Proteomic profiling of mdx-4cv serum reveals highly elevated levels of the inflammation-induced plasma marker haptoglobin in muscular dystrophy

Murphy, Sandra; Dowling, Paul; Zweyer, Margit; Henry, Michael; Mundegar, Rustam R.; Swandulla, Dieter; Ohlendieck, Kay

doi:10.3892/ijmm.2017.2952

Cited by 34 publications

(48 citation statements)

References 87 publications

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“…Biochemical and proteomic studies of the inflammatory pathology of dystrophic muscles have established that progressive muscle wasting is reflected by an altered rate of protein release into the circulatory system and other significant plasma fluctuations (Hathout et al 2016). Serum analysis using both conventional gel electrophoresis in combination with densitometric scanning (John and Purdom 1989) and label-free LC-MS/MS (Murphy et al 2017) revealed a significantly increased concentration of the inflammation-inducible plasma marker haptoglobin in muscular dystrophy. Since this acute phase response protein is a reliable marker of inflammation that is usually associated with tissue damage (Wang et al 2001), dystrophinopathies appear to be closely linked to sterile inflammation.…”

Section: Discussionmentioning

confidence: 99%

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

Murphy

Brinkmeier

Krautwald

et al. 2017

J Muscle Res Cell Motil

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The almost complete loss of the membrane cytoskeletal protein dystrophin and concomitant drastic reduction in dystrophin-associated glycoproteins are the underlying mechanisms of the highly progressive neuromuscular disorder Duchenne muscular dystrophy. In order to identify new potential binding partners of dystrophin or proteins in close proximity to the sarcolemmal dystrophin complex, proteomic profiling of the isolated dystrophin-glycoprotein complex was carried out. Subcellular membrane fractionation and detergent solubilisation, in combination with ion exchange, lectin chromatography and density gradient ultracentrifugation, was performed to isolate a dystrophin complex-enriched fraction. Following gradient gel electrophoresis and on-membrane digestion, the protein constituents of the dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which act as cytolinker proteins and possibly exist in close proximity to the dystrophin complex in the sarcolemma membrane. Interestingly, comparative immunoblotting showed a significant reduction in desmoglein in dystrophin-deficient mdx skeletal muscles, reminiscent of the pathobiochemical fate of the dystrophin-associated core proteins in muscular dystrophy. Comparative membrane proteomics was used to correlate this novel finding to large-scale changes in the dystrophic phenotype. A drastic increase in the extracellular stabilizers biglycan and fibronectin was shown by both mass spectrometric analysis and immunoblotting. The reduced expression of desmoglein in dystrophin-deficient skeletal muscles, and simultaneous increase in components of the extracellular matrix, suggest that muscular dystrophy is associated with plasmalemmal disintegration, loss of cellular linkage and reactive myofibrosis.

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Section: Discussionmentioning

confidence: 99%

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

Murphy

Brinkmeier

Krautwald

et al. 2017

J Muscle Res Cell Motil

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“…The samples were desalted prior to analysis using C18 spin columns (Thermo Scientific) and 500ng was loaded onto an Ultimate 3000 NanoLC system (Dionex Corporation, Sunnyvale, CA, USA) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The raw data were analysed using Progenesis QI for Proteomics software (version 3.1; Non-Linear Dynamics, a Waters company, Newcastle upon Tyne, UK) as previously described, 61 with following modifications: the filter mass peaks with charge states from+1 to+6 was applied to the MS/MS data files, peptides were identified using taxonomy: Homo sapiens in the SwissProt database and peptides with XCorr scores>1.9 for +1 ions,>2.2 for +2 ions and>3.75 for +3 ions or more (from Sequest HT) were selected.…”

Section: Methodsmentioning

confidence: 99%

Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells

et al. 2017

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Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated β-galactosidase (SA-β-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-β-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

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“…In comparison, the CD4:CD8 ratio for the group of dogs was 1.74, consistent with our results for Golden Retriever dogs. Other animal model of muscular dystrophy presented a large number of increased versus decreased protein species in mdx-4cv serum by comparative spectrometry mass tolls (Murphy et al 2017).…”

Section: Discussionmentioning

confidence: 99%

Immunophenotyping lymphocyte and acute phase proteins in canine X-linked muscular dystrophy

Abreu

Monteiro

Souza

et al. 2018

An. Acad. Bras. Ciênc.

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Duchenne Muscular Dystrophy (DMD) is the most common X-linked muscular disease affecting humans. The Golden Retriever Muscular Dystrophy model (GRMD) is consider the most suitable for several studies. This assay aims to quantify lymphocyte subpopulations CD4, CD5, and CD8, and standardize, the serum electrophoretic profile, to understand their contribution to the pathologic process in normal Golden Retriever dogs (GR group) and dystrophic´s (GRMD group), through the umbilical cord blood, in dogs aged from 2 to 3 months (GR II and GRMD II), and in dogs over 1 year of age (GR III and GRMD III). No significant differences were observed between the CD8+ lymphocyte subpopulations of the groups studied. The CD4+ and CD5+ lymphocyte subpopulations were significantly higher in the GRMD III group compared to the GR III group. Twenty-two different proteins in the gel were identified. The serum concentrations of the proteins belonging to the GR I and GRMD I groups were significantly lower than those of the other groups. We show that expression of acute phase proteins are worst during the aging of the dogs. We hope to expand knowledge to better understand the GRMD model and the translational data.

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Proteomic profiling of mdx-4cv serum reveals highly elevated levels of the inflammation-induced plasma marker haptoglobin in muscular dystrophy

Cited by 34 publications

References 87 publications

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells

Immunophenotyping lymphocyte and acute phase proteins in canine X-linked muscular dystrophy

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