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Aim Alport syndrome (AS) is the second most common hereditary kidney disease caused by mutations in collagen IV genes. Patients present with microhaematuria that progressively leads to proteinuria and end stage renal disease. Currently, no specific treatment exists for AS. Using mass spectrometry based proteomics, we aimed to detect early alterations in molecular pathways implicated in AS before the stage of overt proteinuria, which could be amenable to therapeutic intervention. Methods Kidneys were harvested from male Col4a3 −/− knock out and sex and age‐matched Col4a3 +/+ wild‐type mice at 4 weeks of age. Purified peptides were separated by liquid chromatography and analysed by high resolution mass spectrometry. The Cytoscape bioinformatics tool was used for function enrichment and pathway analysis. PPARα expression levels were evaluated by immunofluorescence and immunoblotting. Results Proteomic analysis identified 415 significantly differentially expressed proteins, which were mainly involved in metabolic and cellular processes, the extracellular matrix, binding and catalytic activity. Pathway enrichment analysis revealed among others, downregulation of the proteasome and PPAR pathways. PPARα protein expression levels were observed to be downregulated in Alport mice, supporting further the results of the discovery proteomics. Conclusion This study provides additional evidence that alterations in proteins which participate in cellular metabolism and mitochondrial homeostasis in kidney cells are early events in the development of chronic kidney disease in AS. Of note is the dysregulation of the PPAR pathway, which is amenable to therapeutic intervention and provides a new potential target for therapy in AS., SUMMARY AT A GLANCE Alterations in proteins which participate in cellular metabolism and mitochondrial homeostasis in kidney cells, are early events in the development of CKD in Alport syndrome. In addition, pathway enrichment analysis revealed among others, dysregulation of the proteasome, protein digestion and absorption pathways as well as PPAR pathway in AS compared to control mice. Targeting the PPAR pathway could be a potential therapeutic approach in AS.

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2′O-Methyl (2′OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2′OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2′OMe AON and LNA/2′OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2′OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

Background Approximately 50% of systemic lupus erythematosus (SLE) patients develop nephritis, which is among the most severe and frequent complications of the disease and a leading cause of morbidity and mortality. Despite intensive research, there are still no reliable lupus nephritis (LN) markers in clinical use that can assess renal damage and activity with a high sensitivity and specificity. To this end, the aim of this study was to identify new clinically relevant tissue-specific protein biomarkers and possible underlying molecular mechanisms associated with renal involvement in SLE, using mass spectrometry (MS)-based proteomics. Methods Kidneys were harvested from female triple congenic B6.NZMsle1/sle2/sle3 lupus mice model, and the respective sex- and age-matched C57BL/6 control mice at 12, 24 and 36 weeks of age, representing pre-symptomatic, established and end-stage LN, respectively. Proteins were extracted from kidneys, purified, reduced, alkylated and digested by trypsin. Purified peptides were separated by liquid chromatography and analysed by high-resolution MS. Data were processed by the Progenesis QIp software, and functional annotation analysis was performed using DAVID bioinformatics resources. Immunofluorescence and multiple reaction monitoring (MRM) MS methods were used to confirm prospective biomarkers in SLE mouse strains as well as human serum samples. Results Proteomic profiling of kidney tissues from SLE and control mice resulted in the identification of more than 3800 unique proteins. Pathway analysis revealed a number of dysregulated molecular pathways that may be mechanistically involved in renal pathology, including phagosome and proximal tubule bicarbonate reclamation pathways. Proteomic analysis supported by human transcriptomic data and pathway analysis revealed Coronin-1A, Ubiquitin-like protein ISG15, and Rho GDP-dissociation inhibitor 2, as potential LN biomarkers. These results were further validated in other SLE mouse strains using MRM-MS. Most importantly, experiments in humans showed that measurement of Coronin-1A in human sera using MRM-MS can segregate LN patients from SLE patients without nephritis with a high sensitivity (100%) and specificity (100%). Conclusions These preliminary findings suggest that serum Coronin-1A may serve as a promising non-invasive biomarker for LN and, upon validation in larger cohorts, may be employed in the future as a screening test for renal disease in SLE patients.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited, multisystemic disorder characterized clinically by delayed muscle relaxation and weakness. The disease is caused by a CTG repeat expansion in the 3' untranslated region (3' UTR) of the DMPK gene, which leads to the expression of a toxic gain-of-function mRNA. The expanded CUG repeat mRNA sequesters the MBNL1 splicing regulator in nuclear-retained foci structures, resulting in loss of protein function and disruption of alternative splicing homeostasis. In this study, we used CAG repeat antisense oligonucleotides (ASOs), composed of locked nucleic acid (LNA)- and 2'-O-methyl (2'OMe)-modified bases in a chimeric design, to alleviate CUGexpanded-mediated toxicity. Chimeric 14-18mer LNA/2'OMe oligonucleotides, exhibiting an LNA incorporation of ∼33%, significantly ameliorated the misregulated alternative splicing of Mbnl1-dependent exons in primary DM1 mouse myoblasts and tibialis anterior muscles of DM1 mice. Subcutaneous delivery of 14mer and 18mer LNA/2'OMe chimeras in DM1 mice resulted in high levels of accumulation in all tested skeletal muscles, as well as in the diaphragm and heart tissue. Despite the efficient delivery, chimeric LNA/2'OMe oligonucleotides were not able, even at a high-dosage regimen (400 mg/kg/week), to correct the misregulated splicing of Serca1 exon 22 in skeletal muscles. Nevertheless, oligonucleotide doses were well-tolerated as determined by histological and plasma biochemistry analyses. Our results provide proof of concept that inhibition of MBNL1 sequestration by systemic delivery of a steric-blocking ASO is extremely challenging, considering the large number of target sites that need to be occupied per RNA molecule. Although not suitable for DM1 therapy, chimeric LNA/2'OMe oligonucleotides could prove to be highly beneficial for other diseases, such as Duchenne muscular dystrophy, that require inhibition of a single target site per RNA molecule.

Background Pathogenesis and aetiology of systemic sclerosis (SSc) are currently unclear, thus rendering disease prognosis, diagnosis and treatment challenging. The aim of this study was to use paired skin biopsy samples from affected and unaffected areas of the same patient, in order to compare the proteomes and identify biomarkers and pathways which are associated with SSc pathogenesis. Methods Biopsies were obtained from affected and unaffected skin areas of SSc patients. Samples were cryo-pulverised and proteins were extracted and analysed using mass spectrometry (MS) discovery analysis. Differentially expressed proteins were revealed after analysis with the Progenesis QIp software. Pathway analysis was performed using the Enrichr Web server. Using specific criteria, fifteen proteins were selected for further validation with targeted-MS analysis. Results Proteomic analysis led to the identification and quantification of approximately 2000 non-redundant proteins. Statistical analysis showed that 169 of these proteins were significantly differentially expressed in affected versus unaffected tissues. Pathway analyses showed that these proteins are involved in multiple pathways that are associated with autoimmune diseases (AIDs) and fibrosis. Fifteen of these proteins were further investigated using targeted-MS approaches, and five of them were confirmed to be significantly differentially expressed in SSc affected versus unaffected skin biopsies. Conclusion Using MS-based proteomics analysis of human skin biopsies from patients with SSc, we identified a number of proteins and pathways that might be involved in SSc progression and pathogenesis. Fifteen of these proteins were further validated, and results suggest that five of them may serve as potential biomarkers for SSc.

Additional file 1: Supplemental methods, Tables and Figures. Supplemental methods: Further details about sample preparation for mass spectrometry analysis, untargeted LC-MS/MS proteomics and targeted proteomic analysis. Table S1.1: MRM parameters for the synthesized peptides of the selected mouse kidney target proteins. Table S1.2: MRM parameters for the synthesized peptides of human serum target and immunodepleted proteins. Table S1.3: Spearman’s correlation analysis between CORO1A serum levels and clinical parameters in LN patients. Table S1.4: Two sample Wilcoxon rank-sum (Mann-Whitney) correlation analysis between CORO1A serum levels and clinical parameters in LN patients. Figure S1: Representative chromatograms of target peptides used in the targeted MRM-MS proteomic analysis for the mouse experiments. Figure S2: Representative chromatograms of target peptides used in the targeted MRM-MS proteomic analysis for the human experiments. Figure S3: Coefficient of variation of MRM assays for both mouse and human experiments. Figure S4: MRM-MS analysis of candidate protein biomarkers in different SLE mouse strains at pre-nephritic stage. Figure S5: Concentration levels of serum Amyloid P-component protein in SLE, LN patients and healthy controls. Figure S6: Representative images of H + E staining of the validation set.

ObjectiveTo evaluate the therapeutic effects of omega-3 (ω3) fatty acids in the retina of aged mice when the blood arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is maintained between 1.0 and 1.5.Methods and analysisAged (24-month-old) wild-type C57BL/6J mice were allocated to two groups: ω3 treated and untreated. Treatment with ω3 was by daily gavage administration of EPA and docosahexaenoic acid for 60 days. Gas chromatography was used to identify and quantify fatty acids in the blood and retina. To count lipofuscin granules and measure the photoreceptor layer, eyecups were examined histologically using transmission electron microscopy and light microscopy. We also analysed eyecups using mass spectrometry-based proteomics.ResultsAA levels were lower, and EPA levels were higher, in the blood and retinas of the ω3-treated group than in the untreated group, resulting in a lower AA/EPA ratio. The ω3-treated group also showed significantly fewer lipofuscin granules and a thicker outer nuclear layer than the untreated group. Proteomic analysis revealed significantly greater expression of myelin basic protein, myelin regulatory factor-like protein, myelin proteolipid protein and glial fibrillar acidic protein in the ω3-treated group than in the untreated group. Three different pathways were significantly affected by ω3 treatment: fatty acid elongation, biosynthesis of unsaturated fatty acids and metabolic pathways.ConclusionTwo months of ω3 supplementation (when the blood AA/EPA~1.0–1.5) in aged mice reduced lipofuscin granule formation in the retina and protected the photoreceptor layer, suggesting that ω3 supplementation slows normal age-related retinal degeneration.

Currently, most of the neurodegenerative diseases lack effective therapeutic drugs. The present burden of these diseases on society highlights the need to develop novel approaches for their treatment. The detection of novel indications for existing drugs, known as drug repurposing, is a promising alternative approach to the traditional drug-discovery process that can be used to identify therapeutic compounds. This chapter provides a comprehensive review of the various drug-repurposing methods focusing on the computational approach. The available computational methods, namely structure-based virtual screening, ligand-based, transcriptomics-based, GWAS-based, network-based, integration-based, machine learning-based, and literature-based methods, are discussed along with their advantages and disadvantages. In addition, case studies utilizing existing and available tools are presented. Finally, the challenges of drug repurposing are discussed including the validation methods and the investigation of drug combinations.

Background Huntington’s disease (HD) is a rare, inherited autosomal dominant neurodegenerative disorder. A multisource data integration approach was developed by our group, regarding its ability to drive clusters of connected pathways. Aims Multisource integration approach to identify clusters of connected pathways. Identification of pathways involved in HD. Methodology HD data was retrieved from Malacards, which contains genes, pathways, variants, drugs and differently expressed genes. Additional databases and tools such as DrugBank, MirTarBase, MicroCosm, TargetScan and GeneMania were used for extraction of information of known gene targets for each drug, microRNAs (miRNA). Genetic interactions and protein-protein interactions respectively. A super network was then constructed combining the different information sources, from this a gene-specific score was acquired through the combination of both the topology of the super network and the gene-specific information gene-set enrichment analysis was performed, using EnrichR on the top scored genes for the disease and the top resulting pathways were mapped on a KEGG reference network. Results/outcomes Some of the pathways identified through publicly available datasets were cAMP, TGF-beta, Ca2+, PPAR and VEGF. These are likely to play a role in contributing to disease pathogenesis and clinical characteristics of HD. Conclusions The following integration method can be used to further enrich our knowledge of the different pathways involved in a disease and therefore, facilitate research in the field of personalized and precision medicine.

Background Huntington’s disease (HD) is an inherited autosomal dominant neurodegenerative disorder. Common pathological molecular mechanisms contributing to HD include reactive oxygen species (ROS), mitochondrial dysfunction and neuro-inflammation.1 Exposure to the Mediterranean Diet (MD) provides neuroprotection and has anti-oxidant and anti-inflammatory properties.2–4 Adherence to the MD could positively influence age of onset and progression of HD. Aims to address gaps in understanding on how neurodegeneration mechanisms influence phenoconversion and clinical characteristics of HD, particularly age of onset to investigate how adherence to the Mediterranean diet may modify these associations to delay phenoconversion. Methods This is a case-control study, where HD cases, pre-symptomatic, early symptomatic and later stage and age-matched controls will be recruited. Questionnaires will be used to collect data. A blood sample will be collected for untargeted metabolomic investigation into the pathways involved in HD and pathways affected by the MD. An attempt was made to integrate existing publically available data on HD, to identify pathways which, play a role in disease pathology of HD. Enrichment analysis was performed and seven clusters with pathways involved in HD were obtained.5 Results/outcomes Some of the pathways identified through publicly available datasets were cAMP, TGF-beta, Ca2+, and VEGF. These are likely to play a role in contributing to disease pathogenesis of HD. Conclusions Given the existing results and those that will emerge from the untargeted metabolomic analysis, we hope to identify pathways that can differentiate between different HD disease stages and whose aberrant changes can be positively influenced by adherence to the MD. References . Mo C, Hannan A, Renoir T. Environmental factors as modulators of neurodegeneration: Insights from gene–environment interactions in Huntington’s disease. Neuroscience & Biobehavioral2015;52:178–192. . Sofi F, Macchi C, Casini A. Mediterranean diet and minimizing neurodegeneration. Curr Nutr Rep2013;2:75–80. . Valls-Pedret C, et al. Mediterranean diet and age-related cognitive decline. JAMA Internal Medicine2015;175(7):1094. . Casamenti F, Stefani M. Olive polyphenols: New promising agents to combat aging-associated neurodegeneration. Expert Review of Neurotherapeutics2016;17(4):345–358. . Andrea C, Kakouri, Christiana C, Christodoulou, et al. Revealing clusters of connected pathways through multisource data integration in Huntington’s disease and Spastic Ataxia (paper is currently under review) 2018.

In the healthcare sector, phytocompounds are known to be beneficial by contributing or alleviating a variety of diseases. Studies have demonstrated the progressive effects of phytocompounds on immune-related diseases and to exhibit anticancer effects. Graviola tree is an evergreen tree with its extracts (leafs and seeds) been reported having anticancer properties, but the precise target of action is not clear. Using an in silico approach, we predicted that annonacin, an Acetogenin, the active agent found in Graviola leaf extract (GLE) to potentially act as a novel inhibitor of both sodium/potassium (NKA) and sarcoplasmic reticulum (SERCA) ATPase pumps. We were able to validate and confirm the in silico studies by showing that GLE inhibited NKA and SERCA activity in intact cells. In the present study, we also demonstrated the antiproliferative and anticancer effects of GLE in a variety of cancer cell lines with limited toxic effects on non-transformed cells. Moreover, our results revealed that known inhibitors of both NKA and SERCA pumps could also promote cell death in several cancer cell lines. In addition, a mouse xenograft cancer model showed GLE as able to reduce tumor size and progression. Finally, bioprofiling studies indicated a strong correlation between overexpression of both NKA and SERCA gene expression vs. survival rates. Overall, our results demonstrated that GLE can promote selective cancer cell death via inhibiting NKA and SERCA, and thus can be considered as a potential novel treatment for cancer. After molecular analysis of GLE by liquid chromatography–mass spectrometry and ESI–QTOF–MS analysis, it was found that the MS spectrum of the high abundant chromatographic peak purified sample highly consisted of annonacin.

Background Systemic Sclerosis (SSc) is an autoimmune connective tissue rheumatic disease characterised by three main hallmarks; vasculopathy, immune system abnormalities and fibrosis. It is considered a multisystemic and heterogeneous disease as many organs of the body may be affected and symptoms vary among patients. Up to date, SSc is untreated and its etiology and pathogenesis remain unclear. Early prognosis and diagnosis of the disease are challenging. Objectives The aim of this study was to analyse the proteomic profile of SSc patients in order to gain insights into the mechanisms implicated in disease pathogenesis and also discover new biomarkers that would facilitate early prognosis, more accurate diagnosis and therapeutic targeting of SSc. Methods Human biopsies were obtained from ten affected and three non-affected skin areas of SSc patients and have been classified based on histological criteria. Biopsies were cryo-pulverised and proteins were extracted, purified, reduced, alkylated and digested by trypsin. Purified peptides were analysed on a Waters Synapt G2Si HDMS instrument operated in ion mobility mode using a UDMSE approach. Data were processed by the Progenesis QIp and functional annotation analysis was carried out using multiple bioinformatics resources. Results Proteomic analysis led to the identification and quantification of approximately 1500 non-redundant proteins per sample. About 400 of these proteins, including interferons and interleukins, are differentially expressed between affected and non-affected samples. Functional annotation analysis of these proteins showed that they are involved in multiple pathways including, antigen processing and presentation, complement, ubiquitin mediated proteolysis and Notch signalling, which are known to be associated with autoimmune diseases and fibrosis. Conclusions Using a Mass Spectrometry-based proteomic approach for the analysis of SSc human skin biopsies, we identified a number of proteins that might be involved in the development and pathogenesis of SSc. Interestingly, some of these proteins are differentially expressed in specific histological groups and thus could be considered as potential biomarkers for specific SSc stages. Acknowledgements This work has received support from the EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant n° 115565, the Cyprus Institute of Neurology and Genetics and Universite catholique de Louvain. Disclosure of Interest None declared

Purpose To evaluate the therapeutic effects of omega-3 (ω-3) fatty acids in the ABCA4-/- model of Stargardt's disease. Monitoring the blood level of eicosapentaenoic acid (EPA) and arachidonic acid (AA), served to adjust the treatment dosage (AA/EPA = 1-1.5). Methods Eight-month-old mice were allocated to different groups: A) wild type (129S1), B) ABCA4-/- untreated, C) ABCA4-/- treated with ω-3. Treatment was daily administered by gavage for 3 months. Eye cups from each group were histologically examined using confocal and transmission electron microscopy. Proteomic analysis and N-retinylidene-N-retinylethanolamine (A2E) quantification was carried our using liquid chromatography mass spectrometry (LC-MS/MS). Blood and retinal fatty acids analysis was performed using gas chromatography (GC). Results A significant decrease in melanolipofuscin granules in the retinal pigment epithelium (RPE) was observed in the treatment group, in parallel with a reduction in A2E level, a major component of RPE lipofuscin. Proteomic analysis indicated decreased levels of complement 3 (C3) in the treated group. The outer nuclear layer (ONL) thickness was significantly greater in group C (75.66 ± 4.8 μm), compared to group A (61.40 ± 1.84 μm) and B (56.50 ± 3.24 μm). Increased EPA and decreased AA levels were observed in both blood and retinas in the treatment group. Conclusions Supplementation with ω-3 fatty acids (when AA/EPA = 1-1.5) suggests a protective mechanism in the ABCA4-/- animal model of Stargadt's disease.

Advances in mass spectrometry technologies have created new opportunities for discovering novel protein biomarkers in systemic lupus erythematosus (SLE). We performed a systematic review of published reports on proteomic biomarkers identified in SLE patients using mass spectrometry‐based proteomics and highlight their potential disease association and clinical utility. Two electronic databases, MEDLINE and EMBASE, were systematically searched up to July 2015. The methodological quality of studies included in the review was performed according to Preferred Reporting Items for Systematic Reviews and Meta‐analyses guidelines. Twenty‐five studies were included in the review, identifying 241 SLE candidate proteomic biomarkers related to various aspects of the disease including disease diagnosis and activity or pinpointing specific organ involvement. Furthermore, 13 of the 25 studies validated their results for a selected number of biomarkers in an independent cohort, resulting in the validation of 28 candidate biomarkers. It is noteworthy that 11 candidate biomarkers were identified in more than one study. A significant number of potential proteomic biomarkers that are related to a number of aspects of SLE have been identified using mass spectrometry proteomic approaches. However, further studies are required to assess the utility of these biomarkers in routine clinical practice.

The ubiquitin-binding, three-helix bundle domains of the proteins ubiquilin 1 (UQ1) and hHR23A both exhibited remarkably high, but discrete, ammonium ion adduction when electrosprayed from aqueous ammonium acetate. The degree of adduction was highly charge state dependent with, unusually, the lowest charge states (+3 for UQ1 and +4 for hHR23A) showing almost no adducts and the highest charge states (+5 for UQ1 and +6 for hHR23A) exhibiting adduction with two ammonium cations as the most abundant form. As the charge state of protein ions produced by electrospray ionisation (ESI) is related to solvent-accessible surface area we inferred that the ammonium-carrying ions were of a more open conformation than their protonated counterparts. This was confirmed by ESI-travelling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS), which showed that, although the purely protonated ions were compact, their equivalents bearing one or two ammonium adducts exhibited populations of significantly larger collisional cross section (CCS). We postulate that complexation with the ammonium cation may disrupt a key salt bridge(s) in the compact structure. A similar effect is observed with mono-sodium ion adduction, but this is diminished with each additional sodium ion in the complex to produce more compact structures.

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.

The benefits of lowering protein ion charge states in electrospray ionization (ESI) have attracted recent interest. We describe a simple approach to decrease protein charge states by exposure of electrospray droplets to neutral solvent vapor such as acetonitrile. The technique allows detection of weak noncovalent complexes, provides preferred charge states for tandem mass spectrometry (MS/MS) dissociation of protein complexes, and has the added benefit of reducing common adducts, such as alkali metals, without the addition of solution additives or the requirement for a secondary spray.

cope : A high adherence to the Mediterranean diet (MD) was previously associated with a decreased risk of breast cancer (BC) among Greek-Cypriot women. Additionally, particular polymorphisms were shown to modulate this MD-BC association. Herein, we aimed to investigate the effect of polymorphisms-MD interactions on the levels of specific metabolites that could be related to dietary adherence or enzymatic activity, which is itself modulated by polymorphisms. Methods and results : Greek-Cypriot women who were BC controls and had the lowest or the highest MD adherence (vegetables, fruit, legumes, fish) as assessed by principal component analysis (n = 564) were included. Participants were previously genotyped for 9 polymorphisms of the one-carbon metabolism, oxidative stress and xenobiotic metabolism. The serum levels of 14 metabolites that are key players in the aforementioned pathways were measured by UPLC-MS/MS. ANCOVA was used to assess polymorphism-MD interactions on metabolites’ levels within a multivariate linear regression model. Statistically significant interactions between GSTM1 deletion polymorphism and MD on flavin mononucleotide (FMN) and on 5-methyltetrahydrofolate (5-MTHF) concentrations were observed. The MTHFR rs1801133 interacted significantly with MD on 5-MTHF concentration. Conclusion : Serum levels of FMN and 5-MTHF were shown to be influenced by interactions between GSTM1 deletion or MTHFR (rs1801133) polymorphisms and a dietary pattern, characteristic of MD. This article is protected by copyright. All rights reserved

Introduction. Detection of the very earliest stages of breast cancer remain challenging and the differentiation of those carcinomas that develop into aggressive invasive forms from those that remain benign has not been achieved. Consequently, removal of the tumor by total mastectomy, local excision (LE) plus adjuvant radiotherapy (RT), or LE alone remain the main treatments, even if they prove unnecessary. Previously, using proteomics analysis, we identified proteins that were significantly changed in expression between matched normal and tumor tissues from Cypriot patients with different stages of the disease. Heat shock protein 10 (HSP10), which is responsible for the mitochondrial protein folding, was one of the proteins identified. A range of methods were used to verify HSP10 as a possible biomarker for breast cancer. Materials and Methods. Protein extracts from breast cancer cell lines and breast tissue biopsies from Cypriot patients with different stages of the disease were analysed by (i) Western Blotting (WB) and (ii) multiple reaction monitoring mass spectrometry (MRM MS) using two proteotypic peptides unique to HSP10, in proteins extracted form fresh frozen as well as from FFPE breast tissues. Tissue microarray analysis (TMA) was carried out on core biopsies from the Leeds Breast Tissue Bank. Results. WB indicated that HSP10 was ubiquitously and evenly expressed across breast cancer cell lines representative of different phenotypes. WB and MRM MS revealed that expression was significantly increased in invasive carcinoma compared to normal tissue, but was patient specific for fibroadenoma and DCIS cases. TMA of 360 samples indicated a strong correlation between staining and type of breast cancer (invasive ductal and lobular), grade and ER positive phenotype. Discussion. Proteomics analysis using iTRAQ quantitation indicated an average of 1.6 fold increase in HSP10 in invasive carcinomas compared to matched normal tissue. By comparison, WB and MRM MS analysis indicated a significantly amplified discrimination in the expression of HSP10 as has been observed with other biomarker candidates. HSP10 protein levels were in agreement with mRNA results observed in breast cancer cell lines reported in the Cancer Cell Line Encyclopaedia. In conclusion, HSP10 represents an excellent biomarker candidate for further validation. Citation Format: Sadr-ul Shaheed, Andreas Hadjisavvas, Kleitos Sokratous, Paul Loadman, Chris Sutton, Kyriacos Kyriacou. Analysis of HSP10 as a putative biomarker of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2015-1576

Ubiquitin (Ub) is able to form polymeric isopeptide-linked chains through condensation of any of its seven lysine (Lys) residues with the C-terminus of an adjacent Ub monomer. Electrospray ionisation mass spectrometry (ESI-MS) of commercial in vitro-generated Lys48-linked di-Ub (Lys48-Ub2) revealed a major population of cyclised dimer. The absence of a free C-terminus in this population was confirmed by an inability to bind the zinc finger ubiquitin-binding domain (ZnF-UBP) of USP5/isopeptidase-T. Endogenous Ub2 purified from skeletal muscle and cultured mammalian cells was found to contain cyclic Lys48-Ub2, demonstrating that cyclisation of poly-Ub can also occur in vivo.

Ubiquitination, a fundamental post-translational modification (PTM) resulting in the covalent attachment of ubiquitin (Ub) to a target protein, is currently implicated in several key cellular processes. Although ubiquitination was initially associated with protein degradation, it is becoming increasingly evident that proteins labeled with polyUb chains of specific topology and length are activated in an ever-expanding repertoire of specific cellular processes. In addition to their involvement in the classical protein degradation pathways they are involved in DNA repair, kinase regulation and nuclear factor-κB (NF-κB) signaling. The sorting and processing of distinct Ub signals is mediated by small protein motifs, known as Ub-binding domains (UBDs), which are found in proteins that execute disparate biological functions. The involvement of UBDs in several biological pathways has been revealed by several studies which have highlighted the vital role of UBDs in cellular homeostasis. Importantly, functional impairment of UBDs in key regulatory pathways has been related to the development of pathophysiological conditions, including immune disorders and cancer. In this review, we present an up-to-date account of the crucial role of UBDs and their functions, with a special emphasis on their functional impairment in key biological pathways and the pathogenesis of several human diseases. The still under-investigated topic of Ub-UBD interactions as a target for developing novel therapeutic strategies against many diseases is also discussed.

Hereditary ATTR V30M amyloidosis is a lethal autosomal dominant sensorimotor and autonomic neuropathy caused by deposition of aberrant transthyretin (TTR). Immunohistochemical examination of sural nerve biopsies in patients with amyloidotic neuropathy show co-aggregation of TTR with several proteins; including apolipoprotein E, serum amyloid P and components of the complement cascade. Complement activation and macrophages are increasingly recognized to play a crucial role in amyloidogenesis at the tissue bed level. In the current study we test the effect of two C5a receptor agonists and a C5a receptor antagonist (PMX53) on disease phenotype in ATTR V30M mice. Our results indicate that amyloid deposition was significantly reduced following treatment with the C5a receptor agonists, while treatment with the antagonist resulted in a significant increase of amyloid load. Administration of the C5a receptor agonists triggered increased recruitment of phagocytic cells resulting in clearance of amyloid deposits.