messengers. The objective of this study was to elucidate the role of proteins secreted from synovial sarcoma cells in the regulatory network underlying pazopanib response. We performed a comprehensive analysis of expression of proteins secreted from four synovial sarcoma cell lines (SYO-1, HS-SYII, 1273/99, and YaFuSS) using mass spectroscopy. Comparison of up-regulated proteins in cells, extracellular vesicles-free conditioned media, and extracellular vesicles revealed significantly up-regulated Wnt in synovial sarcoma vesicles. Furthermore, we compared protein signatures of cells, conditioned media, and extracellular vesicles before and after pazopanib treatment. Interestingly, protein signatures of extracellular vesicles showed robust changes in Wnt signaling pathways in response to pazopanib. Our findings provide insight into the potential role of Wnt, a protein secreted from the extracellular vesicles of synovial sarcoma cells, making it a potential candidate for use in sarcoma diagnosis.

Keywords:  Extracellular vesicle; Mass spectrometry; Pazopanib; Synovial sarcoma

DOI:  https://doi.org/10.1016/j.bbrc.2018.10.012
Front Cell Infect Microbiol. 2018 ;8 390

Quantitative Proteomics Reveals Antibiotics Resistance Function of Outer Membrane Proteins in Aeromonas hydrophila.

Zujie Yao, Lina Sun, Yuqian Wang, Ling Lin, Zhuang Guo, Dong Li, Wenxiong Lin, Xiangmin Lin.

  Outer membrane proteins (OMPs) play essential roles in antibiotic resistance, particularly in Gram-negative bacteria; however, they still have many unidentified functions regarding their behavior in response to antibiotic stress. In the current work, quantitative tandem mass tag labeling-based mass spectrometry was used to compare the outer membrane related proteins between an oxytetracycline-resistant (OXY-R) and its original control stain (OXY-O) in Aeromonas hydrophila. Consequently, a total of 261 commonly altered proteins in two biological repeats were identified including 29 proteins that increased and 28 that decreased. Gene ontology analysis showed that the expression of transport proteins was significantly reduced, and translation-related proteins were downregulated in the OXY-R strain. After using western blotting to validate selected altered proteins, eight OMP-related genes were knocked out and their roles in antibiotic resistance were further evaluated. The survival assays showed that some mutants such as ΔAHA_4281, ΔAHA_2766, ΔAHA_2282, ΔAHA_1181, and ΔAHA_1280 affected the susceptibility of A. hydrophila to antimicrobials. Moreover, the minimum inhibitory concentration assay showed that these candidate mutants also respond differently to other types of antibiotics. Our results reveal several novel outer membrane related proteins of A. hydrophila that play important roles in antibiotic resistance, and as such, may be helpful for screening studies to identify novel drug targets.

Keywords:  Aeromonas hydrophila; outer membrane protein; oxytetracycline resistance; quantitative proteomics; sarcosine-insoluble proteins

DOI:  https://doi.org/10.3389/fcimb.2018.00390
Front Cell Neurosci. 2018 ;12 397

Diverse Protein Profiles in CNS Myeloid Cells and CNS Tissue From Lipopolysaccharide- and Vehicle-Injected APPSWE/PS1ΔE9 Transgenic Mice Implicate Cathepsin Z in Alzheimer's Disease.

Camilla Thygesen, Laura Ilkjær, Stefan J Kempf, Anne Louise Hemdrup, Christian Ulrich von Linstow, Alicia A Babcock, Sultan Darvesh, Martin R Larsen, Bente Finsen.

  Neuroinflammation, characterized by chronic activation of the myeloid-derived microglia, is a hallmark of Alzheimer's disease (AD). Systemic inflammation, typically resulting from infection, has been linked to the progression of AD due to exacerbation of the chronic microglial reaction. However, the mechanism and the consequences of this exacerbation are largely unknown. Here, we mimicked systemic inflammation in AD with weekly intraperitoneal (i.p.) injections of APPSWE/PS1ΔE9 transgenic mice with E. coli lipopolysaccharide (LPS) from 9 to 12 months of age, corresponding to the period with the steepest increase in amyloid pathology. We found that the repeated LPS injections ameliorated amyloid pathology in the neocortex while increasing the neuroinflammatory reaction. To elucidate mechanisms, we analyzed the proteome of the hippocampus from the same mice as well as in unique samples of CNS myeloid cells. The repeated LPS injections stimulated protein pathways of the complement system, retinoid receptor activation and oxidative stress. CNS myeloid cells from transgenic mice showed enrichment in pathways of amyloid-beta clearance and elevated levels of the lysosomal protease cathepsin Z, as well as amyloid precursor protein, apolipoprotein E and clusterin. These proteins were found elevated in the proteome of both LPS and vehicle injected transgenics, and co-localized to CD11b+ microglia in transgenic mice and in primary murine microglia. Additionally, cathepsin Z, amyloid precursor protein, and apolipoprotein E appeared associated with amyloid plaques in neocortex of AD cases. Interestingly, cathepsin Z was expressed in microglial-like cells and co-localized to CD68+ microglial lysosomes in AD cases, and it was expressed in perivascular cells in AD and control cases. Taken together, our results implicate systemic LPS administration in ameliorating amyloid pathology in early-to-mid stage disease in the APPSWE/PS1ΔE9 mouse and attract attention to the potential disease involvement of cathepsin Z expressed in CNS myeloid cells in AD.

Keywords:  Alzheimer’s disease; amyloid precursor protein; apolipoprotein E; cathepsin Z; microglia; perivascular cells; quantitative proteomics; systemic inflammation

DOI:  https://doi.org/10.3389/fncel.2018.00397
Int J Mol Sci. 2018 Nov 18. pii: E3635. [Epub ahead of print]19(11):

A Proteomic Approach for Understanding the Mechanisms of Delayed Corneal Wound Healing in Diabetic Keratopathy Using Diabetic Model Rat.

Tetsushi Yamamoto, Hiroko Otake, Noriko Hiramatsu, Naoki Yamamoto, Atsushi Taga, Noriaki Nagai.

  Diabetes mellitus is a widespread metabolic disorder, and long-term hyperglycemia in diabetics leads to diabetic keratopathy. In the present study, we used a shotgun liquid chromatography/mass spectrometry-based global proteomic approach using the cornea of streptozotocin-induced diabetic (STZ) rats to examine the mechanisms of delayed corneal wound healing in diabetic keratopathy. Applying a label-free quantitation method based on spectral counting, we identified 188 proteins that showed expression changes of >2.0-fold in the cornea of STZ rats. In particular, the level of lumican expression in the cornea of STZ rats was higher than that of the normal rats. In the cornea of the normal rat, the expression level of lumican was elevated during the wound healing process, and it returned to the same expression level as before cornea injury after the wound was healed completely. On the other hand, a high expression level of lumican in the cornea of STZ rats was still maintained even after the wound was healed completely. In addition, adhesion deficiency in corneal basal cells and Bowman's membrane was observed in the STZ rat. Thus, abnormally overexpressed lumican may lead to adhesion deficiency in the cornea of STZ rats.

Keywords:  corneal wound healing streptozotocin-induced diabetic rat; diabetic keratopathy; lumican; shotgun proteomics

DOI:  https://doi.org/10.3390/ijms19113635
Clin Chem. 2018 Nov 21. pii: clinchem.2018.291922. [Epub ahead of print]

Development and Validation of Apolipoprotein AI-Associated Lipoprotein Proteome Panel for the Prediction of Cholesterol Efflux Capacity and Coronary Artery Disease.

Zhicheng Jin, Timothy S Collier, Darlene L Y Dai, Virginia Chen, Zsuzsanna Hollander, Raymond T Ng, Bruce M McManus, Robert Balshaw, Sophia Apostolidou, Marc S Penn, Cory Bystrom.

BACKGROUND: Cholesterol efflux capacity (CEC) is a measure of HDL function that, in cell-based studies, has demonstrated an inverse association with cardiovascular disease. The cell-based measure of CEC is complex and low-throughput. We hypothesized that assessment of the lipoprotein proteome would allow for precise, high-throughput CEC prediction.
METHODS: After isolating lipoprotein particles from serum, we used LC-MS/MS to quantify 21 lipoprotein-associated proteins. A bioinformatic pipeline was used to identify proteins with univariate correlation to cell-based CEC measurements and generate a multivariate algorithm for CEC prediction (pCE). Using logistic regression, protein coefficients in the pCE model were reweighted to yield a new algorithm predicting coronary artery disease (pCAD).
RESULTS: Discovery using targeted LC-MS/MS analysis of 105 training and test samples yielded a pCE model comprising 5 proteins (Spearman r = 0.86). Evaluation of pCE in a case-control study of 231 specimens from healthy individuals and patients with coronary artery disease revealed lower pCE in cases (P = 0.03). Derived within this same study, the pCAD model significantly improved classification (P < 0.0001). Following analytical validation of the multiplexed proteomic method, we conducted a case-control study of myocardial infarction in 137 postmenopausal women that confirmed significant separation of specimen cohorts in both the pCE (P = 0.015) and pCAD (P = 0.001) models.
CONCLUSIONS: Development of a proteomic pCE provides a reproducible high-throughput alternative to traditional cell-based CEC assays. The pCAD model improves stratification of case and control cohorts and, with further studies to establish clinical validity, presents a new opportunity for the assessment of cardiovascular health.

DOI: https://doi.org/10.1373/clinchem.2018.291922
Data Brief. 2018 Dec;21 1236-1245

Dataset on the comparative proteomic profiling of mouse saliva and serum from wild type versus the dystrophic mdx-4cv mouse model of dystrophinopathy.

Sandra Murphy, Margit Zweyer, Rustam R Mundegar, Dieter Swandulla, Kay Ohlendieck.

The comparative proteomic data presented in this article provide supporting information to the related research article "Proteomic identification of elevated saliva kallikrein levels in the mdx-4cv mouse model of Duchenne muscular dystrophy " (Murphy et al., 2018). Here we provide additional datasets on the comparative proteomic analysis of saliva and serum proteins and the mass spectrometric identification of kallikrein isoform Klk-1 in wild type versus mdx-4cv saliva specimens. The data article presents the systematic identification of the assessable saliva proteome and the differential presence of proteins in saliva versus serum samples. Representative mass spectrometric scans of unique peptides that were employed to identify the kallikrein isoform Klk-1 in wild type versus mdx-4cv saliva specimens are provided. The dataset contains typical saliva-associated marker proteins, including alpha-amylase and albumin, as well as distinct isoforms of cystatin, serpin, kallikrein, cathepsin, glutathione transferase, carbonic anhydrase, mucin, pyruvate kinase, and aldolase.

DOI: https://doi.org/10.1016/j.dib.2018.10.082
J Proteome Res. 2018 Nov 21.

ASSESSING AUTOMATED SAMPLE PREPARATION TECHNOLOGIES FOR HIGH-THROUGHPUT PROTEOMICS OF FROZEN WELL CHARACTERIZED TISSUES FROM SWEDISH BIOBANKS.

Magdalena Kuras, Lazaro Hiram Betancourt, Melinda Rezeli, Jimmy Rodriguez, Marcell Szasz, Qimin Zhou, Tasso Miliotis, Roland Andersson, György Marko-Varga.

Large cohorts of carefully collected clinical tissue materials play a central role in acquiring sufficient depth and statistical power to discover disease-related mechanisms and biomarkers of clinical significance. Manual preparation of such large sample cohorts requires experienced laboratory personnel. This carries other possible downsides such as low throughput, high risk of errors and low reproducibility. In this work, three automated technologies for high-throughput proteomics of frozen sectioned tissues were compared. The instruments evaluated included the Bioruptor for tissue disruption and protein extraction; the Barocycler, which is able to disrupt tissues and digest the proteins; and the AssayMAP Bravo, a micro-chromatography platform for protein digestion, peptide desalting and fractionation. Wide varieties of tissue samples from rat spleen, malignant melanoma and pancreatic tumors were used for the assessment. The three instruments displayed reproducible and consistent results, as was proven by high correlations and low coefficients of variation between technical replicates and even more importantly, between replicates that were processed in different batches or at different time points. The results from this study allowed us to integrate these technologies into an automated sample preparation workflow for large-scale proteomic studies that are currently ongoing. Data are available via ProteomeXchange with identifiers PXD010296 and PXD011295.

DOI: https://doi.org/10.1021/acs.jproteome.8b00792
NPJ Breast Cancer. 2018 ;4 36

mTORC1 is a key mediator of RON-dependent breast cancer metastasis with therapeutic potential.

Najme Faham, Ling Zhao, Alana L Welm.

Metastasis is the biggest challenge in treating breast cancer, and it kills >40,000 breast cancer patients annually in the US. Aberrant expression of the RON receptor tyrosine kinase in breast tumors correlates with poor prognosis and has been shown to promote metastasis. However, the molecular mechanisms that govern how RON promotes metastasis, and how to block it, are still largely unknown. We sought to determine critical effectors of RON using a combination of mutational and pharmacologic strategies. High-throughput proteomic analysis of breast cancer cells upon activation of RON showed robust phosphorylation of ribosomal protein S6. Further analysis revealed that RON strongly signals through mTORC1/p70S6K, which is mediated predominantly by the PI3K pathway. A targeted mutation approach to modulate RON signaling validated the importance of PI3K/mTORC1 pathway for spontaneous metastasis in vivo. Finally, inhibition of mTORC1 with an FDA-approved drug, everolimus, resulted in transient shrinkage of established RON-dependent metastases, and combined blockade of mTORC1 and RON delayed progression. These studies have identified a key downstream mediator of RON-dependent metastasis in breast cancer cells and revealed that inhibition of mTORC1, or combined inhibition of mTORC1 and RON, may be effective for treatment of metastatic breast cancers with elevated expression of RON.

DOI: https://doi.org/10.1038/s41523-018-0091-5
BMC Mol Biol. 2018 Nov 21. 19(1): 13

Integration of transcriptome and proteome profiles in glioblastoma: looking for the missing link.

Jean-Michel Lemée, Anne Clavreul, Marc Aubry, Emmanuelle Com, Marie de Tayrac, Jean Mosser, Philippe Menei.

   BACKGROUND: Glioblastoma (GB) is the most common and aggressive tumor of the brain. Genotype-based approaches and independent analyses of the transcriptome or the proteome have led to progress in understanding the underlying biology of GB. Joint transcriptome and proteome profiling may reveal new biological insights, and identify pathogenic mechanisms or therapeutic targets for GB therapy. We present a comparison of transcriptome and proteome data from five GB biopsies (TZ) vs their corresponding peritumoral brain zone (PBZ). Omic analyses were performed using RNA microarray chips and the isotope-coded protein label method (ICPL).
RESULTS: As described in other cancers, we found a poor correlation between transcriptome and proteome data in GB. We observed only two commonly deregulated mRNAs/proteins (neurofilament light polypeptide and synapsin 1) and 12 altered biological processes; they are related to cell communication, synaptic transmission and nervous system processes. This poor correlation may be a consequence of the techniques used to produce the omic profiles, the intrinsic properties of mRNA and proteins and/or of cancer- or GB-specific phenomena. Of interest, the analysis of the transcription factor binding sites present upstream from the open reading frames of all altered proteins identified by ICPL method shows a common binding site for the topoisomerase I and p53-binding protein TOPORS. Its expression was observed in 7/11 TZ samples and not in PBZ. Some findings suggest that TOPORS may function as a tumor suppressor; its implication in gliomagenesis should be examined in future studies.
CONCLUSIONS: In this study, we showed a low correlation between transcriptome and proteome data for GB samples as described in other cancer tissues. We observed that NEFL, SYN1 and 12 biological processes were deregulated in both the transcriptome and proteome data. It will be important to analyze more specifically these processes and these two proteins to allow the identification of new theranostic markers or potential therapeutic targets for GB.

Keywords:  Glioblastoma; Molecular biology; NEFL; Proteomics; SYN1; TOPORS; Transcriptomics

DOI:  https://doi.org/10.1186/s12867-018-0115-6
Neurobiol Dis. 2018 Nov 20. pii: S0969-9961(18)30750-2. [Epub ahead of print]

SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

Vietxuan Phan, Dan Cox, Silvia Cipriani, Sally Spendiff, Stephan Buchkremer, Emily O'Connor, Rita Horvath, Hans Hilmar Goebel, Denisa Hathazi, Hanns Lochmüller, Tatjana Straka, Rüdiger Rudolf, Joachim Weis, Andreas Roos.

   BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown.
METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose.
RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms.
CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.

Keywords:  Marinesco-Sjögren syndrome; Neuromuscular junction; PNS pathology; SIL1; Woozy

DOI:  https://doi.org/10.1016/j.nbd.2018.11.019
Proteomics. 2018 Nov 19. e1800157

Extracellular Vesicles from Neurosurgical Aspirates Identifies Chaperonin Containing TCP1 Subunit 6A (CCT6A) as a Potential Glioblastoma Biomarker with Prognostic Significance.

Susannah Hallal, Benjamin P Russell, Heng Wei, Maggie Lee, Christopher W Toon, Joanne Sy, Brindha Shivalingam, Michael E Buckland, Kimberley L Kaufman.

  Glioblastoma, WHO-grade IV glioma, carries a dismal prognosis owing to its infiltrative growth and limited treatment options. Glioblastoma-derived extracellular vesicles (EVs; 30-1000nm membranous particles) influence the microenvironment to mediate tumour aggressiveness and carry oncogenic cargo across the blood-brain-barrier into the circulation. As such, EVs are biomarker reservoirs with enormous potential for assessing glioblastoma tumours in situ. Neurosurgical aspirates are rich sources of EVs, isolated directly from glioma microenvironments. Quantitative LC-MS/MS compared EV proteomes enriched from glioblastoma (n = 15) and glioma grade II-III (n = 7) aspirates and identified 298 differentially-abundant proteins (p-value<0.00496). Along with previously reported glioblastoma-associated biomarkers, levels of all eight subunits of the key molecular chaperone, T-complex protein 1 Ring complex (TRiC), were higher in glioblastoma-EVs, including CCT2, CCT3, CCT5, CCT6A, CCT7 and TCP1 (p<0.00496). Analogous increases in TRiC transcript levels and DNA copy numbers were detected in silico; CCT6A had the greatest induction of expression and amplification in glioblastoma and showed a negative association with survival (p = 0.006). CCT6A is co-localised with EGFR at 7p11.2, with a strong tendency for co-amplification (p<0.001). Immunohistochemistry corroborated the CCT6A proteomics measurements and indicated a potential link between EGFR and CCT6A tissue expression. Putative EV-biomarkers described here should be further assessed in peripheral blood. This article is protected by copyright. All rights reserved.

Keywords:  Biomarker; CCT6A; EGFR; Extracellular vesicles; Glioblastoma

DOI:  https://doi.org/10.1002/pmic.201800157
J Cell Mol Med. 2018 Nov 18.

TSG101 promotes the proliferation, migration and invasion of hepatocellular carcinoma cells by regulating the PEG10.

Zhiyi Liu, Zilu Tian, Kuan Cao, Bin Zhang, Quan Wen, Xinyu Zhou, Weibin Yang, Tao Wang, Hengliang Shi, Renhao Wang.

  The tumour susceptibility gene 101 (TSG101) is reported to play important roles in the development and progression of several human cancers. However, its potential roles and underlined mechanisms in human hepatocellular carcinoma (HCC) are still needed to be further clarified. In the present study, we reported that knock down of TSG101 suppressed the proliferation, migration and invasion of HCC cells, while overexpression of TSG101 facilitated them. Molecularly, the results revealed that knock down of TSG101 significantly decreased the cell cycle related regulatory factor p53 and p21. In another point, knock down of TSG101 also obviously decreased the level of metallopeptidase inhibitor TIMP1 (Tissue inhibitors of metalloproteinases 1), which results in inhibition of MMP2, MMP7 and MMP9. In contrast, overexpression of TSG101 had opposite effects. The iTRAQ proteomics analysis identified that oncogenic protein PEG10 (Paternally expressed gene 10) might be a potential downstream target of TSG101. Further investigation showed that TSG101 interacted with PEG10 and protected it from proteasomal degradation thereby regulating the expression of p53, p21 and MMPs. Finally, we found that both TSG101 and PEG10 proteins are up-regulated and presented a direct correlation in HCC patients. In conclusion, these results suggest that TSG101 is up-regulated in human HCC patients, which may accelerate the proliferation, migration and invasion of HCC cells through regulating PEG10.

Keywords:  HCC; MMPs; PEG10; TSG101; p21; p53

DOI:  https://doi.org/10.1111/jcmm.13878
Eur J Med Chem. 2018 Oct 11. pii: S0223-5234(18)30863-8. [Epub ahead of print]162 423-434

The use of proteomic technologies to study molecular mechanisms of multidrug resistance in cancer.

Yi Cao, Ziyin Li, Lianzhi Mao, Hehe Cao, Jingjing Kong, Bin Yu, Changmin Yu, Wenzhen Liao.

  Multidrug resistance (MDR), defined as the cross-resistance of cancer cells toward a broad range of chemotherapeutic agents, is a universal and intractable problem in chemotherapy. The understanding of MDR mechanisms is essential to discover the potential biomarkers for predicting multidrug resistance and more importantly, tackling and preventing multidrug resistance. Multiple technologies have been used to study MDR mechanisms including comparative genomic hybridization, DNA array, differential display RT-PCR and various immunoassays. Compared with these approaches, proteomic technologies allow a high through-put analysis of protein detection, protein quantification and protein interaction with high accuracy. With the rapid development of proteomic studies in recent years, proteomic technologies have made substantial contributions to the characterization of MDR mechanisms including MDR-related protein detection and quantification, as well as the characterization of drug-transporter binding sites. This review offers a comprehensive illustration of MDR, proteomic technologies and the discoveries made in understanding MDR mechanisms using proteomic approaches.

Keywords:  Cancer; Molecular mechanisms; Multidrug resistance; Proteomic techniques

DOI:  https://doi.org/10.1016/j.ejmech.2018.10.001
Life Sci Alliance. 2018 May;1(2): e201800070

Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity.

Hannelore Hartmann, Daniel Hornburg, Mareike Czuppa, Jakob Bader, Meike Michaelsen, Daniel Farny, Thomas Arzberger, Matthias Mann, Felix Meissner, Dieter Edbauer.

Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)149 and nucleolar (PR)175-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Overexpressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule-like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomes may chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis.

DOI: https://doi.org/10.26508/lsa.201800070
J Exp Clin Cancer Res. 2018 Nov 19. 37(1): 277

Compound kushen injection suppresses human acute myeloid leukaemia by regulating the Prdxs/ROS/Trx1 signalling pathway.

Yanxia Jin, Qian Yang, Li Liang, Lu Ding, Yuxing Liang, Dongdong Zhang, Balu Wu, Tian Yang, Hailing Liu, Tingting Huang, Hui Shen, Honglei Tu, Yunbao Pan, Yongchang Wei, Yi Yang, Fuling Zhou.

   BACKGROUND: The increase in the levels of reactive oxygen species (ROS) in acute myeloid leukemia (AML) patients has been previously described; thus, it is important to regulate ROS levels in AML.
METHODS: Flow cytometry were used to assess the in vitro effect of compound kushen injection (CKI). Quantitative proteomics were used to analyse the mechanism. The AML patient-derived xenograft (PDX) model were used to evaluate the in vivo effect of CKI.
RESULTS: We found that intracellular ROS levels in AML cells were decreased, the antioxidant capacity were increased when treated with CKI. CKI inhibited the proliferation of AML cells and enhanced the cytotoxicity of AML cells, which has few toxic effects on haematopoietic stem cells (HSCs) and T cells. At the single-cell level, individual AML cells died gradually by CKI treatment on optofluidic chips. CKI promoted apoptosis and arrested cell cycle at G1/G0 phase in U937 cells. Furthermore, higher peroxiredoxin-3 (Prdx3) expression levels were identified in CKI-treated U937 cells through quantitative proteomics detection. Mechanically, the expression of Prdx3 and peroxiredoxin-2 (Prdx2) was up-regulated in CKI-treated AML cells, while thioredoxin 1 (Trx1) was reduced. Laser confocal microscopy showed that the proteins Prdx2 could be Interacted with Trx1 by CKI treatment. In vivo, the survival was longer and the disease was partially alleviated by decreased CD45+ immunophenotyping in peripheral blood in the CKI-treated group in the AML PDX model.
CONCLUSIONS: Antioxidant CKI possess better clinical application against AML through the Prdxs/ROS/Trx1 signalling pathway.

Keywords:  Acute myeloid leukaemia; Compound kushen injection; Peroxiredoxin-2; Peroxiredoxin-3; Reactive oxygen species

DOI:  https://doi.org/10.1186/s13046-018-0948-3
Mol Pharm. 2018 Nov 19.

Cluster of Differentiation 46 is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28).

Hiroki Kuroda, Masanori Tachikawa, Yuta Yagi, Mina Umetsu, Armania Nurdin, Eisuke Miyauchi, Michitoshi Watanabe, Yasuo Uchida, Tetsuya Terasaki.

Brain metastasis is a frequent complication of cancer, and may be mediated at least in part by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. Uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37°C was significantly reduced by 95.6% at 4°C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors; integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).

DOI: https://doi.org/10.1021/acs.molpharmaceut.8b00985
Life Sci Alliance. 2018 Oct;1(5): e201800190

Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth.

Jason R Pitarresi, Xin Liu, Alex Avendano, Katie A Thies, Gina M Sizemore, Anisha M Hammer, Blake E Hildreth, David J Wang, Sarah A Steck, Sydney Donohue, Maria C Cuitiño, Raleigh D Kladney, Thomas A Mace, Jonathan J Chang, Christina S Ennis, Huiqing Li, Roger H Reeves, Seth Blackshaw, Jianying Zhang, Lianbo Yu, Soledad A Fernandez, Wendy L Frankel, Mark Bloomston, Thomas J Rosol, Gregory B Lesinski, Stephen F Konieczny, Denis C Guttridge, Anil K Rustgi, Gustavo Leone, Jonathan W Song, Jinghai Wu, Michael C Ostrowski.

The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3β (GSKβ), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.

DOI: https://doi.org/10.26508/lsa.201800190
Sci Signal. 2018 Nov 20. pii: eaar5680. [Epub ahead of print]11(557):

Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways.

Jimmy Van den Eynden, Ganesh Umapathy, Arghavan Ashouri, Diana Cervantes-Madrid, Joanna Szydzik, Kristina Ruuth, Jan Koster, Erik Larsson, Jikui Guan, Ruth H Palmer, Bengt Hallberg.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements in ALK trigger the activation of the encoded receptor and its downstream signaling pathways. ALK mutations have been identified in both familial and sporadic neuroblastoma cases as well as in 30 to 40% of relapses, which makes ALK a bona fide target in neuroblastoma therapy. Tyrosine kinase inhibitors (TKIs) that target ALK are currently in clinical use for the treatment of patients with ALK-positive non-small cell lung cancer. However, monotherapy with the ALK inhibitor crizotinib has been less encouraging in neuroblastoma patients with ALK alterations, raising the question of whether combinatorial therapy would be more effective. In this study, we established both phosphoproteomic and gene expression profiles of ALK activity in neuroblastoma cells exposed to first- and third-generation ALK TKIs, to identify the underlying molecular mechanisms and identify relevant biomarkers, signaling networks, and new therapeutic targets. This analysis has unveiled various important leads for novel combinatorial treatment strategies for patients with neuroblastoma and an increased understanding of ALK signaling involved in this disease.

DOI: https://doi.org/10.1126/scisignal.aar5680
J Natl Cancer Inst. 2018 Nov 20.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

Elie Hatem, Sandy Azzi, Nadine El Banna, Tiantian He, Amélie Heneman-Masurel, Laurence Vernis, Dorothée Baïlle, Vanessa Masson, Florent Dingli, Damarys Loew, Bruno Azzarone, Pierre Eid, Giuseppe Baldacci, Meng-Er Huang.

Background: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing.
Methods: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided.
Results: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations.
Conclusion: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

DOI: https://doi.org/10.1093/ije/djy149
Asian Pac J Allergy Immunol. 2018 Oct 15.

Proteomic Identification of Allergenic Proteins of Morus alba L. Pollenexacerbation.

Demirhan Çetereisi, Nurgül Karlioglu, Asli Gelincik, Sylvie Michelland, Evren Önay-Uçar, Belkis Ertek, Nilgün Akdeniz, Sacide Erden, Bahattin Çolkoglu, Ünal Akkemik, Günnur Deniz, Murat Pekmez, Hèlène Flamant-Waret, Sylvia Lehmann, Sandrine Bourgoin-Voillard, Michel Sève, Suna Büyüköztürk, Nazli Arda.

BACKGROUND: Tree pollens are well-known aeroallergens all over the world. Little is known about the allergenicity of Morus alba (white mulberry) pollen.
OBJECIVE: We aimed to explore the potential allergens of this pollen and its clinical relevance in tree pollen allergic patients living in Istanbul, Turkey.
METHODS: Twenty three seasonal allergic rhinitis patients with a confirmed tree pollen allergy and 5 healthy control subjects underwent skin prick and nasal provocation tests with M.alba pollen extract. The pollen extract was then resolved by gel electrophoresis, and immunoblotted with sera from patients/control individuals to detect the potential allergenic proteins. The prevalent IgE binding proteins from 1D-gel were analyzed by MALDI-TOF/TOF.
RESULTS: Eleven out of 23 patients were reactive to the extract with skin prick tests. Seven of those patients also reacted positively to the nasal provocation tests. The most common IgE-binding pollen proteins were detected between 55-100 kDa, and also at molecular weights lower than 30 kDa for some patients. Mass spectrometry analyses revealed that the principal IgE-binding protein was methionine synthase (5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase), which is then proposed as a novel allergen in M.alba pollen.
CONCLUSION: This study provides the first detailed information for the potential allergens of Morus alba pollen of Istanbul. Methionine synthase with an apparent molecular weight of 80 to 85 kDa has been recognized as one of the allergens in Morus alba pollen for the first time.

DOI: https://doi.org/10.12932/AP-120418-0296
Proc Natl Acad Sci U S A. 2018 Nov 19. pii: 201810413. [Epub ahead of print]

ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay.

Marisa Kamelgarn, Jing Chen, Lisha Kuang, Huan Jin, Edward J Kasarskis, Haining Zhu.

  Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by preferential motor neuron death. Approximately 15% of ALS cases are familial, and mutations in the fused in sarcoma (FUS) gene contribute to a subset of familial ALS cases. FUS is a multifunctional protein participating in many RNA metabolism pathways. ALS-linked mutations cause a liquid-liquid phase separation of FUS protein in vitro, inducing the formation of cytoplasmic granules and inclusions. However, it remains elusive what other proteins are sequestered into the inclusions and how such a process leads to neuronal dysfunction and degeneration. In this study, we developed a protocol to isolate the dynamic mutant FUS-positive cytoplasmic granules. Proteomic identification of the protein composition and subsequent pathway analysis led us to hypothesize that mutant FUS can interfere with protein translation. We demonstrated that the ALS mutations in FUS indeed suppressed protein translation in N2a cells expressing mutant FUS and fibroblast cells derived from FUS ALS cases. In addition, the nonsense-mediated decay (NMD) pathway, which is closely related to protein translation, was altered by mutant FUS. Specifically, NMD-promoting factors UPF1 and UPF3b increased, whereas a negative NMD regulator, UPF3a, decreased, leading to the disruption of NMD autoregulation and the hyperactivation of NMD. Alterations in NMD factors and elevated activity were also observed in the fibroblast cells of FUS ALS cases. We conclude that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation, both of which likely contribute to motor neuron death.

Keywords:  RNA-protein granules; amyotrophic lateral sclerosis; fused in sarcoma; nonsense-mediated decay; protein translation

DOI:  https://doi.org/10.1073/pnas.1810413115