Quantitative analysis of the seminal plasma proteome in secondary hypogonadism

In the grey zone of testosterone levels between 8 and 12 nmol/L, the usefulness of therapy is controversial; as such, markers of tissue action of androgens may be helpful in adjusting clinical decisions. To better understand the effect of the hypothalamic-pituitary-testicular axis on male accessory secretion, we performed a proteomic quantitative analysis of seminal plasma in patients with secondary hypogonadism, before and after testosterone replacement therapy (TRT). Ten male patients with postsurgical hypogonadotrophic hypogonadism were enrolled in this study, and five of these patients were evaluated after testosterone treatment. Ten men with proven fertility were selected as a control group. An aliquot of seminal plasma from each individual was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLC-nano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer. The label-free quantitative analysis was performed via Precursor Ions Area Detector Node. Eleven proteins were identified as decreased in hypogonadic patients versus controls, which are primarily included in hydrolase activity and protein binding activity. The comparison of the proteome before and after TRT comes about within the discovery of six increased proteins. This is the primary application of quantitative proteomics pointed to uncover a cluster of proteins reflecting an impairment not only of spermatogenesis but of the epididymal and prostate epithelial cell secretory function in male hypogonadism. The identified proteins might represent putative clinical markers valuable within the follow-up of patients with distinctive grades of male hypogonadism

Identification of Novel Putative Urinary Markers of Endometriosis by High-Resolution Quantitative Proteomics

Endometriosis is a chronic gynecological inflammatory disease characterized by the presence of functional endometrial glands..

Analysis of the GFP-labelled β-dystroglycan interactome in HEK-293 transfected cells reveals novel intracellular networks

Highlights•HEK-293 cells were transfected with β-dystroglycan fused to GFP and the protein predominantly localized at the nuclear envelope.•Protein complexes bound to β-dystroglycan were investigated using immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis.•Novel potential interaction networks involving β-dystroglycan were identified in cytoskeleton, endoplasmatic reticulum, mitochondria and nucleus.•Lamin-Associated Polypeptide-1B (LAP1B) was identified as a novel protein that interacts with β-dystroglycan.AbstractDystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane β-DG whose cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. β-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases.To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with β-DG, HEK-293 cells were transiently transfected with a plasmid carrying the β-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing β-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG.Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis, led to the identification of an interactome formed by 313 exclusive protein matches for β-DG binding. A series of already known β-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel β-DG interactors and their related networks, were identified in diverse subcellular compartments such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamin-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with β-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires β-DG for a proper nuclear localization.These results expand the role of β-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy

Top-down proteomic profiling of human saliva in multiple sclerosis patients

Multiple sclerosis is a chronic disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration which is of undetermined origin. To date a single diagnostic test of multiple sclerosis does not exists and novel biomarkers are demanded for a more accurate and early diagnosis. In this study, we performed the quantitative analysis of 119 salivary peptides/proteins from 49 multiple sclerosis patients and 54 healthy controls by a mass spectrometry-based top-down proteomic approach. Statistical analysis evidenced different levels on 23 proteins: 8 proteins showed lower levels in multiple sclerosis patients with respect to controls and they were mono- and di-oxidized cystatin SN, mono- and di-oxidized cystatin S1, mono-oxidized cystatin SA and mono-phosphorylated statherin. 15 proteins showed higher levels in multiple sclerosis patients with respect to controls and they were antileukoproteinase, two proteoforms of Prolactin-Inducible Protein, P-C peptide (Fr.1-14, Fr. 26-44, and Fr. 36-44), SV1 fragment of statherin, cystatin SN Des1-4, cystatin SN P11 → L variant, and cystatin A T96 → M variant. The differences observed between the salivary proteomic profile of patients suffering from multiple sclerosis and healthy subjects is consistent with the inflammatory condition and altered immune response typical of the pathology. Data are available via ProteomeXchange with identifier PXD009440

Marked differences in the submandibular salivary proteome between Sardinian alcohol-preferring and Sardinian alcohol-non preferring rats revealed by an integrated top-down-bottom-up proteomic platform

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation

Disclosing the molecular profile of the human amniotic mesenchymal stromal cell secretome by filter-aided sample preparation proteomic characterization

Abstract Background The secretome of mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) has been extensively studied for its in vitro immunomodulatory activity as well as for the treatment of several preclinical models of immune-related disorders. The bioactive molecules within the hAMSCs secretome are capable of modulating the immune response and thus contribute to stimulating regenerative processes. At present, only a few studies have attempted to define the composition of the secretome, and several approaches, including multi-omics, are underway in an attempt to precisely define its composition and possibly identify key factors responsible for the therapeutic effect. Methods In this study, we characterized the protein composition of the hAMSCs secretome by a filter-aided sample preparation (FASP) digestion and liquid chromatography-high resolution mass spectrometry (LC–MS) approach. Data were processed for gene ontology classification and functional protein interaction analysis by bioinformatics tools. Results Proteomic analysis of the hAMSCs secretome resulted in the identification of 1521 total proteins, including 662 unique elements. A number of 157 elements, corresponding to 23.7%, were found as repeatedly characterizing the hAMSCs secretome, and those that resulted as significantly over-represented were involved in immunomodulation, hemostasis, development and remodeling of the extracellular matrix molecular pathways. Conclusions Overall, our characterization enriches the landscape of hAMSCs with new information that could enable a better understanding of the mechanisms of action underlying the therapeutic efficacy of the hAMSCs secretome while also providing a basis for its therapeutic translation

Mitochondria, ciglitazone and liver: a neglected interaction in biochemical pharmacology.

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors now actively investigated for their involvement in lipid and glucidic metabolism, immune regulation and cell differentiation. Drugs binding and activating PPARs are therefore attracting attention for their potential therapeutic role in various diseases like type 2 diabetes, dyslipidemias, atherosclerosis, obesity (i.e., metabolic syndrome). Agonists of these receptors have been already used in therapeutic protocols: fibrates (PPAR-alpha ligands) are being used in hyperlipidemias, and thiazolidinediones (mainly PPAR-gamma ligands) are being employed as insulin sensitizers. The latter drugs introduction into therapy, however, showed very soon some unwanted effects (hepatotoxicity at first and myocardiotoxicity later on) which confirmed some contradictory data already suggested by pre-clinical trial-experiments. In this study we show that some PPAR ligands impair mitochondrial oxidative metabolism in human liver cell line mainly by deranging NADH oxidation. Intriguingly, the PPAR-gamma ligand ciglitazone caused a dose-dependent inhibition of NADH-cytochrome c reductase that resulted, at a drug concentration of 50 microM, of about 60% (P<0.001), while other PPAR ligands with different receptor affinity - positive controls like clofibrate (0.7 mM), gemfibrozil (0.23 mM) and bezafibrate (1 mM) - reduced the activity of mitochondrial Complex I by about 20% (P<0.01, P<0.01 and P<0.05, respectively). The induced mitochondrial dysfunction imposed a series of metabolic compensatory adaptations characterized by a significant shift to anaerobic glycolysis. These findings underline the undervalued non-genomic effects of PPAR ligands and can provide a better understanding of the pharmacotoxicological profiles of these drugs and of their roles in the therapy of diabetes mellitus

Multiple Herpes simplex virus-1 (HSV-1) reactivations induce protein oxidative damage in mouse brain. Novel mechanisms for Alzheimer’s disease progression

Compelling evidence supports the role of oxidative stress in Alzheimer’s disease (AD) pathophysiology. Interestingly, Herpes simplex virus-1 (HSV-1), a neurotropic virus that establishes a lifelong latent infection in the trigeminal ganglion followed by periodic reactivations, has been reportedly linked both to AD and to oxidative stress conditions. Herein, we analyzed, through biochemical and redox proteomic approaches, the mouse model of recurrent HSV-1 infection we previously set up, to investigate whether multiple virus reactivations induced oxidative stress in the mouse brain and affected protein function and related intracellular pathways. Following multiple HSV-1 reactivations, we found in mouse brains increased levels of oxidative stress hallmarks, including 4-hydroxynonenal (HNE), and 13 HNE-modified proteins whose levels were found significantly altered in the cortex of HSV-1-infected mice compared to controls. We focused on two proteins previously linked to AD pathogenesis, i.e., glucose-regulated protein 78 (GRP78) and collapsin response-mediated protein 2 (CRMP2), which are involved in the unfolded protein response (UPR) and in microtubule stabilization, respectively. We found that recurrent HSV-1 infection disables GRP78 function and activates the UPR, whereas it prevents CRMP2 function in mouse brains. Overall, these data suggest that repeated HSV-1 reactivation into the brain may contribute to neurodegeneration also through oxidative damage