Analysis of a 26-kb region linked to the Mhc in zebrafish: genomic organization of the proteasome component beta/transporter associated with antigen processing-2 gene cluster and identification of five new proteasome beta subunit genes.

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for beta subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-gamma. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became associated with it.     

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.     

Dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) of ectothermic tetrapods originated in multiple independent evolutionary events

The proteasome subunit beta type 8 gene (PSMB8) encodes one of the beta subunits of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex class I molecules. Dimorphic alleles of the PSMB8 gene, termed A and F types, based on the deduced 31st amino acid residue of the mature protein have been reported from various vertebrates. Phylogenetic analysis revealed the presence of dichotomous ancient lineages, one comprising the F-type PSMB8 of basal ray-finned fishes, and the other comprising the A-type PSMB8 of these animals and both the F- and A-type PSMB8 of Xenopus and acanthopterygians, indicating that evolutionary history of the PSMB8 dimorphism was not straightforward. We analyzed the PSMB8 gene of five reptile and one amphibian species and found both the A and F types from all six. Phylogenetic analysis indicated that the PSMB8 F type was apparently regenerated from the PSMB8 A type at least five times independently during tetrapod evolution. Genomic typing of wild individuals of geckos and newts indicated that the frequencies of the A- and F-type alleles are not highly biased in these species. Phylogenetic analysis of each exon of the reptile PSMB8 gene suggested interallelic sequence homogenization as a possible evolutionary mechanism for the apparent recurrent regeneration of PSMB8 dimorphism in tetrapods. An extremely strong balancing selection acting on PSMB8 dimorphism was implicated in an unprecedented pattern of allele evolution.     

Evolution of dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) in basal ray-finned fish

The proteasome subunit beta type 8 (PSMB8) gene encodes a catalytic subunit of immunoproteasome that plays a central role in the processing of antigenic peptides presented by major histocompatibility complex class I molecules. The A- and F-type alleles defined by the 31st amino acid residue determining cleaving specificity have been identified from ray-finned fish, amphibia, and reptiles. These two types show extremely long-term trans-species polymorphism in Polypteriformes, Cypriniformes, and Salmoniformes, suggesting the presence of very ancient lineages termed A and F. To elucidate the evolution of the PSMB8 dimorphism in basal ray-finned fish, we analyzed Pantodon buchholzi (Osteoglossiformes), seven species of Anguilliformes, and Hypomesus nipponensis (Osmeriformes). Both A and F lineage sequences were identified from P. buchholzi and H. nipponensis, confirming that these two lineages have been conserved by basal ray-finned fish. However, both the A- and F-type alleles found in Anguilliformes species belonged to the F lineage irrespective of their types. This apparently suggests that the A lineage was lost in the common ancestor of Anguilliformes, and recovery of the A type within the F lineage occurred in Anguilliformes. The apparent loss of the F lineage and recovery of the F type within the A lineage have already been reported from tetrapods and higher teleosts. However, this is the first report on the reverse situation and reveals the dynamic evolution of the PSMB8 dimorphism.     

Comparative genomic analysis of the major histocompatibility complex class I region in the teleost genus <Emphasis Type="Italic">Oryzias</Emphasis>

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. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the MHC class I region of the Japanese pufferfish (Fugu rubripes)

A BAC map of the Japanese pufferfish (Fugu) MHC class I region was constructed using a mixture of sequence scanning and sequence-tagged site mapping methodologies. The Fugu MHC class Ia genes are linked to genes which are found within the human classical MHC class II and extended class II regions, a situation which has been found in the MHC of all teleosts mapped so far. The 300-kb contig comprises 24 MHC-related genes and is bounded by six non-MHC genes, which are thought to represent an evolutionary breakpoint within the region. Comparative analysis with both human and zebrafish MHC maps indicates two blocks of genes (KNSL2, ZNF297, DAXX, TAPBP, FLOTILLIN; and PSMB8, PSMB10, PSMB9, ABCB3, FABGL, BRD2, COL11A2, RXRB) which have remained linked over 400 million years and may represent an ancestral arrangement of the vertebrate MHC. Zebrafish and Fugu diverged between 100-200 million years ago and differences exist between these two fish species. The position and number of MHC class Ia genes is not conserved between species, there is an inversion of a block of nine genes centering on the PSMB cluster, and additional genes are present in zebrafish coding for a transport-associated protein and a beta proteasome subunit. The extent of these differences has implications for the extrapolation of fish model organism data to commercial aquaculture species. The data presented here represent the most extensive analysis of a fish MHC class Ia region described so far and clearly delimit the extent of this region in Fugu and, potentially, all teleosts.     

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     

Enhanced Inflammatory Potential of CD4+ T-Cells That Lack Proteasome Immunosubunit Expression,in a T-Cell Transfer-Based Colitis Model

Proteasomes play a fundamental role in intracellular protein degradation and therewith regulate a variety of cellular processes. Exposure of cells to (pro)inflammatory cytokines upregulates the expression of three inducible catalytic proteasome subunits, the immunosubunits, which incorporate into newly assembled proteasome complexes and alter the catalytic activity of the cellular proteasome population. Single gene-deficient mice lacking one of the three immunosubunits are resistant to dextran sulfate sodium (DSS)-induced colitis development and, likewise, inhibition of one single immunosubunit protects mice against the development of DSS-induced colitis. The observed diminished disease susceptibility has been attributed to altered cytokine production and CD4⁺ T-cell differentiation in the absence of immunosubunits. To further test whether the catalytic activity conferred by immunosubunits plays an essential role in CD4⁺ T-cell function and to distinguish between the role of immunosubunits in effector T-cells versus inflamed tissue, we used a T-cell transfer-induced colitis model. Naïve wt or immunosubunit-deficient CD4⁺ T-cells were adoptively transferred into RAG1^−/− and immunosubunit-deficient RAG1^−/− mice and colitis development was determined six weeks later. While immunosubunit expression in recipient mice had no effect on colitis development, transferred immunosubunit-deficient T- cells were more potent in inducing colitis and produced more proinflammatory IL17 than wt T-cells. Taken together, our data show that modifications in proteasome-mediated proteolysis in T-cells, conferred by lack of immunosubunit incorporation, do not attenuate but enhance CD4⁺ T-cell-induced inflammation.     

Comparative genomic analysis of the proteasome β5t subunit gene: implications for the origin and evolution of thymoproteasomes

The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t subunit, designated PSMB11, was identified in the cartilaginous fish, the most divergent group of jawed vertebrates compared to the other jawed vertebrates, but not in jawless vertebrates or invertebrates. Interestingly, teleost fish have two copies of apparently functional PSMB11 genes, designated PSMB11a and PSMB11b, that encode β5t subunits with distinct amino acids in the S1 pocket. BLAST searches of genome databases suggest that birds such as chickens, turkey, and zebra finch lost the PSMB11 gene, and have neither thymoproteasomes nor immunoproteasomes. In mammals, reptiles, amphibians, and teleost fishes, the PSMB11 gene (the PSMB11a gene in teleost fish) is located next to the PSMB5 gene coding for the β5 subunit of the standard 20S proteasome, indicating that the PSMB11 gene arose by tandem duplication from the evolutionarily more ancient PSMB5 gene. The general absence of introns in PSMB11 and an unusual exon–intron structure of jawed vertebrate PSMB5 suggest that PSMB5 lost introns and duplicated in tandem in a common ancestor of jawed vertebrates, with PSMB5 subsequently gaining two introns and PSMB11 remaining intronless.     

Molecular cloning and expression analysis of two lipopolysaccharide-induced TNF-α factors (LITAFs) from rock bream,Oplegnathus fasciatus

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α factor (LITAF) plays an important role controlling the expression of TNF-α and the other cytokine genes in the presence of LPS. However, two LITAF homologues have not been characterized in fish. In this study, we cloned two distinct LITAF (RbLITAF1 and RbLITAF2) cDNAs from rock bream (Oplegnathus fasciatus) and characterized their expression profiles after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding regions of RbLITAF1 and RbLITAF2 cDNAs were 492 bp and 417 bp, encoding 153 and 138 amino acid residues, respectively. The genes consisted of a LITAF domain. RbLITAF1 was highly expressed in the spleen and heart of healthy rock bream, whereas RbLITAF2 was highly expressed in the gill, intestine and stomach. In spleen, the gene expression of RbLITAF1 and RbLITAF2 were increased until 5 days post-infection (dpi), and then decreased at 7 dpi. In kidney, E. tarda and RSIV infection led to induction of the RbLITAF1 gene at 1 dpi, RbLITAF2 gene was down-regulated after pathogen infection. These results suggest that RbLITAFs may be involved in the LITAF-mediated immune response and regulate systemic immune responses against pathogen infection.     

Regulation of immunoproteasome subunit expression in vivo following pathogenic fungal infection

The proteasome catalytic beta subunits LMP2, LMP7, and MECL-1 and two proteasome activator proteins, PA28 alpha and beta, are induced following exposure to IFN-gamma in vitro. Induction of these immunosubunits and the PA28 alpha/beta hetero-oligomer alters proteasome catalytic functions and specificity and enhances production of certain MHC class I epitopes. We sought to determine whether and to what extent proteasome subunit composition is regulated in vivo and to elucidate the mechanisms of such regulation. We analyzed basal expression levels of these inducible genes in normal, IFN-gamma-deficient, and Stat-1-deficient mice. Mice of all three genotypes display constitutive expression of the immunosubunits and PA28, demonstrating that basal expression in vivo is independent of endogenous IFN-gamma production. However, basal expression levels are reduced in Stat-1(-/-) mice, demonstrating a role for Stat-1 independent of IFN-gamma signaling. To demonstrate that IFN-gamma can induce these genes in vivo, mice were infected with Histoplasma capsulatum. Elevated expression of these genes followed the same time course as IFN-gamma expression in infected mice. IFN-gamma-deficient mice did not display elevated protein expression following infection, suggesting that other inflammatory cytokines produced in infected mice are unable to influence proteasome expression. Cytokines other than IFN-gamma also failed to influence proteasome gene expression in vitro in cell lines that had no basal expression of LMP2, LMP7, or MECL-1. Thus, both in vitro and in vivo data demonstrate that IFN-gamma is essential for up-regulation, but not constitutive expression, of immunoproteasome subunits in mice.     

Identification of the Immunoproteasome as a Novel Regulator of Skeletal Muscle Differentiation

While many of the molecular details of myogenesis have been investigated extensively, the function of immunoproteasomes (i-proteasomes) in myogenic differentiation remains unknown. We show here that the mRNA of i-proteasome subunits, the protein levels of constitutive and inducible proteasome subunits, and the proteolytic activities of the 20S and 26S proteasomes were significantly upregulated during differentiation of skeletal muscle C2C12 cells. Knockdown of the i-proteasome catalytic subunit PSMB9 by short hairpin RNA (shRNA) decreased the expression of both PSMB9 and PSMB8 without affecting other catalytic subunits of the proteasome. PSMB9 knockdown and the use of i-proteasome-specific inhibitors both decreased 26S proteasome activities and prevented C2C12 differentiation. Inhibition of the i-proteasome also impaired human skeletal myoblast differentiation. Suppression of the i-proteasome increased protein oxidation, and these oxidized proteins were found to be more susceptible to degradation by exogenous i-proteasomes. Downregulation of the i-proteasome also increased proapoptotic proteins, including Bax, as well as cleaved caspase 3, cleaved caspase 9, and cleaved poly(ADP-ribose) polymerase (PARP), suggesting that impaired differentiation is likely to occur because of significantly increased apoptosis. These results demonstrate for the first time that i-proteasomes, independent of constitutive proteasomes, are critical for skeletal muscle differentiation of mouse C2C12 cells.     

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

The grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.     

Identification of the causative gene of a transparent phenotype of juvenile red sea bream Pagrus major

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T₄) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T₄ immersion. Loss-of-function of duoxa by CRISPR–Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans.Subject terms: Agricultural genetics, Animal breeding     

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.