Characterization of a novel affinity binding ligand for tyrosine nitrated peptides from a phage-displayed peptide library.

As an important post-translational modification in response to oxidative and nitrosative stress, protein tyrosine nitration is deeply involved in many physiological and pathological processes. Identifying tyrosine nitration in proteins is challenging due to its low abundance.Consequently, pre-separation and enrichment of tyrosine-nitrated peptides (TNPs) are necessary before submitting them to mass spectrometry analysis. However, the most popularly used anti-nitrotyrosine antibody pull-down method showed limitations like sequence preference and unspecific binding. Therefore, developing novel affinity purification materials for TNPs is of significance. In the present study, we screened the phage-displayed 12-mer randomized peptide library for affinity binding peptide of the synthetic standard TNP (sTNP, sequence: H2N-GGGGY*GGG-COOH) and identified a peptide named NT-1 (H2N-TLWPFDLWLKTR-COOH) as a promising candidate. NT-1 at extremely low concentration (3 nM) in solutions could be efficiently captured by immobilized sTNP as determined by pull-down and subsequent MALDI-TOF MS analysis. Surface plasmon resonance (SPR) measurement confirmed that NT-1 possesseed a good selectivity, showing more than 100-fold higher binding affinity with TNP than its non-nitrated counterpart. Moreover, NT-1 could efficiently capture various types of TNPs in solutions even in the presence of 1000-fold excessive amount of trypsinized BSA fragments. Most importantly, NT-1 showed superiority to commercially used nitrotyrosine antibody as the former captured more TNPs, with less sequence preference. In summary, our study provided NT-1 as a novel affinity binding ligand for TNPs and should be useful in developing an alternative enrichment strategy for TNPs.

Insight into the proteomic profiling of exosomes secreted by human OM-MSCs reveals a new potential therapy.

Mesenchymal stromal cells (MSCs) have been used for the treatment of neuronal injury and neurodegenerative diseases. Their underlying mechanism may involve increased secretion of paracrine factors, which promotes tissue repair. Presently, exosomes have been regarded as important components of paracrine secretion and paracrine factors. MSC exosomes represent a promising opportunity to develop novel cell-free therapy approaches. In this study, exosomes from nasal olfactory mucosa MSCs (OM-MSCs) were extracted and purified using ultracentrifugation, resulting in exosome diameters of 40-130 nm. Similar to other exosomes, OM-MSC exosomes were CD63- and CD81-positive and calnexin-negative. Functionally, OM-MSC exosomes promoted human brain microvascular endothelial cell (HBMEC) proliferation and migration. The present study analyzed the OM-MSC exosome paracrine proteome. A total of 304 exosome-associated proteins were identified by LC-MS/MS, including plasminogen activator inhibitor 1 (SERPINE 1), insulin-like growth factor binding protein family members (IGFBP 4 and 5), epidermal growth factor receptor (EGFR), neurogenic locus notch homolog protein 2 (NOTCH 2), apolipoprotein E (APOE), and heat shock protein HSP90-beta (HSP90AB1). These molecules are known to be important in neurotrophic, angiogenesis, cell growth, differentiation, apoptosis, and inflammation and are highly correlated with the mechanism of tissue repair and neural restoration. These observations may provide a basis for further evaluation of OM-MSC exosome potential as a novel therapeutic modality.

Changes in the mitochondrial proteome in human hepatocytes in response to alpha-amanitin hepatotoxicity.

Amanitin-induced apoptosis is proposed to have a significant effect on the pathogenesis of liver damage. However, few reports have focused on proteome changes induced by α-amanitin (α-AMA). Here, we evaluated changes in mitochondrial proteins of hepatocytes in response to 2 µM α-AMA, a concentration at which α-AMA-induced cell damage could be rescued at cellular level by common clinical drugs. We found 56 proteins were differentially expressed in an α-AMA-treated group. Among them, 38 proteins were downregulated and 18 were upregulated. Downregulated functional proteins included importer TOMM40, respiratory chain component cytochrome C, and metabolic enzymes of citrate acid cycle such as malate dehydrogenase, which localize on the mitochondrial outer membrane, inner membrane and matrix respectively. Immunoblot analysis showed that α-AMA decreased mitochondrial import receptor subunit TOMM40 and cytochrome c accompanied by an increase in the cytosol although their total protein levels were not affected significantly. The mitochondrial membrane potential was also destroyed by α-AMA and was restored by the clinical drug silibinin. Immunofluorescence suggested that mitochondrial morphology did not change. Taken together, our results provide further insights into the toxic mechanism of α-AMA on hepatocytes.