PSMA7 inhibits the tumorigenicity of A549 human lung adenocarcinoma cells

The gene for proteasome subunit alpha type-7 (PSMA7) is located in chromosomal 20q13.33, a region frequently amplified in tumor. In this study, we employed A549 human lung adenocarcinoma cells and showed that PSMA7 inhibits the proliferation, tumorigenicity and invasion of A549 cells in vitro. Moreover, both gain and loss of function studies demonstrated that PSMA7 modulates the tumorigenicity of A549 cells in a xenograft nude mice model. In conclusion, these results identify inhibitory effects associated with PSMA7 that affect the tumorigenicity of A549 cells, suggesting PSMA7 as a potential tumor biomarker.     

Identification of collaborative activities with oxidative phosphorylation in bipolar disorder

Bipolar disorder (BD) is a psychiatric disease considered to polygenic with multiple factors in genetics, each of which is not dominant but collaborative during pathogenic progression. We describe a method that estimates the collaborative contribution to the disease between a certain well-studied pathway and the other candidate pathway using Gene Set Enrichment Analysis (GSEA). We describe a modified GSEA (improved derivation) to identify genes that are significantly and differentially expressed between disease and non-disease states and that are consistently co-expressed with a target pathway which is deeply related to disease etiology. The modified GSEA uses available gene expression data to identify molecular mechanism (ubiquitin-proteasome and inflammatory response) associated with the disease. We believe that this approach could reveal hidden relations between a certain well-studied pathway and the other candidate pathway known in literature.

Abbreviations

ATP5I - ATP synthase H+ transporting mitochondrial F0 complex subunit E, ATP5J - ATP synthase H+ transporting mitochondrial F0 complex subunit F6, BAD - Bcl-2-associated death promoter, BAX - Bcl-2-associated x protein, Bcl-2 - B-cell lymphoma 2, BDNF - brain derived neurotrophic factor, COX5B - Cytochrome c oxidase subunit Vb, COX7A2 - cytochrome c oxidase subunit VIIa polypeptide 2, DLK - dual leucine zipper-bearing kinase, GABA - Gamma aminobutyric acid, IL-8 - Interleukin 8, NDUFA1 - NADH dehydrogenase 1 alpha subcomplex 1, NDUFB2 - NADH dehydrogenase1 beta subcomplex 2, NDUFS4 - NADH dehydrogenase Fe-S protein 4, NGF - nerve growth factor, PPP2R5C - protein phosphatase 2 regulatory subunit B gamma, PSMA3 - proteasome subunit alpha type 3, PSMA7 - proteasome subunit alpha type 7, PSMB1 - proteasome subunit beta type 1, PSMB6 - proteasome subunit beta type 6, PSMB7 - proteasome subunit beta type 7, PSMC2 - proteasome 26S subunit ATPase 2, PSMC5 - proteasome 26S subunit ATPase 5, SLC6A4 - solute carrier family 6 member 4, TNFa - tumor necrosis factor a, UBE2A - ubiquitinconjugating enzyme E2A, UCRC - ubiquinol-cytochrome c reductase complex, UFC1 - ubiquitin-fold modifier conjugating enzyme 1, UQCRQ - ubiquinol-cytochrome c reductase complex III subunit VII, USP14 - ubiquitin specific protease 14.     

Parkin interacts with the proteasome subunit alpha4

Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.     

Interaction between c-Abl and Arg tyrosine kinases and proteasome subunit PSMA7 regulates proteasome degradation

Proteasome-mediated proteolysis is a primary protein degradation pathway in cells. The present study demonstrates that c-Abl and Arg (abl-related gene) tyrosine kinases associate with and phosphorylate the proteasome PSMA7 (alpha4) subunit at Tyr-153. Consequently, proteasome-dependent proteolysis is compromised. Notably, cells expressing a phosphorylation mutant of PSMA7(Y153F) display impaired G1/S transition and S/G2 progression, highlighting the biological significance of tyrosine phosphorylation of a proteasome subunit as an important cellular regulatory control.     

Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1α activity via the proteasome pathway

The calcineurin (CN) B subunit (CNB) is the regulatory subunit of CN, which is the only serine/threonine-specific protein phosphatase regulated by Ca²⁺/CaM. It has been shown to have potential as an anticancer agent, and has a positive effect on the phagocytic index and coefficient. We report here that CNB binds to proteasome subunit alpha type 7 (PSMA7) and inhibits the transactivation activity of hypoxia-inducible factor-1α (HIF-1α) via the proteasome pathway. In addition, we show that CNB represses the expression of vascular endothelial growth factor (VEGF), which is regulated by HIF-1α. These results indicate that CNB modulates cellular proteasome activity via a specific interaction with PSMA7. This may provide a molecular basis for its anticancer and antiviral activities.     

Molecular Cloning, Mapping, and Expression Analysis of the EIF4A2 Gene in Pig

A full-length cDNA clone encoding the eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), was cloned from the fetal skeletal cDNA library from the pig (Sus scrofa). EIF4A2 is a highly conserved gene for one of the protein-synthesis initiation factors involved in the binding of mRNA to the ribosome. Based on this cDNA sequence, the deduced protein of 407 amino acids contains the characteristic motifs shared by the DEAD-box supergene family. The genomic nucleotide sequence of this gene was determined and a single nucleotide polymorphism located in the 5′ untranslated region was genotyped. The porcine EIF4A2 was expressed in all tissues examined but in variable amounts. The EIF4A2 expression level in muscle was upregulated through embryonic and neonatal development until adult, suggesting that porcine EIF4A2 was possibly involved in translation regulation of other muscle-related genes in muscle formation and development. In addition, we mapped porcine EIF4A2 to q4.1 of SSC13, in agreement with comparative mapping data.     

Porcine alpha-1-antitrypsin (PI): cDNA sequence, polymorphism and assignment to chromosome 7q2.4- > q2.6

A cDNA clone encoding the complete coding sequence for porcine alpha-1-antitrypsin (or α₁-protease inhibitor, PI) was isolated and its DNA sequence determined. The cDNA is assumed to encode alpha-1-antitrypsin on the basis of its sequence similarity to the corresponding cDNAs for human, baboon, rat, mouse, sheep and cow. The porcine cDNA clone was used in conjunction with BamHI, KpnI, MspI, SacI, TuqI and XbaI to develop restriction fragment length polymorphism-based genetic markers for linkage mapping in pigs. The cDNA has also been used to map the porcine PI locus to chromosome 7q2.4- > q2.6 by radioactive in situ hybridization. Thus, the PI locus has been added to the developing physical and genetic maps of the porcine genome.     

X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex

The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.     

Chemical structure-dependent gene expression of proteasome subunits via regulation of the antioxidant response element

Antioxidants possess potent ability to regulate gene expression beyond their specific antioxidant activity. Genomic analysis reveals that three phenolic antioxidants, probucol, BO-653, and tBHQ, all of which have a phenoxyl group with one or two tert-butyl groups at the ortho-position, inhibit both the mRNA and protein levels of proteasome alpha-subunits in human endothelial cells. The chemical structure required for the gene regulation was studied by using derivatives of BO-653 and other antioxidants. It was found that the phenoxyl group and tert-butyl group at the ortho-position of the compounds were critical for down-regulation of the proteasome gene. Two antioxidant responsive elements (AREs) were identified in the promoter region of proteasome alpha subunit 3 (PSMA3). Results from promoter truncation analysis revealed that the proximal ARE region was necessary for the down-regulation of the expression of PSMA3. Electrophoretic mobility shift assays revealed that BO-653-mediated induction of DNA-binding to an upstream promoter region of PSMA3 containing the ARE motif was blocked by antibody against c-Jun but not Nrf2. These results indicate that the suppression of the proteasome alpha subunits expression by phenolic antioxidants is strictly dependent on both their chemical structure and the ARE consensus region in the promoter, which may be negatively regulated by AP-1.     

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.     

The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

The complete primary structure of mouse 20S proteasomes

 The proteasome is a large multicatalytic proteinase that plays a role in the generation of peptides for presentation by major histocompatibility complex class I molecules. The 20S proteolytic core of mammalian proteasomes is assembled from a group of 17 protein subunits that generate a distinctive pattern of spots upon two-dimensional gel electrophoresis. The genes for most of these subunits have been cloned from humans and rats. We isolated cDNA clones for the mouse orthologues of ten of the subunits [PSMA1 (C2), PSMA2 (C3), PSMA3 (C8), PSMA4 (C9), PSMA5 (ZETA), PSMA6 (IOTA), PSMA7 (C6-I), PSMB2 (C7-I), PSMB3 (C10-II), and PSMB5 (X)] to complete the cloning of all of the mouse subunits. Using antisera raised against these subunits or their orthologues, we verified the identity of these proteins by two-dimensional NEPHGE-PAGE. Received: 8 March 1999 / Accepted: 8 April 1999     

猪核糖体蛋白基因RPLP0的cDNA全长、染色体定位和多态性分析

RPLP0基因编码酸性核糖体磷蛋白大亚基P0, 是核糖体60S亚基的组成成分之一。从本室构建的猪胚胎骨骼肌cDNA文库中分离得到猪RPLP0基因的全长cDNA,并提交 GenBank 数据库。比较猪 RPLP0 基因和人及小鼠同源基因的cDNA序列和蛋白质序列,结果表明该基因在 3 个物种中具有高的相似性。用 PCR-RFLP 方法在猪 RPLP0基因cDNA 545处检测到 C→A 的单碱基突变,为 Csp6Ⅰ的酶切位点。统计分析结果表明 3 种基因型 (AA,AC,CC)在外来品种杜洛克,大约克, 长白和中国地方品种通城猪,小梅山,玉山猪中的分布各不相同。同时使用体细胞杂种板(SCHP)和辐射杂种板(IMpRH) 对 RPLP0 基因进行染色体定位,该基因被定位于 SSC 14q22-q24 并且和SW1321微卫星标志紧密连锁 (25cR, LOD = 14.54)。     

Measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in peripheral blood mononuclear cells: Optimisation and application to samples from a case-control study on cancers of the oesophagus and cardia

Antioxidants possess potent ability to regulate gene expression beyond their specific antioxidant activity. Genomic analysis reveals that three phenolic antioxidants, probucol, BO-653, and tBHQ, all of which have a phenoxyl group with one or two tert-butyl groups at the ortho-position, inhibit both the mRNA and protein levels of proteasome α-subunits in human endothelial cells. The chemical structure required for the gene regulation was studied by using derivatives of BO-653 and other antioxidants. It was found that the phenoxyl group and tert-butyl group at the ortho-position of the compounds were critical for down-regulation of the proteasome gene. Two antioxidant responsive elements (AREs) were identified in the promoter region of proteasome α subunit 3 (PSMA3). Results from promoter truncation analysis revealed that the proximal ARE region was necessary for the down-regulation of the expression of PSMA3. Electrophoretic mobility shift assays revealed that BO-653-mediated induction of DNA-binding to an upstream promoter region of PSMA3 containing the ARE motif was blocked by antibody against c-Jun but not Nrf2. These results indicate that the suppression of the proteasome α subunits expression by phenolic antioxidants is strictly dependent on both their chemical structure and the ARE consensus region in the promoter, which may be negatively regulated by AP-1.     

Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.     

Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.