Theodoros Roumeliotis

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.

Resistance of naturally secreted α-synuclein to proteolysis

Recent evidence suggests that specific extracellular α‐synuclein (α‐syn) strains are implicated in the progression of Parkinsons disease (PD) pathology. It is plausible that deregulation in the normal processing of secreted α‐syn may be a causative risk factor for PD. To date, the degradation mechanisms involved have received very little attention. Here, we sought to investigate factors that regulate extracellular α‐syn levels. We show, for the first time, that cell‐secreted α‐syn forms are resistant to direct proteolysis by kallikrein‐related peptidase 6 (KLK6), an extracellular enzyme known to cleave recombinant α‐syn. This differential susceptibility appears to be partially due to the association of secreted α‐syn with lipids. We further provide evidence that secreted α‐syn can be cleaved by KLK6 indirectly through activation of a secreted metalloprotease, suggestive of the involvement of a proteolytic cascade in the catabolism of secreted α‐syn. Our results clearly suggest that physiological modifications affect the biochemical behavior of secreted α‐syn and provide novel insights into mechanisms and potential targets for therapeutic interventions.—Ximerakis, M., Pampalakis, G., Roumeliotis, T. I., Sykioti, V.‐S., Garbis, S. D., Stefanis, L., Sotiropoulou, G., Vekrellis, K. Resistance of naturally secreted α‐synuclein to proteolysis. FASEB J. 28, 3146–3158 (2014). www.fasebj.org

Muscle lim protein isoform negatively regulates striated muscle actin dynamics and differentiation

Muscle lim protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine‐rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, whereas aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP‐b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N‐terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically, it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/cofilin‐2‐mediated F‐actin dynamics. In differentiated striated muscles, MLP‐b localizes to the sarcomeres and binds directly to Z‐disc components, including α‐actinin, T‐cap and MLP. The findings of the present study unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, as well as in differentiated striated muscles as a contributor to sarcomeric integrity.

Increased circulating resistin levels in early-onset breast cancer patients of normal body mass index correlate with lymph node negative involvement and longer disease free survival: a multi-center POSH cohort serum proteomics study

BackgroundEarly-onset breast cancer (EOBC) affects about one in 300 women aged 40 years or younger and is associated with worse outcomes than later onset breast cancer. This study explored novel serum proteins as surrogate markers of prognosis in patients with EOBC.MethodsSerum samples from EOBC patients (stages 1–3) were analysed using agnostic high-precision quantitative proteomics. Patients received anthracycline-based chemotherapy. The discovery cohort (n = 399) either had more than 5-year disease-free survival (DFS) (good outcome group, n = 203) or DFS of less than 2 years (poor outcome group, n = 196). Expressed proteins were assessed for differential expression between the two groups. Bioinformatics pathway and network analysis in combination with literature research were used to determine clinically relevant proteins. ELISA analysis against an independent sample set from the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) cohort (n = 181) was used to validate expression levels of the selected target. Linear and generalized linear modelling was applied to determine the effect of target markers, body mass index (BMI), lymph node involvement (LN), oestrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 status on patients’ outcome.ResultsA total of 5346 unique proteins were analysed (peptide FDR p ≤ 0.05). Of these, 812 were differentially expressed in the good vs poor outcome groups and showed significant enrichment for the insulin signalling (p = 0.01) and the glycolysis/gluconeogenesis (p = 0.01) pathways. These proteins further correlated with interaction networks involving glucose and fatty acid metabolism. A consistent nodal protein to these metabolic networks was resistin (upregulated in the good outcome group, p = 0.009). ELISA validation demonstrated resistin to be upregulated in the good outcome group (p = 0.04), irrespective of BMI and ER status. LN involvement was the only covariate with a significant association with resistin measurements (p = 0.004). An ancillary in-silico observation was the induction of the inflammatory response, leucocyte infiltration, lymphocyte migration and recruitment of phagocytes (p < 0.0001, z-score > 2). Survival analysis showed that resistin overexpression was associated with improved DFS.ConclusionsHigher circulating resistin correlated with node-negative patients and longer DFS independent of BMI and ER status in women with EOBC. Overexpression of serum resistin in EOBC may be a surrogate indicator of improved prognosis.

Marine omega-3 fatty acid supplementation in non-alcoholic fatty liver disease: Plasma proteomics in the randomized WELCOME* trial

BACKGROUND & AIMSnNon-alcoholic fatty liver disease (NAFLD) is a liver condition characterised by liver fat accumulation and often considered to be the liver manifestation of metabolic syndrome. The aim ofxa0this study was to examine in patients with NAFLD the system-wide effects of treatment with docosahexaenoic acidxa0+xa0eicosapentaenoic acid (DHAxa0+xa0EPA) versus placebo on the plasma proteome.nnnMETHODSnPlasma from patients that participated in a 15-18 months randomised, double-blind placebo-controlled trial testing the effects of 4xa0g DHAxa0+xa0EPA daily was analysed using depletion-free quantitative proteomics.nnnRESULTSnBioinformatics interpretation of the proteomic analysis showed that DHAxa0+xa0EPA treatment affected pathways involving blood coagulation, immune/inflammatory response and cholesterol metabolism (pxa0<xa00.05). Two key proteins of cardiovascular risk, prothrombin and apolipoprotein B-100, were shown to decrease as a result of DHAxa0+xa0EPA supplementation [Prothrombin: Males DHAxa0+xa0EPA Mean iTRAQ log2ratio (SD)xa0=xa0-0.13 (0.20) pxa0=xa00.05, Females DHAxa0+xa0EPA Mean iTRAQ log2ratio (SD)xa0=xa0-0.48 (0.35) pxa0=xa00.03; Apo B-100: Males DHAxa0+xa0EPA Mean iTRAQ log2ratio (SD)xa0=xa0-0.24 (0.16) pxa0=xa00.01, Females DHAxa0+xa0EPA Mean iTRAQ log2ratio (SD)xa0=xa0-0.15 (0.05) pxa0=xa00.02].nnnCONCLUSIONSnPlasma proteomics applied in a randomised, placebo-controlled trial showed that high dose DHAxa0+xa0EPA treatment in patients with NAFLD affects multiple pathways involved in chronic non-communicable diseases.