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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.

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.

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.

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.

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

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.