Proteasome Subunit Alpha Type Research Articles

Calcineurin stimulates the expression of inflammatory factors in RAW 264.7 cells by interacting with proteasome subunit alpha type 6

Calcineurin is the only Ca2+-dependent serine/threonine-specific protein phosphatase and is considered a potential regulator of many intracellular signaling events. In this study we identified a novel interaction between calcineurin and the 20S proteasome subunit PSMA6 that increased intracellular proteasomal activity. Using RAW 264.7 macrophage cells, we demonstrated that expression of inflammatory factors was induced by calcineurin, and suppressed by the calcineurin inhibitor FK506. We also found that these calcineurin-activated processes result from activation of NF-κB, and that the interaction of calcineurin with PSMA6 stimulates transcription by NF-κB via degradation of IκB by the ubiquitin–proteasome pathway. These findings indicate that calcineurin is required for expression of inflammatory factors, and reveal a novel process of calcineurin-mediated activation of NF-κB.

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 Ca 2+/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.

Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on epigenetics and can help us to gain new insight into the time of departure as well as caste trajectory influencing elements at the molecular level.

Schistosoma japonicum: Cloning, expression and characterization of a gene encoding the α5-subunit of the proteasome

The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and CD 4 + cells were significantly higher ( P < 0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis.

Cryptosporidium parvum: Radiation-induced alteration of the oocyst proteome

Cryptosporidium parvum is a waterborne protozoan parasite that is found intracellularly in host animals, including humans, and causes severe diarrhea, which can lead to the death of an immunocompromised individual. Previously, we found that this organism is highly radioresistant as it can productively infect mice after exposure to a 10-kGy dose of γ-radiation. To understand how C. parvum avoids radiation damage, we characterized its protein expression patterns 6, 24, and 48 h after a 10-kGy dose of γ-radiation using two-dimensional PAGE. The gels showed 10 silver-stained spots that increased or decreased in size following γ-irradiation. Five proteins contained in these spots were identified using MALDI-TOF MS peptide fingerprinting, and two of these showed an increase in expression after γ-irradiation. These proteins were identified by LC–MS/MS as proteasome subunit alpha type 4 (NTN hydrolase fold) and thioredoxin peroxidase-like protein. The roles of these two upregulated proteins as related to the radioresistance of C. parvum remain to be evaluated.

Protein expression profiling of mouse thymoma cells upon exposure to the trichothecene deoxynivalenol (DON): Implications for its mechanism of action

The objective of this work was to investigate whether proteomic analysis of thymoma cells treated with the trichothecene deoxynivalenol (DON) as compared to non-treated (control) cells would reveal differential protein expression, and thus would contribute to a better understanding of the mechanisms of its toxicity. For that purpose the mouse thymoma cell line EL4 was exposed to 0.5 μM DON for 6 hr. A total of 30 proteins were affected after exposure of EL4 cells to DON. Most of these proteins were up-regulated and included key metabolic enzymes (e.g., fatty acid synthase, aldose reductase, carbamoyl phosphate synthetase, glucose-6-phosphate isomerase), chaperones (e.g., HSP9AB1 and HSP70), enzymes implicated in protein folding (PDI and ERO1-lα), and proteins involved in protein degradation (ubiquitin-conjugating enzyme (E1) and proteasome subunit alpha type-1). In addition, an IgE-binding protein with a molecular weight of 60 kDa and My-binding protein 1a (MYBBP1A), a transcription factor, were found to be up-regulated by DON. The observed up-regulation of MYBBP1A, a known repressor of a number of transcription factors such as PGC-1α, C-myb, and p65 of the NF-κB family, suggests that this protein might play a role in the mechanism of DON toxicity.

Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.)

Turmeric rhizome (Curcuma domestica Linn.) contains proteases and has proteolytic activity. Curcumin from turmeric rhizomes has been used for healing many ailments, including cancer. The purpose of this study was to purify turmeric protease and to research their biochemical characteristics [corrected]. Cysteine protease from C. domestica has been purified to homogeneity using acetone precipitation followed by preparatory native polyacrylamide gel electrophoresis (PAGE). This protocol resulted in six fold purification with 28% final recovery. The purified turmeric protease showed a prominent single peak and band on high-performance liquid chromatography and sodium dodecyl sulfate-PAGE, respectively, and an estimated molecular weight of 43 KDa, and exhibited optimal activity between 37 and 60 degrees C. The protease activity of the turmeric protease was significantly inhibited by iodoacetic acid. The turmeric protease had higher alanine and glutamate content and cleaved synthetic peptides N-Cbz-Ile-Pro and N-Cbz-Phe-Leu in a time-dependent manner. Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight mass spectroscopy revealed peptide matches to proteasome subunit alpha type 3 of Oryza sativa ssp. japonica (Rice). The turmeric protease showed antifungal activity at 10 microg mL(-1) towards pathogens Pythium aphanidermatum, Trichoderma viride and Fusarium sp. Cysteine addition significantly activated turmeric protease. The protease inhibition test suggested that turmeric protease belonged to the cysteine type. The biochemical characteristics of turmeric protease described in this paper can provide useful information for potential end uses of turmeric protease for pharmaceutical industry applications such as therapeutics.

Proteomic Analysis of the Response of Human Endothelial Cell Line EA.hy926 to 1800 GSM Mobile Phone Radiation

Background: We have earlier shown that exposure of human endothelial cell line EA.hy926 to 900 MHz GSM mobile phone radiation causes changes in the expres sion of numerous proteins. Here, we have examined the ef fects of 1800 MHz GSM mobile phone signal on the proteome of the same cell line. Results: EA.hy926 cells were exposed for one hour to 1800 MHz GSM signal, simulating mobile phone talkin g conditions, at an average specific absorption rate (SAR) of 2.0 W/kg at 37±0.3°C. Sham samples were produced simultaneously in the same conditions but without t he radiation exposure. Cells were harvested immediatel y after 1-hour exposure to the radiation, and proteins were extr a cted a nd sepa r a ted using 2-dimensiona l electrophoresis (2DE). In total, 10 experimental re plicates were generated from both exposed and sham samples. About 900 protein spots were detected in the 2DE-ge ls using PDQuest software and eight of them were found to be differentially expressed in exposed cells (p<0.0 5, t-test). Three out of these eight proteins were identified u sing Maldi-ToF mass spectrometry (MS). These proteins ar e: spermidine synthase (SRM), 78 kDa glucose-regulated protein (55 kDa fragment) (GRP78) and proteasome subunit alpha type 1 (PSA1). Due to the lack of the availability of commercial antibodies we were able to further examine expression of only GRP78. Using SDS - PAGE and western blot method we were not able to co nfirm the result obtained for GRP78 using 2DE. Additional ly, we have not seen any effect of 1800GSM exposure on the expression of vimentin and Hsp27 - proteins that we re affected by the 900 MHz GSM exposure in our earlier studies. Conclusions: Our results suggest that the 900GSM and 1800GSM exposures might affect the expression of so me proteins in the EA.hy926 cell line. The observed he re discrepancy between the expression changes of GRP78 detected with 1DE and 2DE confirms the importance o f validation of the results obtained with 2DE using o ther methods, e.g. western blot.

Molecular Characterization and Tissue Expression of Ovine PSAM6 Gene from Muscle Full-Length cDNA Library of Black-Boned Sheep

An ovine PSMA6 gene was obtained from muscle full-length cDNA library of black-boned sheep. The sequences for the PSAM6 gene of Romney sheep and Yunling black goat were also generated in this study. Sequence analysis revealed that nucleotide sequence of this gene was not homologous to any of the known sheep genes, and its open reading frame encodes a protein that contains the putative conserved domain of proteasome subunit alpha type 6 (PSAM6). The nucleotide sequence had higher identity with other animals. However, one mutation of A to G at the site of 383 bp, leading to an amino acid mutation of Asn to Ser, was found only in the black-boned sheep. Tissue expression analysis indicated that this gene was generally expressed in most tissues and differently expressed in tissues of black-boned sheep. This the first report of the ovine PSAM6 gene.

Down-Regulation of 14-3-3 Isoforms and Annexin A5 Proteins in Lung Adenocarcinoma Induced by the Tobacco-Specific Nitrosamine NNK in the A/J Mouse Revealed by Proteomic Analysis

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in the A/J mouse model. Here we identified and validated, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry and immunoblotting, proteins that are differentially expressed in the lungs of mice treated with NNK versus vehicle control treatment. We also determined whether protein levels in the lungs of NNK-treated mice could be further modulated by the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC). The proteins identified in this study are SEC14-like 3, dihydropyrimidinase-like 2, proteasome subunit alpha type 5, annexin A5, 14-3-3 protein isoforms (theta, epsilon, sigma, and zeta), Rho GDP dissociation inhibitor alpha, myosin light polypeptide 6, tubulin-alpha-1, vimentin, Atp5b protein, alpha-1-antitrypsin, and Clara cell 10 kDa protein (CC10). Among those proteins, we demonstrated for the first time that 14-3-3 isoforms (theta, epsilon, and sigma) and annexin A5 were significantly down-regulated in mouse lung adenocarcinoma induced by NNK and were recovered by p-XSC. These proteins are involved in a variety of biological functions that are critical in lung carcinogenesis. Identification of these proteins in surrogate tissue in future studies would be highly useful in early detection of lung adenocarcinoma and clinical chemoprevention trials.

PSMA7, A Potential Biomarker of Diseases

Proteasome subunit alpha type 7(PSMA7) is an alpha-type subunit of the 20S proteasome core complex and participates in degrading proteins through ubiquitin-proteasome pathway (UPP) which plays an important role in the regulation of cell proliferation or cell cycle control, transcriptional regulation, immune and stress response, cell differentiation, and apoptosis. Previous studies have demonstrated that PSMA7 can be a target interacting with some important proteins involved in transcription factor regulation, cell cycle transition, viral replication and even tumor initiation and progression, suggesting that PSMA7 could be a potential target for the development of clinical diagnosis and new therapeutic drugs. Here, we review the recent studies on PSMA7 involved in many different cellular processes, ranging from the cell cycle process to antigen processing and tumorigenesis.

The −8 UTR C/G polymorphism of PSMA6 gene is associated with susceptibility to myocardial infarction in type 2 diabetic patients

Aims Controversial results have been provided regarding the potential role of proteasome subunit alpha type 6 gene ( PSMA6) in determining susceptibility to myocardial infarction (MI). In addition, no data in Caucasians have been provided. We aimed to: (a) validate in a Caucasian population the association between PSMA6 gene polymorphism and MI; (b) investigate the potential association between PSMA6 gene variants and MI in type 2 diabetics; (c) evaluate a potential functional role of PSMA6 allelic variants. Methods 224 subjects (73 patients with MI and 151 controls) were genotypized for variants of PSMA6 gene. In 36 patients, biopsy specimens were taken from the left ventricle area of presumed ischemia for ubiquitin–proteasome activity quantification. Results Allele frequency and genotype distribution of all PSMA6 gene polymorphisms studied did not differ between patients with MI and controls. When the analysis was restricted to type 2 diabetics ( n = 119), a different rs_1048990 C/G allele frequency and genotype distribution was found having diabetic subjects with MI ( n = 34) higher G allele frequency compared to controls ( n = 85), ( p = 0.02). Myocardial ubiquitin levels ( r = 0.75; p < 0.001) and proteasome 20S ( r = 0.7; p < 0.01) activity significantly correlated with plasma glucose even after adjusting for covariates. Type 2 diabetic subjects had higher myocardial ubiquitin levels ( p < 0.001) and proteasome 20S activity ( p < 0.001) compared to non-diabetics. Subjects carriers the allele G at rs_1048990 loci had higher myocardial ubiquitin levels and proteasome 20S activity. Conclusion PSMA6 rs_1048990 polymorphism may contribute to MI susceptibility in type 2 diabetes. Up-regulation of ubiquitin–proteasome pathway may be a potential mechanism for explaining such association.

Identification of proteins undergoing tyrosine phosphorylation during mouse sperm capacitation

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 beta chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function.

The hippocampal protein machinery varies over the estrous cycle

Information about estrous cycle (EC) and sex-dependent protein levels is limited. Cognitive functions vary over the EC and the aim of this study was to investigate rat protein fluctuations in the hippocampus, the main cognitive brain area for learning and memory, in the individual phases of the EC and in males and indeed protein fluctuations may reflect functional variation over the EC. Sprague-Dawley rats were used in the studies and estrous phases were determined. Hippocampi were taken, proteins extracted, run on 2-DE, and identified using MALDI-TOF/TOF and nano-LC-ESI-MS/MS; protein levels were quantified using Proteomweaver software. Levels of protein synthetic machinery components transcriptional activator protein PUR(α,β), elongation factor 2, heterogeneous nuclear ribonucleoprotein K, chaperones 78 kDa glucose-regulated protein, heat shock cognate 71 kDa protein, Hsp 105, stress-70 protein, peptidyl-prolyl cis-trans isomerase A, prefoldin subunit 2, T-complex protein 1 subunit alpha and subunit delta, and degradation principle proteasome subunit alpha type 1 and ubiquitin carboxyl-terminal hydrolase isozyme L1, were different between sex and phase of the EC. We suggest that differences in the protein synthetic, chaperoning, and degradation machinery indicate different function in the individual EC phases. Results herein are relevant for further design of studies in the hippocampus at the protein level and interpretation of previous studies because EC phases will have to be respected and taken into account.

Proteomic Analysis of Pycnogenol Effects in RAW 264.7 Macrophage Reveals Induction of Cathepsin D Expression and Enhancement of Phagocytosis

Pycnogenol, polyphenolic compounds extracted from the pine bark, is beneficial for human health. To understand more of its effects, the present study is to explore the protein expression pattern induced by pycnogenol in RAW 264.7 cells. Global analysis using two-dimensional gel electrophoresis indicated that treatment with pycnogenol induces upregulation of four proteins, whose identities were revealed by mass spectrometry as cathepsin D, keratinocyte lipid-binding protein, proteasome subunit alpha type 1, and annexin IV. The pycnogenol effect displayed a time- and concentration-dependent manner. Unlike pycnogenol, N-acetyl cysteine and vitamin C had no effect on cathepsin D expression. Further studies showed that cathepsin D induction is correlated with an increase of lysosomal staining and enhancement of phagocytosis. These results reveal the novel effects of pycnogenol on protein expression and phagocytic functions and illustrate the advantage of proteomics-based strategy in unveiling the molecular basis of phytochemicals.

Identification of Novel Molecular Candidates for Acute Liver Failure in Plasma of BALB/c Murine Model

In an effort to identify proteins involved in the disease process of acute liver failure (ALF), we investigated changes in the plasma proteome associated with d-galactosamine/lipopolysaccharide (GalN/LPS) treatment of BALB/c mice. The plasma samples from mice with ALF and control were screened for potential differences using two-dimensional electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectrometry or matrix associated laser desorption ionization-time-of-flight mass spectrometry. The expression levels of candidate protein named phosphatidylethanolamine binding protein (PEBP) in plasma and liver, brain tissues were confirmed by western blot and RT-PCR analyses. Results were confirmed in plasma samples of human beings. Seven proteins existed in plasma of GalN/LPS-treatment animals only but not in controls. They included PEBP, regucalcin, Cu/Zn superoxide dismutase, glyoxalase 1, malate dehydrogenase, proteasome subunit alpha type 1, and HPMS haptoglobin precursor. Two proteins, proteasome subunit alpha type 5 and apolipoprotein A-I precursor, were up-regulated by GalN/LPS, and one protein, HPMS haptoglobin precursor, was down-regulated by this treatment. Western blot analysis confirmed the results that PEBP protein levels increased significantly in plasma and liver tissues only in ALF mice, but not in surviving mice treated with GalN/LPS. Further analysis revealed that GalN/LPS also induced up-regulation of PEBP mRNA levels in liver tissues. Importantly, plasma obtained from ALF patients, but not from healthy volunteers or from hepatitis patients, also contained detectable levels of PEBP. The present study show that PEBP may be a potential plasma biomarker for ALF diagnosis and participate in the pathphysiological process of ALF.

Validation of the Association Between the Gene Encoding Proteasome Subunit .ALPHA. Type 6 and Myocardial Infarction in a Japanese Population

Recently, a large case-control study (2,851 cases and 2,592 controls) reported that a functional single nuclear polymorphism (SNP) in the proteasome subunit alpha type 6 gene (PSMA6) conferred a risk of myocardial infarction (MI) in a Japanese population. The SNP (exon 1, -8C/G) is located in the 5' untranslated region of exon 1, and the risk-conferring allele G appears to enhance the transcription of PSMA6, which may exaggerate inflammation through activation of nuclear factor-kappa beta protein. The frequency of the risk conferring genotype (GG) in cases was reported to be greater than that in controls (12.4% vs 8.9%). The purpose of the present study was to validate this observation in our study population. Subjects with MI (n=433) were recruited from the outpatient clinic of the National Cardiovascular Center. Control subjects (n=2,186) were recruited from the Suita study. The frequencies of the GG genotype did not significantly differ between the control (9.8%) and MI groups (10.6%). Moreover, this genotype was not associated with C reactive protein levels in the Suita study. However, the GG genotype was significantly associated with greater intima-media thickness (n=2,051, p=0.015) after adjusting for blood pressure, sex, body mass index and age in the Suita study. The reported genotype in PSMA6 appears not to contribute appreciably to MI, but may contribute slightly to atherosclerosis in the present study population.

Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteins

The nitric oxide-mediated Tyr-nitration of endogenous proteins is associated with several pathological and physiological processes. In order to investigate the presence – and potential roles – of Tyr-nitration in the human pituitary, a large-format two-dimensional gel separation plus a Western blot against a specific anti-3-nitrotyrosine antibody were used to separate and detect nitroproteins from a human pituitary proteome. The nitroproteins were subjected to in-gel trypsin digestion, and high-sensitivity vacuum matrix-assisted laser desorption/ionization (vMALDI) linear ion-trap tandem mass spectrometry was used to analyze the tryptic peptides. Those MS/MS data were used to determine the amino acid sequence and the specific nitration site of each tryptic nitropeptide, and were matched to corresponding proteins with Bioworks TuboSEQUEST software. Compared to our previous study, 16 new nitrotyrosine-immunoreactive positive Western blot spots were found within the area pI 3.0–10 and M r 10–100 kDa. Four new nitroproteins were discovered: the stanniocalcin 1 precursor—involved in calcium and phosphate metabolism; mitochondrial co-chaperone protein HscB, which might act as a co-chaperone in iron–sulfur cluster assembly in mitochrondria; progestin and adipoQ receptor family member III—a seven-transmembrane receptor; proteasome subunit alpha type 2—involved in an ATP/ubiquitin-dependent non-lysosomal proteolytic pathway. Those data demonstrate that nitric oxide-mediated Tyr-nitration might participate in various biochemical, metabolic, and pathological processes in the human pituitary.

A functional SNP in PSMA6 confers risk of myocardial infarction in the Japanese population

Inflammation is now considered critical in the pathogenesis of myocardial infarction. One of the mechanisms regulating the inflammatory process is the ubiquitin-proteasome system. We investigated whether variants of the 20S proteasome are associated with susceptibility to myocardial infarction and found a common SNP (minor allele frequency of 0.35) in the proteasome subunit alpha type 6 gene (PSMA6) conferring risk of myocardial infarction in the Japanese population (chi(2) = 21.1, P = 0.0000044, 2,592 affected individuals versus 2,851 controls). We replicated this association in another panel of myocardial infarction and control subjects, although its relevance to other ethnic groups remains to be clarified. The SNP, located in the 5' untranslated region of exon 1 in this gene, enhanced the transcription of PSMA6. Moreover, suppression of PSMA6 expression using short interfering RNA in cultured cells reduced activation of the transcription factor NF-kappaB by stabilizing phosphorylated IkappaB. Our results implicate this PSMA6 SNP as a previously unknown genetic risk factor for myocardial infarction.

Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry

The aim of this study was to characterize endogenous nitroproteins, and those proteins that interact with nitroproteins, in a human pituitary nonfunctional adenoma so as to clarify the role of protein nitration in adenomas. A nitrotyrosine affinity column (NTAC) was used to preferentially enrich and isolate endogenous nitroproteins and nitroprotein–protein complexes from a tissue homogenate that was prepared from a human pituitary nonfunctional pituitary adenoma. The preferentially enriched endogenous nitroproteins and nitroprotein–protein complexes were subjected to trypsin digestion, desalination, and tandem mass spectrometry analysis. Nine nitroproteins (Rho-GTPase-activing protein 5, leukocyte immunoglobulin-like receptor subfamily A member 4 precursor, zinc finger protein 432, cAMP-dependent protein kinase type I-beta regulatory subunit, sphingosine-1-phosphate lyase 1, centaurin beta 1, proteasome subunit alpha type 2, interleukin 1 family member 6, and rhophilin 2) and three proteins (interleukin 1 receptor-associated kinase-like 2, glutamate receptor-interacting protein 2, and ubiquitin) that interacted with nitroproteins were discovered. The nitration site of each nitroprotein was located onto the functional domain where nitration occurred, and each nitroprotein was related to a corresponding functional system. Those data indicate that protein nitration might be an important molecular event in the formation of a human pituitary nonfunctional adenoma.