PSMB8 Research Articles

PSMB8 (LMP7) but not PSMB9 (LMP2) gene polymorphisms are associated to pigeon breeder's hypersensitivity pneumonitis

Hypersensitivity Pneumonitis (HP) is a lung inflammatory disorder caused by inhalation of organic particles by a susceptible host. However, only a small proportion of individuals exposed to HP-associated antigens develop the disease, suggesting that additional host/environmental factors may play a role. We have previously found that genetic susceptibility associated to the major histocompatibility complex (MHC) plays an important role in this disease. The low molecular weight proteosome (LMP, currently named PSMB) genes code for subunits of the proteosome, a multimeric enzymatic complex that degrades proteins into peptides in order to be presented in the MHC class I pathway. We hypothesized that polymorphisms in PSMB8 or PSMB9 genes could be involved in the susceptibility to HP. Thus, in this study we analyzed the polymorphic site at amino acid position 60 (Arg/His) of the fourth exon in the PSMB9 gene and the amino acid position 49 (Gln/Lys) in the second exon of PSMB8 gene in 50 Mexican patients with HP and 50 healthy ethnically matched controls. PSMB typing was performed using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Our results demonstrated that HP patients had a significant increase of the PSMB8 KQ genotype frequency (OR = 7.25, CI = 2.61-21.3; p = 0.000034). No differences were found in the distribution of PSMB9 alleles/genotypes. However, PSMB9-RH/PSMB8 KQ haplotype was significantly increased in HP patients (OR = 6.77, CI = 1.34-65.31, p < 0.02). These findings suggest that PSMB8 KQ genotype could increase the risk to develop hypersensitivity pneumonitis.

The Novel Proteasome Inhibitor 5-Amino-8-Hydroxyquinole (5AHQ) Overcomes Bortezomib Resistance in Malignant Hematological Cell Line Models Harboring Mutations in the PSMB5 Gene.

Abstract 940The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome, thereby interfering in the degradation of proteins involved in cell-cycling, NF-kB activation, apoptosis induction and micro-environmental interactions. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy.Previously we reported the development of three different BTZ-resistant cell line models, including the AML THP-1 cell-line (Oerlemans & Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, ASH 2008) after chronic exposure to stepwise increasing concentrations of BTZ. The aim of our current study was to further elucidate the molecular basis of BTZ resistance in these cell lines and investigate whether the resistant phenotype could be overcome by the second generation proteasome inhibitor 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008). When added to intact cells, cell extracts, or isolated rabbit reticulocyte proteasomes (gift of A Navon, Weizmann Institute of Science, Israel), 5AHQ inhibited the chymotrypsin-like enzymatic activity of the proteasome with IC50's of 6.9 ± 0.05 uM, 4.2 ± 0.6 uM, and 2.1 ± 0.18 μM, respectively.Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2-3 fold higher IC50 concentrations) and subsequently challenged to higher concentrations of BTZ (up to 500 nM) to provoke higher resistance levels. Sequencing of the PSMB5 gene, encoding for the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones. An overview of the mutation site in the PSMB5 gene, the amino acid position at which substitutions take place at the beta 5 subunit protein level, and the functional implication of each mutation are depicted in the table below.Cell lineMutationBTZ-resistant subcloneAmino acid changeFunctional effect on beta 5 subunit proteinTHP-1 (AML)NoneTHP1/BTZ7NoneG322ATHP1/BTZ30-200Ala49ThrBTZ binding pocketG322A G311TTHP1/BTZ500Ala49Thr Met45Ileenzyme-substrate interactions (hydrolysis of hydrophobic peptide bonds)CEM-C7 (ALL)G332TCEM/BTZ7Cys52Phesubstrate/inhibitor binding domainG332T C323TCEM/BTZ 30-200Cys52Phe Ala49ValBTZ binding pocket8226 (MM)None8226/BTZ7NoneA247G8226/BTZ14-30Thr21AlaBTZ binding pocketG322A8226/BTZ100Ala49ThrBTZ binding pocketSubsequently, we determined whether THP-1, CEM and 8226 cells with low and high levels of BTZ resistance would be sensitive towards the new proteasome inhibitor 5AHQ. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4-7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells.Together, these data indicate that in vitro selection of low and high levels of BTZ resistance in 3 hematological cell lines is accompanied by the introduction of multiple mutations of amino acids, all of which have key positions in BTZ binding within the active site of the proteasome beta 5 subunit. Notably, Ala49 mutations were independently observed in all 3 cell lines selected for BTZ concentrations > 100 nM. Knowledge of specific mutations in the PSMB5 gene that can confer BTZ resistance allows purposive screening for these types of mutations in clinical samples of BTZ refractory patients. In this respect, the notion that 5AHQ can bypass BTZ resistance related to single or multiple mutations in the PSMB5 gene, supports further preclinical development and clinical application of this drug. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw), The Leukemia and Lymphoma Society and the Ontario Institute for Cancer Research through funding from the Ministry of Research and Innovation, Province of Ontario. Disclosures:No relevant conflicts of interest to declare.

Amplification and overexpression of the PSMB5 gene contributes to bortezomib resistance in retreatment of patients with multiple myeloma

e19500 Background: The proteasome inhibitor Bortezomib is a novel anticancer drug that targets primarily the beta5 subunit (PSMB5) of the 26 S proteasome which has also demonstrated efficacy in re-treatment of almost half of relapsed Multiple Myeloma (MM) pts. Recent studies have identified qualitative and quantitative alterations at the level of PSMB5 subunit, which is located at 14q11, and cells that carry these alterations displayed a marked resistance to proteasome inhibition. We investigated the relevance of amplification and PSMB5 overexpression in the clinical outcome of pts with MM during retreatment with bortezomib. Methods: Between January and June 2008 pts with secretory MM on relapse were elegible for bortezomib re-treatment (1.3 or 1.0 mg/mq on days 1,4,8 and 11 of a 21 day cycle) if they had at least a partial remission to a bortezomib based on most recent treatment and a free interval of at least 6 months since the last dose. Bone marrow samples for morphologic analysis, mRNA expression levels of PSMB5, classical cytogenetics, FISH and in situ hybridization using a fluorescein-labeled probe to detect the amplification of PSMB5 gene, were obtained from 26 pts (16 male, 10 female mean age 64 years) before and during re-treatment with Bortezomib. Results: At the time of analyses all pts who received at least two cycles were evaluable. We identified 12 pts refractory to retreatment and five of these pts (3 males, 2 females, mean age 65 years) had a detectable amplification and overexpression of PSMB5 which interestingly appears rapidly following exposure to bortezomib and wanes with time off-therapy, even if it curiously reappeared rapidly after re-exposure to bortezomib. Conclusions: Amplification and overexpression of PSMB5 contributes to bortezomib resistance in clinical practice. These findings do highlight the susceptibility of proteasome units to genetic modifications under constant selective pressure which occur with continued treatment, furthermore, the drug resistance remains dormant but it rapidly revives upon re-exposure to bortezomib. No significant financial relationships to disclose.

Different mutants of PSMB5 confer varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the Jurkat cell line

To investigate the relationship between bortezomib resistance and mutations in the proteasome beta5 subunit (PSMB5) gene. Various bortezomib-resistant lymphoblastic lymphoma/leukemia lines were established by repeated cycles of bortezomib selection. Mutations were detected by sequencing the complementary DNA of the PSMB5 gene. Mutated clones were selected by limited dilution and cultured without bortezomib. Messenger RNA expression levels of PSMB5 in these mutated clones were measured by quantitative reverse transcription polymerase chain reaction. The degree of resistance was determined by cytotoxicity at various bortezomib concentrations. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin from the substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. In addition to the previously reported PSMB5 G322A mutant (Ala49Thr), a C323T mutant (Ala49Val), and G322A, C326T conjoined mutant (Ala49Thr and Ala50Val) were selected and clones containing these mutations (JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/C326T) were obtained. After being cultured without bortezomib for >2 months, no significant difference in PSMB5 messenger RNA levels was detected between these JurkatB cells and parental Jurkat cells. JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/C326T clones displayed 22.0-fold, 39.4-fold, and 66.7-fold resistance, respectively, to bortezomib compared to Jurkat cells. There were no significant differences between the chymotrypsin-like activities of these mutants and Jurkat cells. The inhibitory effect of bortezomib on chymotrypsin-like activity was the weakest in JurkatB-G322A/C326T cells, and the strongest in JurkatB-G322A cells, with JurkatB-C323T cells falling in between. Mutations of the PSMB5 gene resulting in substitutions of Ala49 and Ala50 of PSMB5 protein can confer varying bortezomib resistance.

Comparative genomic analysis of the major histocompatibility complex class I region in the teleost genus Oryzias

The major histocompatibility complex (MHC) class I region of teleosts harbors a tight cluster of the class IA genes and several other genes directly involved in class I antigen presentation. Moreover, the dichotomous haplotypic lineages (termed d- and N- lineages) of the proteasome subunit beta genes, PSMB8 and PSMB10, are present in this region of the medaka, Oryzias latipes. To understand the evolution of the Oryzias MHC class I region at the nucleotide sequence level, we analyzed bacterial artificial chromosome clones covering the MHC class I region containing the d- lineage of Oryzias luzonensis and the d- and N- lineages of Oryzias dancena. Comparison among these three elucidated sequences and the published sequences of the d- and N- lineages of O. latipes indicated that the order and orientation of the encoded genes were completely conserved among these five genomic regions, except for the class IA genes, which showed species-specific variation in copy number. The PSMB8 and PSMB10 genes showed trans-species dimorphism. The remaining regions flanking the PSMB10, PSMB8, and class IA genes showed high degrees of sequence conservation at interspecies as well as intraspecies levels. Thus, the three independent evolutionary patterns under apparently distinctive selective pressures are recognized in the Oryzias MHC class I region.

Dichotomous Haplotypic Lineages of the Immunoproteasome Subunit Genes, PSMB8 and PSMB10, in the MHC Class I Region of a Teleost Medaka, Oryzias latipes

Sequence comparison of the medaka, Oryzias latipes, major histocompatibility complex (MHC) class I region between two inbred strains, the HNI (derived from the Northern Population) and the Hd-rR (from the Southern Population), revealed a approximately 100 kb highly divergent segment encompassing two MHC class IA genes, Orla-UAA and Orla-UBA, and two immunoproteasome beta subunit genes, PSMB8 and PSMB10. To elucidate the genetic diversity of this region, we analyzed polymorphisms of the PSMB8 and PSMB10 genes using wild populations of medaka from three genetically different groups: the Northern Population, the Southern Population, and the China-West Korean Population. A total of 1,245 specimens from 10 localities were analyzed, and all the PSMB8 and PSMB10 alleles were classified into the N (fixed in the HNI strain) or the d (fixed in the Hd-rR strain) lineage. Polymerase chain reaction analysis of the region from PSMB8 to PSMB10 indicated that the two allelic lineages of these genes are segregating together constituting dichotomous haplotypic lineages. Both haplotypic lineages were identified in all three groups, although the frequency of d haplotypic lineage (73-100%) was much higher than that of N haplotypic lineage (0-27%) in all analyzed populations. The two allelic lineages of the PSMB8 gene showed curious substitutions at the 31st and 53rd residues of the mature peptide, which are likely involved in formation of the S1 pocket, suggesting that these alleles have a functional difference in cleavage specificity. These results indicate that the two medaka MHC haplotypic lineages encompassing the PSMB8 and PSMB10 genes are maintained in wild populations by a balancing selection.

Acquisition of Mutations in the PSMB5 Gene during Chronic Exposure of Human Acute Lymphoblastic Leukemia CCRF-CEM Cells to the Proteasome Inhibitor Bortezomib

Despite the good prognosis of children suffering from acute lymphoblastic leukemia (ALL), approximately 25% of the patients develop a relapse. The proteasome inhibitor bortezomib (BTZ) is a representative of a novel generation of therapeutic drugs that revealed promising activity in patients with multiple myeloma (MM) and certain types of lymphomas, and is being studied in the treatment of relapsed ALL. Whereas most research on the mechanism of action of BTZ has focused on MM or lymphoma, the present study was designed to examine the activity of BTZ in an ALL model and in particular to assess the propensity to develop resistance to BTZ after prolonged drug exposure. Previously, we have shown (Franke et al., ASH 2007) that BTZ-resistant subclones of the human T-ALL cell line CCRF-CEM could emerge relatively rapid (within 2–3 months) after chronic exposure to stepwise increasing concentrations (0.1–200 nM) of BTZ. These BTZ-resistant cells (CEM/BTZ) showed cross-resistance against other proteasome inhibitors while remaining completely sensitive to a broad spectrum of other anticancer drugs. Subsequent analysis of these BTZ-resistant subclones showed an upregulation of the catalytic proteasomal β-subunits at both mRNA and protein level. In the present study we further elucidated the molecular basis for BTZ-resistance in a panel of CEM/BTZ cells selected at 7 nM BTZ (CEM/BTZ7) up to 200 nM BTZ (CEM/BTZ200). Since the proteasome β5 subunit is the primary target of BTZ, we screened whether mutations in the PSMB5 gene could be involved in conferring resistance to BTZ. Indeed, sequencing of exon 2 of the PSMB5 gene for CEM/BTZ7 cells revealed a single G to T nucleotide shift at position 332 of this gene. In the mature and functional β5 proteasome subunit, this mutation introduces a Cys to Phe substitution at amino acid 52, residing in a highly conserved substrate/inhibitor binding domain of the β5 subunit. In this context, it was of interest to note that CEM/BTZ7 cells already displayed a marked level (28-fold) of cross-resistance to 4A6, a 6-mer peptide that specifically targets the proteasome β5 catalytic subunit, thus suggesting that this mutation is responsible for diminished inhibitor binding. Upon screening for mutations in CEM cells with higher BTZ resistance levels, an additional mutation in the PSMB5 gene was revealed in CEM/BTZ30 cells. Herein, an additional C to T nucleotide shift at position 323 of the PSMB5 gene was identified, introducing an Ala to Val amino acid substitution at amino acid 49 of the mature and functional β5 subunit protein. This amino acid also resides in a highly conserved substrate/inhibitor binding domain of the β5 subunit. Both Cys52Phe and Ala49Val mutations were retained, and no others revealed, in BTZ-resistant CEM cells up to CEM/BTZ200 cells. Of note, recently we have identified an Ala49Thr mutation in the BTZ binding domain of the proteasome β5 subunit in a BTZ-resistant subclone of the human acute myeloid leukemic cell line THP-1 (Oerlemans & Franke, Blood 2008). Moreover, an Ala49Thr mutation was also recently described in a BTZ-resistant Jurkat cell line (Lü et al., J Pharmacol Exp Ther 2008). Together, this study demonstrates that mutations in the PSMB5 gene are provoked relatively fast in ALL cells, already at low levels of BTZ resistance. Furthermore, given the notion that independent studies in 3 different leukemia cell line models demonstrated that BTZ-resistance is associated with a mutation introducing amino acid substitutions at position Ala49 in the highly conserved substrate/inhibitor binding domain of the β5 subunit, this position may be considered as a hot spot for mutations in the PSMB5 gene through which acquired resistance to BTZ may be facilitated. Analysis of mutational status of the PSMB5 gene in future clinical BTZ studies in ALL patients should further confirm the clinical relevance of our finding, and might be exploited in the development of next generation proteasome inhibitors, designed to overcome resistance.

Acquired Resistance to Bortezomib in Human RPMI-8226 Multiple Myeloma Cells: Molecular Characterization, Cross-Resistance with Other Proteasome Inhibitors but Marked Sensitization to Glucocorticoids

The proteasome inhibitor bortezomib (BTZ, PS-341, Velcade®) has proven to be an effective drug in the treatment of multiple myeloma (MM) patients, both in first line as in refractory disease. However, emergence of drug resistance, for which the molecular mechanism(s) remain elusive, can hamper its clinical efficacy. Recently, we showed (Oerlemans & Franke, Blood 2008) that resistance to BTZ in human THP1 acute myeloid leukemia cells was conferred by an acquired mutation in the PSMB5 gene. This mutation introduced an amino acid substitution in the highly conserved substrate binding pocket of the β5 subunit of the proteasome, which is the primary target of BTZ. In the present study we investigated whether chronic exposure to BTZ could provoke acquired resistance to BTZ in the human RPMI-8226 MM cell line model and whether a PSMB5 gene mutation would be responsible. To this end, 8226 cells were exposed in vitro to stepwise increasing concentrations of BTZ from 0.1 nM BTZ up to 7nM (8226/BTZ7) over a period of 6 months and up to 30nM (8226/BTZ30) over a period of 12 months. In parallel to these selective concentrations, RPMI-8226 cells were cultured without BTZ (8226/WT). Characterization of 8226/BTZ7 vs 8226/WT cells revealed:2.6-fold resistance to BTZ along with cross-resistance to other peptide-based proteasome inhibitors; MG132 (2.2-fold), MG262 (1.9-fold) and 4A6 (4.9 fold),unchanged sensitivity to other anti-MM drugs, including doxorubicin, melphalan and thalidomide,a marked gain in sensitivity for the glucocorticoids dexamethasone (IC50: 25 nM vs >10 uM, respectively) and prednisolone (IC50: 2 uM vs > 1000 uM, respectively),other than 8226/WT cells, no G2M cell cycle arrest after BTZ exposure in 8226/BTZ7 cells,no significant changes in mRNA and functional activity of the three dominant catalytic subunits of the proteasome; β1, β2 and β5, although at a protein level β5 expression was markedly increased (5–10 fold), andsequencing of the PSMB5 gene in 8226/BTZ7 and 8226/BTZ30 cells, and one clinical sample of plasma cells of a BTZ-resistant plasma cell leukemia patient, showed no evidence for PSMB5 mutations.Microarray experiments were initiated to further elicit possible molecular mechanism(s) of BTZ resistance in 8226/BTZ7 cells. Initial analysis showed upregulation of several genes involved in microenvironment interaction and plasma cell differentiation. In this context, Gene Set Enrichment Analysis (GSEA) showed enrichment in the 8226/BTZ7 for the gene sets related to CD138 upregulation and MYC upregulation, indicative for a more differentiated phenotype of 8226/BTZ7 cells. Consistently, immune phenotypic characterization showed a shift to a dominant CD138dim/CD45+/CD20dim/CD27dim cell population for 8226/WT cells grown in parallel with 8226/BTZ7, which retained a CD138+/CD45−/CD20−/CD27− marker profile. Collectively, these data indicate that, other than in acute leukemia, the onset of acquired resistance to BTZ in 8226 MM cells proceeds very slowly, is associated with the upregulation of a (non-mutated) β5 catalytic proteasome subunit, and goes along with a shift towards a more mature immune phenotype in combination with an enrichment of genes that support cell growth. Notwithstanding this fact, the marked sensitization of BTZ-resistant 8226 MM cells for glucocorticoids may further corroborate the use of these drugs in combination with BTZ.

Overexpression of the PSMB5 gene contributes to bortezomib resistance in T-lymphoblastic lymphoma/leukemia cells derived from Jurkat line

To study the mechanism of bortezomib resistance in JurkatB lines derived from T-lymphoblastic lymphoma/leukemia Jurkat line. Cytotoxicities of popular chemotherapeutic drugs to JurkatB cells were analyzed by trypan blue assay. Functional drug efflux in JurkatB cells was determined by flow cytometry utilizing daunorubicin and the expression of P-glycoprotein (P-gp) was detected by Western blot. mRNA expression levels of proteasome beta5 subunit (PSMB5) were measured by quantitation real-time reverse transcription polymerase chain reaction. In situ hybridization was performed to detect the amplification of PSMB5 gene. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin (AMC) from the substrate N-succinyl-Leu-Leu-Val-Tyr-AMC. Cytogenetic studies were performed using R-banded metaphases and fluorescence in situ hybridization (FISH) analysis. IkappaB-alpha levels were detected by Western blot. No cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide was found in JurkatB cells. No evidence of drug efflux was found in JurkatB cells and the expression of P-gp was negative. The PSMB5 mRNA was overexpressed in highly resistant JurkatB5 and JurkatB1 lines compared with parental Jurkat, corresponding well with the increase of chymotrypsin-like activity and a karyotype of i(14q). Amplification of PSMB5 gene was demonstrated by in situ hybridization and FISH. The decreased IkappaB-alpha level in JurkatB5 cells after bortezomib treatment indicating an upregulation of nuclear factor-kappaB (NF-kappaB) activity. The mechanism of bortezomib resistance is different from that of multidrug resistance. Overexpression of PSMB5 is an important mechanism for bortezomib resistance in JurkatB lines. NF-kappaB may play a critical role in evading the apoptotic effects.

Proteasome β Subunit Pharmacogenomics: Gene Resequencing and Functional Genomics

The proteasome is a multisubunit cellular organelle that functions as a nonlysosomal threonine protease. Proteasomes play a critical role in the degradation of proteins, regulating a variety of cellular processes, and they are also the target for antineoplastic proteasome inhibitors. Genetic variation in proteasome subunits could influence both proteasome function and response to drug therapy. We resequenced genes encoding the three active proteasome beta subunits using 240 DNA samples from four ethnic groups and the beta 5 subunit gene in 79 DNA samples from multiple myeloma patients who had been treated with the proteasome inhibitor bortezomib. Resequencing was followed by functional studies of polymorphisms identified in the coding region and 3'-flanking region (3'-FR) of PSMB5, the gene encoding the target for clinically useful proteasome inhibitors. Resequencing of 240 DNA samples identified a series of novel ethnic-specific polymorphisms that are not represented in public databases. The PSMB5 3'-FR 1042 G allele significantly increased transcription during reporter gene studies, observations confirmed by genotype-phenotype correlations between single nucleotide polymorphisms (SNP) in PSMB5 and mRNA expression in the 240 lymphoblastoid cell lines from which the resequenced DNA was obtained. Studies with patient DNA samples identified additional novel PSMB5 polymorphisms, including a SNP and an insertion in the 3'-FR. Reporter-gene studies indicated that these two novel polymorphisms might decrease transcription. These results show that nonsynonymous coding SNPs in the PSMB5 gene did not show significant effects on proteasome activity, but SNPs did influence transcription. Future studies might focus on regulatory region polymorphisms.

Point Mutation of the Proteasome β5 Subunit Gene Is an Important Mechanism of Bortezomib Resistance in Bortezomib-Selected Variants of Jurkat T Cell Lymphoblastic Lymphoma/Leukemia Line

To study the mechanism of acquired resistance to bortezomib, a new antitumor drug that is the first therapeutic proteasome inhibitor, we established a series of bortezomib-resistant T lymphoblastic lymphoma/leukemia cell lines, designated the JurkatBs, from the parental Jurkat line via repeated drug selection. There were no significant differences in the growth curves or colony formation between the JurkatB cells and parental Jurkat cells. The effects of bortezomib on cytotoxicity, cell cycle arrest, and induction of apoptosis were decreased in JurkatB cells compared with parental Jurkat cells. A mutation in the proteasome beta5 subunit (PSMB5) gene (G322A), which encodes an amino acid change from Ala to Thr at polypeptide position 108, was detected by sequencing full-length cDNA clones and direct polymerase chain reaction products of the PSMB5 gene. Bortezomib caused less inhibition of chymotrypsin-like activity in resistant cells. When the G322A mutant PSMB5 was retrovirally introduced into parental Jurkat cells, it conferred bortezomib resistance to these cells, resulting in decreased cytotoxicity, apoptosis, and inhibition of chymotrypsin-like activity. The predicted structure of A108T-mutated PSMB5 shows a conformational change that suggests decreased affinity to bortezomib. In short, the G322A mutation of the PSMB5 gene is a novel mechanism for bortezomib resistance.

Association of the Polymorphisms in the PSMA6 (rs1048990) and PSMB5 (rs2230087) Genes with Type 2 Diabetes in Korean Subjects

Background: The 26S ubiquitin-proteasome system (UPS) is a principal proteolytic pathway of intracellular molecules regulating apoptosis, cell cycle, cell proliferation or differentiation, infl ammation and etc. The recent study suggests that the rs1048990 (C/G) polymorphism of the proteasome subunit α type 6 (PSMA6) gene is associated with the increase of the risk of myocardial infarction by the dysregulation of IкB degradation. We hypothesized that 26S UPS is important in the development of insulin resistance and type 2 diabetes (T2DM) by controlling the degradation of IкB and insulin receptor substances as a substrate. We therefore investigated whether the rs1048990 (C/G) polymorphism of PSMA6 gene and the rs2230087 (G/A) polymorphism of proteasome subunit β type 5 gene (PSMB5), that is chymotrypsin-like protease determining the rate of proteolysis, are associated with susceptibility to T2DM in Korean subjects. Methods: We examined the polymorphisms of these genes in 309 diabetic subjects and 170 non-diabetic controls. The polymorphisms of rs1048990 (C/G) and rs2230087 (G/A) were genotyped by real-time PCR. Results: The frequency of the G allele of rs1048990 (C/G) and the A allele of rs2230087 (G/A) polymorphisms was significantly higher in diabetic patients (28% and 13%) compared to that in controls (13% and 1%; P = 0.000 and P = 0.000, respectively). Logistic regression analysis of the rs1048990 (C/G) polymorphism showed that the odds ratio (OR) (adjusted for age, smoking, waist circumference, fasting plasma glucose, systolic blood pressure, HDL-C, triglyceride, and total cholesterol) was 3.93 (95% confidence interval [CI], 2.35-6.59; P = 0.000) for the G allele and 5.09 (95% CI, 2.71-9.57; P = 0.000) for CG and GG genotype when compared with the CC genotype. Logistic regression analysis of the rs2230087 (G/A) polymorphism showed

Amplification and Mutation of PSMB5 Gene in Bortezomib Resistant Lymphoblastic Leukemia Cells Derived from Jurkat Line.

Bortezomib, the first selective proteasome inhibitor in this new category of anti-cancer drug, inhibits chymotrypsin-like activity sited at beta 5 subunit of proteasome (PSMB5). To study the mechanism of proteasome inhibitor resistance in tumor cells, we established a series of bortezomib resistant lymphoblastic leukemia cell lines, named JurkatBs, from Jurkat line by repeated drug induction. There were no significant differences observed in the growth curves, colony formation rates, or cell cycle distribution between the JurkatBs and Jurkat cells. However, the effects of bortezomib, namely cytotoxicity, cell cycle arrest at G2 phase and induction of apoptosis, were decreased significantly in JurkatBs cells. There were no significant differences of intracellular mean fluorescence intensities of daunorubicin between JurkatBs and Jurkat cells (P>0.05) after treated with daunorubicin. Accordingly, the JurkatBs showed no cross-resistance to anthracycline, alkaloid and topoisomerase inhibitor. Quantitative PCR analysis showed no significant overexpression of MDR1 gene in JurkatB1, JurkatB2, JurkatB5 cells in comparison with that of Jurkat cells (22.77±5.58, 1.17±0.23, 8.30±2.62 vs 1.00±0.50; P>0.05). P-gp expression analysis was also negative in JurkatBs and Jurkat cells by Western bloting. By using quantitative PCR, we found that the PSMB5 gene was significantly amplified in JurkatB1 (5.82±0.60) and JurkatB5 cells (6.78±1.21) in comparison with that of Jurkat cells (1±0.49)(P<0.001), but not in JurkatB2 cells(0.16±0.03, P=1.000). A specific chromosome abnormality, i(14q), was found in all JurkatB5 cells (highly resistant to bortezomib), 4/16 JurkatB1 cells (moderately resistant), but not in JurkatB2 cells (slightly resistant). This results suggested that i(14q) may be resulted from the amplification of PMSB5 gene, which is located at 14q11. The chymotrypsin-like activities, determined by measuring the release of the fluorescent AMC from the substrate N- Suc-Leu-Leu-Val- Tyr-AMC, increased significantly in JurkatB1 (relative activity 3.27±0.12) and JurkatB5 cells (5.75±0.22) in comparison with that in Jurkat cells (1.00±0.14; P<0.001), but not in JurkatB2 cells (0.92±0.09; P>0.05). These results were coincident with the amplication of PSMB5, which may partly elucidated the bortezomib resistance in JurkatB cells. We then cloned and sequenced the full-length cDNA product of the PSMB5 gene from JurkatBs and Jurkat cells. A mutation at position 322 (G322A) of PSMB5 gene, causing an an amino acid substitution (Ala108Thr), was found in all selected JurkatB clones. The inhibition of chymotrypsin-like activities in JurartB2 (39.66±2.89) and JurartB5 cells (1.71±3.51), incubated with 10nM bortezomib up to 18h, were decreased significantly in comparison with that of Jurkat cells (86.87±0.97; P<.001), suggesting a decreased binding affinity of bortezomib to the chymotrypsin-like active site caused by Ala108Thr in JurkatB cells, which may resulted in conformation change in β5 subunit. In conclusion, we established bortezomib resistant leukemia cell lines with a different mechanism from that of multi-drug resistance (MDR). Both amplification and G322A mutation of PSMB5 gene are the important mechanisms of bortezomib resistance, by increasing chymotrypsin-like activity, and decreasing binding affinity of bortezomib to the chymotrypsin-like site, respectively.

Cis- and Trans-Acting Elements Regulate the Mouse Psmb9 Meiotic Recombination Hotspot

In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases) called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.

Interchromosomal duplication of major histocompatibility complex class I regions in rainbow trout (Oncorhynchus mykiss), a species with a presumably recent tetraploid ancestry

Salmonid fishes are among the few animal taxa with a probable recent tetraploid ancestor. The present study is the first to compare large (>100 kb) duplicated genomic sequence fragments in such species. Two contiguous stretches with major histocompatibility complex (MHC) class I genes were detected in a rainbow trout BAC library, mapped and sequenced. The MHC class I duplicated regions, mapped by fluorescence in situ hybridization (FISH), were shown to be located on different metaphase chromosomes, Chr 14 and 18. Gene organization in both duplications is similar to that in other fishes, in that the class I loci are tightly linked with the PSMB8, PSMB9, PSMB10 and ABCB3 genes. Whereas one region, Onmy-IA, has a classical MHC class I locus (UBA), Onmy-IB encodes only non-classical class Ib proteins. The nucleotide diversity between the Onmy-IA and Onmy-IB noncoding regions is about 14%. This suggests that the MHC class I duplication event has occurred about 60 mya close to the time of an hypothesized ancestral tetraploid event. The present article is the first convincing report on the co-existence of two closely related MHC class I core regions on two different chromosomes. The interchromosomal duplication and the homology levels are supportive of the tetraploid model.

Natural Selection During Functional Divergence to LMP7 and Proteasome Subunit X (PSMB5) Following Gene Duplication

The LMP7 and PSMB5 genes were created through an ancient gene duplication event of their ancestral locus. These proteins contain an active site of proteolysis, and LMP7 replaces PSMB5 as a component of the 20S proteasome after stimulation of cells by interferon-gamma. Replacement of PSMB5 by LMP7 changes the profile of the products of 20S proteasome processing, predisposing digested peptides for transport to and display by the immune system. The purpose of this study is to investigate evolutionary forces influencing functional divergence between LMP7 and PSMB5 following duplication. Levels of synonymous and nonsynonymous substitution rates are estimated to infer differences in levels of natural selection. Estimates of substitution rates indicate that natural selection elevated rates of nonsynonymous substitution in LMP7 following gene duplication, whereas PSMB5 experienced an increase in substitution rate that was not likely due to diversifying natural selection following duplication. Following initial divergence, nearly neutral mutations have dominated gene evolution in both lineages. The LMP7 gene locus provides a rare example of a protein with specialized function arising from duplication and divergence of a housekeeping protein by way of natural selection.

Structures of two major histocompatibility complex class I genes of the rainbow trout (Oncorhynchus mykiss).

Here we describe two rainbow trout major histocompatibility complex (MHC) class I genes characterized from lambda phage genomic clones prepared from a single fish. Clone GC71 contains all exons except a leader peptide-encoding exon. An open reading frame is maintained, and thus the gene MhcOnmy-U71 could be expressed in this individual. The class I gene found on clone GC41 lacks exons encoding the leader peptide and cytoplasmic domain. This gene, MhcOnmy-U41p, is a pseudogene due to a deletion in the alpha(2) domain-encoding exon causing premature termination. Both the Onmy-U71 and Onmy-U41p genes are distinguished by long introns between the exons encoding the alpha(1) and alpha(2) domains. Clone GC41 also contains the 3' exons of the LMP7/ PSMB8 gene encoding the gamma-interferon-induced proteosome subunit of rainbow trout.

Sequencing of amphioxus PSMB5/8 gene and phylogenetic position of agnathan sequences

Reanalyses of vertebrate PSMB5 and PSMB8 genes were conducted with a newly obtained amphioxus sequence. The phylogenetic analysis indicated that the amphioxus sequence is an outgroup of both vertebrate PSMB5 and PSMB8 genes. The agnathan sequences were previously thought to be closer to PSMB5 than PSMB8 sequences. However, in this study the phylogenetic trees supported the clustering of agnathan sequences with PSMB8 rather than with PSMB5 sequences. In the alignment there are many sites in which the agnathan sequences are more similar to PSMB5 than PSMB8 sequences. Parsimony analysis showed that the amino acid pattern at these sites could be explained by one substitution on the ancestral branch leading to the jawed vertebrate PSMB8 cluster. The number of substitutions required at these sites was the same for different tree topologies with respect to the position of agnathans. The phylogenetic analyses of PSMB6/PSMB9 and PSMB7/PSMB10 genes indicated that all the interferon-γ-inducible forms have evolved two to three times faster than the housekeeping forms in the jawed vertebrate lineage. Hence although the agnathan sequences cluster with the PSMB8 sequences, they have a slower rate of evolution similar to that of the housekeeping forms, and may be functioning as housekeeping forms.

Structural analysis and chromosomal localization of the mouse Psmb5 gene coding for the constitutively expressed beta-type proteasome subunit.

The proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex class I molecules. Accumulated evidence indicates that, upon stimulation with interferon-gamma (IFN-gamma), three beta-type subunits, designated LMP2, LMP7, and PSMB10, are incorporated into the 20S proteasome by displacing the housekeeping beta-type subunits designated PSMB6, PSMB5, and PSMB7, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. In the present study, we determined the organization of the mouse gene Psmb5, coding for the PSMB5 subunit. Psmb5 is made up of three exons, spanning approximately 5 kilobases. Its exon-intron organization differs radically from those of the other IFN-gamma-regulated, beta-type subunit genes including Lmp7 with which Psmb5 is believed to share an immediate common ancestor. The structure of the mouse Psmb5 gene is identical to that of its recently characterized human counterpart. Thus, the unique organization of the gene coding for the PSMB5 subunit appears to have been established before mammalian radiation. As well as the Psmb5 gene, the mouse genome contains a processed pseudogene designated Psmb5-ps. Interspecific backcross mapping showed that Psmb5 maps close to the Gtrgal2 locus on chromosome 14 and that Psmb5-ps is located in the vicinity of the Psme3 locus on chromosome 11. These results were confirmed by fluorescent in situ hybridization analysis that localized Psmb5 to band C2 to proximal D1 of chromosome 14 and Psmb5-ps to band D of chromosome 11.