The greatly increased amounts of accumulated versican and decorin with specific post-translational modifications may be closely associated with the malignant phenotype of pancreatic cancer

Pancreatic carcinoma (PC) is a cancer type with highly malignant growth and dissemination pattern of which the mechanisms are poorly understood. However, the malignant phenotype is closely linked to extracellular matrix (ECM) of which proteoglycans (PGs) and hyaluronan (HA) play a crucial role in the control of tumor progression and metastasis. In this study, we demonstrated that versican and decorin, two different PGs with contradictory roles and functions in the pathobiology of cancer, were the main matrix PGs in PC presenting a great increase 27- and 7-fold, respectively, in comparison to normal pancreas (NP). PC was characterized by the disproportional increase of versican compared to decorin, about 4 to 1, with a concurrent increase of HA, which may be closely associated with the growth and aggressiveness of this carcinoma. Significant specific post-translational modifications were also observed in both versican and decorin regarding the type, hydrodynamic size, sulfation pattern and extent of uronate epimerization of their glycosaminoglycan chains (GAGs). In particular, chondroitin sulphate (CS) was the predominant GAG type in both PC-associated versican and decorin. The CS of PC-decorin was increased 11-fold, compared to NP in which dermatan sulfate (DS) was the predominant GAG type in both PGs. The sulfation pattern of GAG chains was significantly altered in PC, since 6-sulfated disaccharides predominated in both versican and decorin with a marked presence of non-sulfated disaccharides accompanied by lower hydrodynamic sizes of both CS and DS chains compared to NP. In conclusion, all these findings agree with the highly malignant phenotype of this cancer and, thus, more studies need to be addressed on the roles of the post-translational modifications of versican and decorin in the biology of cancer.     

The increased accumulation of structurally modified versican and decorin is related with the progression of laryngeal cancer

Versican and decorin, two proteoglycans (PGs) with contradictory roles in the pathophysiology of cancer, comprise important stromal components in many tumor types and play a crucial role in the progression of cancer. In this study, we provide direct evidence for a significant and stage-related accumulation of versican and decorin in the tumor-associated stroma of laryngeal squamous cell carcinoma (LSCC) in comparison to normal larynx. Both PGs were found to be co-localized within the peritumorous stroma. In addition, the accumulated versican and decorin were markedly modified on both protein core and glycosaminoglycan (GAG) levels. Decorin, which was present under both glycanated and non-glycanated forms, perceptibly increased with the progression of LSCC, compared to the normal larynx. Tumor-associated glycanated decorin was found to contain significant amounts of dermatan sulfate (DS) sequences. Versican was also found to undergo stage-related structural modifications since a marked heterogeneity of protein cores was observed, being intense in late stage of laryngeal cancer. The increased accumulation of both versican and decorin was associated with a significant stage-related increase of the molar ratio of Delta di-mono4S to Delta di-mono6S up to approximately threefold in LSCC compared to the normal ones. The modified chemical structure of both PGs could be associated with the degree of aggressiveness of laryngeal squamous cell carcinomas.     

Human placental calreticulin characterization of domain structure and post-translational modifications.

The domain organization and the post-translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6- to 7-kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N-terminus were observed, but no stable fragments of intermediate size were found. These results show that the C-domain of calreticulin is susceptible to proteolytic cleavage and that the N- and P-domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N-domain, and the third cysteine was found in the reduced form. No post-translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C-terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.     

Isolation and characterization of bovine gingival proteoglycans versican and decorin.

1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunological studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosylated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule.     

Cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties

Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains approximately 21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function.     

Mapping protein post-translational modifications with mass spectrometry

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.     

Proteomic analysis of post-translational modifications

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.     

Identification of multiple post-translational modifications in the porcine brain specific p25alpha

P25α is a protein normally expressed in oligodendrocytes and subcellular relocalization of p25α occurs in multiple system atrophy, Parkinson's disease and Lewy body dementia along with ectopic expression in neurons. Moreover, it accumulates in Lewy body inclusions with aggregated α-synuclein and is a potent stimulator of α-synuclein aggregation. P25α is a phosphoprotein and post-translational modifications (PTMs) may play a role in its disease-related abnormalities. To investigate the spectrum of PTMs on p25α we cloned porcine p25α and isolated the protein from porcine brain. Using several complementary tandem mass spectrometry techniques for peptide mass analysis and amino acid sequencing, a comprehensive analysis of the PTMs on porcine p25α was performed. It was found that porcine p25α is heavily modified with a variety of modifications: phosphorylation, di- and trimethylation, citrullination and a HexNAc group. The modifications are localized within p25α's unfolded terminal domains and suggest that their functional states are regulated. This comprehensive mapping of p25α's PTMs will form the basis for future functional studies and investigations of p25α's potential role as a biomarker.     

The secretory pathway Ca-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells

Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation. In this study the microdomain niche of the Golgi-resident SPCA Ca²⁺/Mn²⁺ pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.     

A five-mRNA signature associated with post-translational modifications can better predict recurrence and survival in cervical cancer

High mortality of patients with cervical cancer (CC) stresses the imperative of prognostic biomarkers for CC patients. Additionally, the vital status of post-translational modifications (PTMs) in the progression of cancers has been reported by numerous researches. Therefore, the purpose of this research was to dig a prognostic signature correlated with PTMs for CC. We built a five-mRNA (GALNTL6, ARSE, DPAGT1, GANAB and FURIN) prognostic signature associated with PTMs to predict both disease-free survival (DFS) (hazard ratio [HR] = 3.967, 95% CI = 1.985-7.927; P < .001) and overall survival (HR = 2.092, 95% CI = 1.138-3.847; P = .018) for CC using data from The Cancer Genome Atlas database. Then, the robustness of the signature was validated using GSE44001 and the Human Protein Atlas (HPA) database. CIBERSORT algorithm analysis displayed that activated CD4 memory T cell was also an independent indicator for DFS (HR = 0.426, 95% CI = 0.186-0.978; P = .044) which could add additional prognostic value to the signature. Collectively, the PTM-related signature and activated CD4 memory T cell can provide new avenues for the prognostic predication of CC. These findings give further insights into effective treatment strategies for CC, providing opportunities for further experimental and clinical validations.     

Partners and post-translational modifications of nuclear lamins

Nuclear intermediate filament networks formed by A- and B-type lamins are major components of the nucleoskeleton that are required for nuclear structure and function, with many links to human physiology. Mutations in lamins cause diverse human diseases (‘laminopathies’). At least 54 partners interact with human A-type lamins directly or indirectly. The less studied human lamins B1 and B2 have 23 and seven reported partners, respectively. These interactions are likely to be regulated at least in part by lamin post-translational modifications. This review summarizes the binding partners and post-translational modifications of human lamins and discusses their known or potential implications for lamin function.     

System level dynamics of post-translational modifications

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Surface accessibility of protein post-translational modifications

Protein post-translational modifications are crucial to the function of many proteins. In this study, we have investigated the structural environment of 8378 incidences of 44 types of post-translational modifications with 19 different approaches. We show that modified amino acids likely to be involved in protein-protein interactions, such as ester-linked phosphorylation, methylarginine, acetyllysine, sulfotyrosine, hydroxyproline, and hydroxylysine, are clearly surface associated. Other modifications, including O-GlcNAc, phosphohistidine, 4-aspartylphosphate, methyllysine, and ADP-ribosylarginine, are either not surface associated or are in a protein's core. Artifactual modifications were found to be randomly distributed throughout the protein. We discuss how the surface accessibility of post-translational modifications can be important for protein-protein interactivity.     

Multiple fibronectin subunits and their post-translational modifications

We report analyses of fibronectin subunit diversity by high resolution one- and two-dimensional gel electrophoresis. We have studied plasma and cellular fibronectins of rats and hamsters. Each form of fibronectin comprises multiple distinguishable subunits and, within each rodent species, all subunits of plasma fibronectin are resolvable from those of cellular fibronectin. Some, but not all, of this heterogeneity is caused by differential glycosylation. Thus, while glycosylated plasma and cellular fibronectins share no common subunits, nonglycosylated forms of these proteins appear to share 2-3 subunits. In addition, there are subunits unique to plasma and to cellular fibronectins in both rats and hamsters, although the pattern of diversity differs slightly between species. All size variants of fibronectin are phosphorylated to varying degrees. However, only some subunits are sulfated, apparently on tyrosine residues in the C-terminal third of the molecule. Comparison of the distribution of sulfate on the various fibronectin subunits with recent results on generation of multiple mRNAs by alternative splicing suggests that tyrosine sulfate is located in a polypeptide segment present in only certain fibronectin subunits. The results reported here provide information on the likely contributions of primary sequence differences and post-translational modifications to the heterogeneity of fibronectin subunits.