preparation-mitochondria-from-ovarian-cancer-tissues-control-ovarian

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

Central South University

Oxidative/nitrative damage is a crucial element among the complex factors that contribute to lung carcinogenesis. Nitric oxide (NO) free radicals, through chemical modifications such as tyrosine nitration, are significantly involved in lung cancinogenesis and metastasis. NO-mediated protein nitration, which is the addition of the nitro group (-NO2) to position 3 of the phenolic ring of a tyrosine residue, is an important molecular event in lung cancer, and has been studied with mass spectrometry. Nitration is involved in multiple biological processes, including signal transduction, protein degradation, energy metabolism, mitochondrial dysfunction, enzyme inactivation, immunogenic response, apoptosis, and cell death. This article reviews the relationship of NO and its derivates and lung cancer, formation and roles of tyrosine nitration in lung cancer, differences of protein nitration between lung cancer and other inflammatory pulmonary diseases, current status of protein nitration and nitroproteomics in lung cancer, and future perspectives to achieve a better understanding of lung carcinogenesis, for biomarker discovery; and for new diagnostic and prognostic monitoring, and therapeutic targets.

Protein tyrosine nitration in lung cancer: current research status and future perspectives.

Human prolactin (hPRL) plays multiple roles in growth, metabolism, development, reproduction, and immunoregulation, which is an important protein synthesized in a pituitary. Two-dimensional gel electrophoresis (2DE) is an effective method in identity of protein variants for in-depth insight into functions of that protein. 2DE, 2DE-based PRL-immunoblot, mass spectrometry, and bioinformatics were used to analyze hPRL variants in human normal (control;  = 8) pituitaries and in five subtypes of pituitary adenomas [NF ( = 3)-, FSH ( = 3)-, LH ( = 3)-, FSH/LH ( = 3)-, and PRL ( = 3)-adenomas]. Six hPRL variants were identified with different isoelectric point (I)-relative molecular mass ( ) distribution on a 2DE pattern, including variants V1 (I 6.1; 26.0 kDa), V2 (I 6.3; 26.4 kDa), V3 (I 6.3; 27.9 kDa), V4 (I 6.5; 26.1 kDa), V5 (I 6.8; 25.9 kDa), and V6 (I 6.7; 25.9 kDa). Compared to controls, except for variants V2-V6 in PRL-adenomas, V2 in FSH-adenomas, and V3 in NF-adenomas, the other PRL variants were significantly downregulated in each subtype of pituitary adenomas. Moreover, the pattern of those six PRL variants was significantly different among five subtypes of pituitary adenomas relative to control pituitaries. Different hPRL variants might be involved in different types of PRL receptor-signaling pathways in a given condition. Those findings clearly revealed the existence of six hPRL variants in human pituitaries, and the pattern changes of six hPRL variants among different subtypes of pituitary adenomas, which provide novel clues to further study the functions, and mechanisms of action, of hPRL in human pituitary and in PRL-related diseases, and the potential clinical value in pituitary adenomas.

Prolactin Variants in Human Pituitaries and Pituitary Adenomas Identified With Two-Dimensional Gel Electrophoresis and Mass Spectrometry.

Protein ubiquitination is an important post-translational modification that is associated with multiple diseases, including pituitary adenomas (PAs). Protein ubiquitination profiling in human pituitary and PAs remains unknown. Here, we performed the first ubiquitination analysis with an anti-ubiquitin antibody (specific to K-ε-GG)-based label-free quantitative proteomics method and bioinformatics to investigate protein ubiquitination profiling between PA and control tissues. A total of 158 ubiquitinated sites and 142 ubiquitinated peptides in 108 proteins were identified, and five ubiquitination motifs were found. KEGG pathway network analysis of 108 ubiquitinated proteins identified four statistically significant signaling pathways, including PI3K-AKT signaling pathway, hippo signaling pathway, ribosome, and nucleotide excision repair. R software Gene Ontology (GO) analysis of 108 ubiquitinated proteins revealed that protein ubiquitination was involved in multiple biological processes, cellular components, and molecule functions. The randomly selected ubiquitinated 14-3-3 zeta/delta protein was further analyzed with Western blot, and it was found that upregulated 14-3-3 zeta/delta protein in nonfunctional PAs might be derived from the significantly decreased level of its ubiquitination compared to control pituitaries, which indicated a contribution of 14-3-3 zeta/delta protein to pituitary tumorigenesis. These findings provided the first ubiquitinated proteomic profiling and ubiquitination-involved signaling pathway networks in human PAs. This study offers new scientific evidence and basic data to elucidate the biological functions of ubiquitination in PAs, insights into its novel molecular mechanisms of pituitary tumorigenesis, and discovery of novel biomarkers and therapeutic targets for effective treatment of PAs.

Quantitative Analysis of Ubiquitinated Proteins in Human Pituitary and Pituitary Adenoma Tissues.

To investigate the existence and their potential biological roles of mitochondrial phosphoproteins (mtPPs) in human ovarian carcinoma (OC), mitochondria purified from OC and control tissues were analyzed with TiO enrichment-based iTRAQ quantitative proteomics. Totally 67 mtPPs with 124 phosphorylation sites were identified, which of them included 48 differential mtPPs (mtDPPs). Eighteen mtPPs were reported previously in OCs, and they were consistent in this study compared to previous literature. GO analysis revealed those mtPPs were involved in multiple cellular processes. PPI network indicated that those mtPPs were correlated mutually, and some mtPPs acted as hub molecules, such as EIF2S2, RPLP0, RPLP2, CFL1, MYH10, HSP90, HSPD1, PSMA3, TMX1, VDAC2, VDAC3, TOMM22, and TOMM20. Totally 32 mtPP-pathway systems (p<0.05) were enriched and clustered into 15 groups, including mitophagy, apoptosis, deubiquitination, signaling by VEGF, RHO-GTPase effectors, mitochondrial protein import, translation initiation, RNA transport, cellular responses to stress, and c-MYC transcriptional activation. Totally 29 mtPPs contained a certain protein domains. Upstream regulation analysis showed that TP53, TGFB1, dexamethasone, and thapsigargin might act as inhibitors, and L-dopa and forskolin might act as activators. This study provided novel insights into mitochondrial protein phosphorylations and their potential roles in OC pathogenesis and offered new biomarker resource for OCs.

Shehua Qian

Central South University

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis