Comparison of proteomes in various human plasma preparations by two-dimensional gel electrophoresis

Serum or plasma can be utilized in a variety of studies targeted toward the discovery of disease biomarkers. In this study, the proteome profiles of plasma samples prepared using various anticoagulants (EDTA, heparin or citrate), were compared with those of serum using two-dimensional electrophoresis (2-DE). Proteins which evidenced different levels in the plasma and serum were screened and identified using ESI-Q-TOF MS/MS. The proteins which became detectable after the removal of fibrinogen from serum were identified as pigment epithelial differentiating factor (four spots), fetuin-like protein, and the hemopexin precursor. In particular, three proteins, pre-serum amyloid P component, plasma glutathione peroxidase precursor, and tetranectin, evidenced increased volume intensity only in the plasma samples prepared with EDTA.     

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.     

Biochemical,physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background

Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H₂O₂ and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).

Methods

Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.

Results

Vu-2-Cys-Prx reduces H₂O₂ using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH₂-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys⁵² residue and the amino acids Pro⁴⁵, Thr⁴⁹ and Arg¹²⁸ are conserved as in other 2-Cys-Prx.

General significance

The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.     

应用蛋白质组学和RNAi技术筛选鼻咽癌细胞中p53功能相关蛋白质

为了阐明鼻咽癌中高表达的p53蛋白聚集与失活的机制,高通量地检测与p53功能相关的蛋白质,首先采用RNA干扰(RNAi)技术稳定沉默鼻咽癌细胞系CNE2的p53基因表达,然后用蛋白质组技术研究稳定沉默该基因对鼻咽癌蛋白质表达谱的影响.通过对稳定干扰p53基因后鼻咽癌细胞系CNE2的蛋白质表达谱改变的研究,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析和电喷雾串联质谱(ESI-Q-TOF-MS)验证鉴定了22个差异表达蛋白质.在这些差异表达蛋白质中,有些是已经报道的p53功能相关蛋白质,如热休克蛋白27(HSP27)、异质性胞核核糖核蛋白K(hnRNPK)、14-3-3σ等,其他可能是新的p53功能相关蛋白质,如eIF4B、TPT1、hnRNPH3、SFRS1等.部分差异表达蛋白质如HSP27、14-3-3σ和GRP75经蛋白质印迹分析技术进行了验证,同时pcDNA3.1-FLAG-p53质粒转染CNE2细胞引起了HSP27、14-3-3σ表达下调,GRP75表达上调.在鼻咽癌细胞中鉴定的22个差异表达蛋白质大致可以分为5类,包括信号传导相关蛋白质、分子伴侣、与转录和翻译相关蛋白质、代谢相关蛋白和细胞结构相关蛋白质,涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为p53功能相关蛋白质,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.     

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.     

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

Background

Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma.

Results

Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology.

Conclusion

These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.     

Purification and characterization of levoglucosan kinase from <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis> YZ-215

A protein named as levoglucosan kinase (EC 2.7.-.-)was purified to homogeneity from a wild isolated strain of Lipomyces starkeyi YZ-215. The protein was purified approximately 30-fold by conventional ammonium sulphate fractionation followed by Resource Q chromatography and two steps of Superdex 200 chromatography, and its physical and kinetic properties were investigated. The purified enzyme showed a molecular weight of 48 kDa by SDS-PAGE and 47.7 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), respectively. The enzyme was stable at pH 7–10 and showed maximum activity at 30°C and pH 9.0. Kinetic constants (apparent K _m values) for levoglucosan and ATP were 68.6 ± 13.7 mM and 0.68 ± 0.06 mM, respectively. After in-gel digestion by trypsin, three peptides were sequenced and analyzed by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Data of the amino acid sequences indicated that this protein might be a novel kinase. The purification of levoglucosan kinase from L. starkeyi YZ-215 represented a fundamental step to provide insights into the efficient utilization of cellulosic pyrolysate by bioconversion.     

Methylation of arginine residues interferes with citrullination by peptidylarginine deiminases in vitro

Peptidylarginine deiminase (PAD) enzymes catalyze the conversion of arginine residues in proteins to citrulline residues. Citrulline is a non-standard amino acid that is not incorporated in proteins during translation, but can be generated post-translationally by the PAD enzymes. Although the existence of citrulline residues in proteins has been known for a long time, only a few proteins have been reported to contain this amino acid under normal conditions. These include the nuclear histones, which also contain a wide variety of other post-translational modifications, as for instance methylation of arginine residues. It has been suggested that citrullination and methylation of arginine residues are competing processes and that PAD enzymes might "reverse" the methylation of arginine residues by converting monomethylated arginine into citrulline. However, conflicting data have been reported on the capacity of PADs to citrullinate monomethylated peptidylarginine. Using synthetic peptides that contain either arginine or methylated arginine residues, we show that the human PAD2, PAD3 and PAD4 enzymes and PAD enzyme present in several mouse tissues in vitro can only convert non-methylated peptidylarginine into peptidylcitrulline and that hPAD6 does not show any deiminating activity at all. A comparison of bovine histones either treated or untreated with PAD by amino acid analysis also supported the interference of deimination by arginine methylation. Taken together, these data indicate that it is unlikely that methyl groups at the guanidino position of peptidylarginine can be removed by peptidylarginine deiminases, which has important implications for the recently reported role of these enzymes in gene regulation.