The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy.
Amino Acid Sequence ; Breast Neoplasms ; drug therapy ; genetics ; pathology ; Cell Line, Tumor ; Cross-Linking Reagents ; administration & dosage ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Mass Spectrometry ; Proteasome Endopeptidase Complex ; drug effects ; Protein Binding ; drug effects ; Protein Isoforms ; genetics ; Protein Subunits ; biosynthesis ; genetics ; Proteomics ; Succinimides ; administration & dosage

Crystallography, X-Ray ; Escherichia coli ; metabolism ; Fibroblast Growth Factors ; chemistry ; genetics ; metabolism ; Heparin ; metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Isoforms ; chemistry ; metabolism ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Sulfates ; chemistry ; metabolism

Plant betaine aldehyde dehydrogenase (BADH) is a physiologically important enzyme in response to salt or drought stress. In this study, two BADH genes (PeBADH1 and PeBADH2) were cloned from Populus euphratica. Both PeBADH1 and PeBADH2 genes encode the proteins of 503 amino acid residues, with a calculated molecular mass of 54.93 kDa and 54.90 kDa, respectively. Reverse transcription PCR showed the divergence of expression pattern between the PeBADH1 and PeBADH2 genes in P. euphratica. The recombinant PeBADH1 and PeBADH2 proteins were overexpressed in E. coli, and purified by Ni-affinity chromatography. The PeBADH2 protein had 1.5-fold higher enzymatic activity towards the substrate aldehyde than PeBADH1 protein. The PeBADH1 protein revealed higher thermal stability than PeBADH2 protein. These results indicated obvious functional divergence between the PeBADH1 and PeBADH2 genes.
Amino Acid Sequence ; Betaine-Aldehyde Dehydrogenase ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; physiology ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; chemistry ; genetics ; Populus ; genetics ; Protein Isoforms ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Substrate Specificity

MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC. Consistently, DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2 (NOS2) and subsequently promotes the synthesis of nitric oxide (NO). NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro; hence, overexpression of DC-CASPIC in DC enhances this stimulation. Collectively, our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.
Alternative Splicing ; Amino Acid Sequence ; Animals ; Base Sequence ; CARD Signaling Adaptor Proteins ; genetics ; metabolism ; Cell Line ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; In Vitro Techniques ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Mitochondria ; metabolism ; Molecular Sequence Data ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; T-Lymphocytes ; immunology ; metabolism ; Trans-Activators ; genetics ; metabolism ; Up-Regulation ; drug effects

Mechano-growth factor (MGF) is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA (330 bp) was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5' end sequence was truncated by primer, then the obtained truncated MGF (des(1-3)MGF) cDNA (321 bp) was subcloned in pET32a(+) vector to construct a prokaryotic recombination expression plasmid. Trx/des(1-3)MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21(DE3) by IPTG induction at 30 degres C. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2+ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des(1-3)MGF was isolated by reverse-phase HPLC. Through these procedures, des(1-3) MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des(1-3)MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des(1-3)MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E1 than that of des(1-3)IGF-1.
Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Insulin-Like Growth Factor I ; Osteoblasts ; metabolism ; Protein Isoforms ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; STAT5 Transcription Factor ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics

Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEK293 cells to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.
Adenoviridae ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Defective Viruses ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Protein Isoforms ; biosynthesis ; genetics ; Receptors, Interleukin ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Virus Cultivation ; methods

OBJECTIVETo find the molecular mechanism of decreasing blood fat effect of Darning capsule on hyperlipemic rat, we study the expression of connexin43 in the myocardium before and after using the capsule.

METHODForty Wistar rats were randomly divided into 5 group: control group, hyperlipemia model group, Daming capsule group of high dose, middle dose and low dose (200, 100, 50 mg kg(-1) d(-1)). Each group had 8 rats. Hyperlipemic rat model was made firstly, the blood was obtained via vena caudalis and the indexes of TC, TG, LDL, HDL and NEFA in the serum were measured. The myocardial total RNA was extracted by Trizol method. To compare the expression of connexin43 in the following groups: hyperlipemia, normal and drug, we used the technique of RT-PCR, immunostaining and microconfoul.

RESULTThe concentrations of TC, TG, LDL and NEFA in hyperlipemic serum were increased (P <0. 05), while that of HDL was decreased (P <0. 05). After treating with Daming capsule, the concentration of the preceding four indexes were decreased and the concentrations of HDL was increased up to nearly normal level. No significant difference was found in the ECG of the three groups. As compared with the normal group, the mRNA expressions of connexin43 in hyperlipemia group was weakened (P <0.05), while that of the drug group was enhanced(P <0.05). The same result in immunostaining was observed.

CONCLUSIONHyperlipemic rat model was successfully established and Daming capsule has the effect of lowering blood lipid. Furthermore, the molecular mechanism of Darning capsule is related with the change of Cx43 closely.

Animals ; Capsules ; Cholesterol ; blood ; Connexin 43 ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fatty Acids, Nonesterified ; blood ; Hyperlipidemias ; blood ; metabolism ; Hypolipidemic Agents ; administration & dosage ; pharmacology ; Male ; Myocardium ; metabolism ; Plants, Medicinal ; chemistry ; Protein Isoforms ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Random Allocation ; Rats ; Rats, Wistar ; Triglycerides ; blood

Cytokine and chemokine receptors play a key role in inflammation caused by rheumatoid arthritis (RA). Two isoforms of human CC chemokine receptor R2 (CCR2), the receptor of monocyte chemoattractant protein 1 (MCP-1), have been identified but their relative expression in fibroblast-like synoviocytes (FLS) and their contribution to inflammatory responses mediated by MCP-1 or inflammatory cytokines in patients with RA remain uncertain. We examined the pattern of expression of two CCR2 isoforms upon stimulation by proinflammatory cytokines and CD40 ligation. FLS were prepared from the synovial tissues of RA patients and cultured in the presence of MCP-1, soluble CD40 ligand (sCD40L), TGF-beta, IL-1beta, IL-18, IL-15, and LPS. CCR2A and CCR2B expression was examined by immunohistochemistry, RT-PCR and western blot analysis. IL-15, TNF-alpha and MCP-1 production was determined by ELISA. Immunohistochemistry showed that CCR2A is highly expressed in RA synovium compared with OA synovium. Transcripts of both CCR2A and CCR2B were detected in FLS. Exogenous MCP-1, CD40L, TGF-beta, and IL-15 significantly increased the expression of CCR2A but not CCR2B. Exposure of FLS to sCD40L caused strong upregulation of CCR2A but not of CCR2B protein expression. MCP-1 increased the proliferation of FLS and the production of IL-15, TNF-alpha, and IL-18. Because CCR2A is the main target of regulation by cytokines and CD40 ligation, the relatively higher expression of CCR2A on the cell surface suggests that this isoform of MCP-1 receptor functions as the principal mediator of inflammatory signals in RA FLS.
Arthritis, Rheumatoid/*metabolism ; CD40 Ligand/*pharmacology ; Cells, Cultured ; Chemokine CCL2/*pharmacology ; Chemokines/biosynthesis ; Fibroblasts/*metabolism ; Humans ; Protein Isoforms ; Receptors, CCR2/*biosynthesis ; Synovial Membrane/*pathology ; Transforming Growth Factor beta/*pharmacology

OBJECTIVETo detect the expression of voltage-gated Na(+) channel (NaCh) isoforms in rat sinoatrial node and explore their functions.

METHODSExpressions of NaCh isoforms Nav1.1, Nav1.2, Nav1.3, Nav1.5, Nav1.6 and Nav1.7 in the rat sinoatrial node were detected by immunohistochemistry. The functional roles of the NaChs were tested by observing the effect of tetrodotoxin, a specific blocker of NaChs, on the intrinsic heart rate of isolated rat working heart.

RESULTSThe tetrodotoxin- sensitive neuronal isoforms Nav1.1, Nav1.6 and Nav1.7 as well as the tetrodotoxin-resistant cardiac isoform Nav1.5 were present in the rat sinoatrial node, and the neuronal isoforms were more abundant than Nav1.5 (P<0.05). The selective blockade of tetrodotoxin-sensitive isoforms (presumably Nav1.1, Nav1.6 and Nav1.7) by 100 nmol/L tetrodotoxin scarcely affected the intrinsic heart rate (0.5-/+2.9%, P>0.05) while blockade of tetrodotoxin-resistant isoform (presumably Nav1.5) by 2 micromol/L tetrodotoxin resulted in an obvious decline in the intrinsic heart rate (22.1-/+2.1%, P<0.001).

CONCLUSIONSNav1.1, Nav1.5, Nav1.6 and Nav1.7 are all present in rat sinoatrial node. Although neuronal isoforms are more abundant, Nav1.5 seems to contribute more to activity of the sinoatrial node.

Animals ; Heart Rate ; drug effects ; physiology ; Immunohistochemistry ; Ion Channel Gating ; drug effects ; physiology ; Male ; NAV1.1 Voltage-Gated Sodium Channel ; NAV1.5 Voltage-Gated Sodium Channel ; NAV1.6 Voltage-Gated Sodium Channel ; Nerve Tissue Proteins ; biosynthesis ; Protein Isoforms ; biosynthesis ; Rats ; Sinoatrial Node ; drug effects ; metabolism ; physiology ; Sodium Channels ; biosynthesis ; Tetrodotoxin ; pharmacology

OBJECTIVETo investigate the feasibility of metallothionein (MT) gene expression level in human peripheral blood lymphocytes (HPBLs) as a biomarker in cadmium exposure.

METHODSThe MT gene expression level in HPBLs of workers exposed to cadmium was examined using RT-PCR technique, and the exposure assessment and effect assessment were conducted in exposed workers.

RESULTSThe basal MT-1A, IE, IF, IX and MT-2A expression level in workers exposed to cadmium were significantly higher than those in the control group (P < 0.05). The basal MT-1A, IE, IF, IX and MT-2A expression level would be significantly increased with the increase of the blood cadmium (BCd) level (P < 0.05). There was a trend of increase for the mRNA expression of the basal MT-1A, 1E, IF, IX, MT-2A, especially for the mRNA expression of MT-1A and MT-2A (P < 0.05) with the increase of the level of the urine cadmium (UCd). There was a good dose-response relationship between basal MT-1A expression and UCd. The basal MT-1A, IE, IF, IX and MT-2A expression level were significantly correlated with BCd (P < 0.05) while the basal MT-1A, IF and MT-2A expression level were significantly correlated with UCd (P < 0.05). There were dose-effect relationships of BCd to the basal MT-1E, MT-1F, MT-1X and MT-2X expression level respectively and there were also dose-effect relationships of UCd, beta(2)-MG and the urine metallothionein to the basal MT-1A expression.

CONCLUSIONThe expression of the MT gene isoforms in HPBLs can serve as the biomarker for the cadmium exposure and MT-1A can also serve as the effective biomarkers for the cadmium-induced renal toxicity.

Adult ; Biomarkers ; metabolism ; Cadmium ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Gene Expression ; Humans ; Lymphocytes ; metabolism ; Male ; Metallothionein ; biosynthesis ; genetics ; Occupational Exposure ; Protein Isoforms ; biosynthesis ; RNA, Messenger ; genetics