Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL. This region mediates the formation of a multiprotein VHL complex containing elongin B, elongin C, cul-2, and Rbx1. This VHL complex is thought to function as an E3 ubiquitin ligase. Here, we report that VHL proteins harboring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. In contrast, wild-type VHL proteins are directly stabilized by associating with both elongins B and C. In addition, elongins B and C are stabilized through their interactions with each other and VHL. Thus, the entire VHL/elongin complex is resistant to proteasomal degradation. Because the elongin-binding domain of VHL is frequently mutated in cancers, these results suggest that loss of elongin binding causes tumorigenesis by compromising VHL protein stability and/or potential VHL ubiquitination functions.

Major histocompatibility complex (MHC) class I KbDb −/− deficient mice possess functional CD8+ T cells and natural killer cells

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb −/− mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although β2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb −/− mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by β2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb −/− mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb −/− mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb −/− animals also have natural killer cells that retain their cytotoxic potential.

Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2 domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5′ end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5′ noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) in Saccharomyces cerevisiae, mutations in the four class C VPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPS gene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditional vps18 allele (vps18tsf) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition, vps18tsf cells accumulated multivesicular bodies, autophagosomes, and other membrane compartments that appear to represent blocked transport intermediates. Overproduction of either Vps16p or the vacuolar syntaxin homologue Vam3p suppressed defects associated with vps18tsf mutant cells, indicating that the class C Vps proteins and Vam3p may functionally interact. Thus we propose that the class C Vps proteins are components of a hetero-oligomeric protein complex that mediates the delivery of multiple transport intermediates to the vacuole.

Gene Conversion of Major Histocompatibility Complex Genes in the Mouse Spermatogenesis is a Premeiotic Event

The molecular genetic mechanism of gene conversion in higher eukaryotes remains unknown. We find it of considerable interest to determine when during spermatogenesis gene conversion occurs. We have therefore purified pachytene spermatocytes and haploid spermatocytes from adult mice and analyzed these fractions for the presence of gene conversion products resulting from the transfer between the major histocompatibility complex class II genes Ebd and Abk in a polymerase chain reaction assay. We have further isolated spermatogenic cells from prepubescent mice and analyzed them for the presence of the same gene conversion products. We can detect gene conversion products in testis cells as early as in 8-d-old mice where the only existing spermatogenic cells are spermatogonia. The frequency of gene conversion products remains the same as the cells reach meiosis in 18-d-old mice, and is unchanged after meiosis is completed in haploid spermatocytes. Gene conversion of this specific fragment therefore appears to be a premeiotic event and, consequently, relies on genetic mechanisms other than normal meiotic recombination.

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.

Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.

Synaptonemal complex (SC) component Zip1 plays a role in meiotic recombination independent of SC polymerization along the chromosomes.

Zip1 is a yeast synaptonemal complex (SC) central region component and is required for normal meiotic recombination and crossover interference. Physical analysis of meiotic recombination in a zip1 mutant reveals the following: Crossovers appear later than normal and at a reduced level. Noncrossover recombinants, in contrast, seem to appear in two phases: (i) a normal number appear with normal timing and (ii) then additional products appear late, at the same time as crossovers. Also, Holliday junctions are present at unusually late times, presumably as precursors to late-appearing products. Red1 is an axial structure component required for formation of cytologically discernible axial elements and SC and maximal levels of recombination. In a red1 mutant, crossovers and noncrossovers occur at coordinately reduced levels but with normal timing. If Zip1 affected recombination exclusively via SC polymerization, a zip1 mutation should confer no recombination defect in a red1 strain background. But a red1 zip1 double mutant exhibits the sum of the two single mutant phenotypes, including the specific deficit of crossovers seen in a zip1 strain. We infer that Zip1 plays at least one role in recombination that does not involve SC polymerization along the chromosomes. Perhaps some Zip1 molecules act first in or around the sites of recombinational interactions to influence the recombination process and thence nucleate SC formation. We propose that a Zip1-dependent, pre-SC transition early in the recombination reaction is an essential component of meiotic crossover control. A molecular basis for crossover/noncrossover differentiation is also suggested.

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

beta2 knockout mice develop parenchymal iron overload: A putative role for class I genes of the major histocompatibility complex in iron metabolism.

Hemochromatosis (HC) is an inherited disorder of iron absorption, mapping within the human major histocompatibility complex (MHC). We have identified a multigene system in the murine MHC that contains excellent candidates for the murine equivalent of the human HC locus and implicate nonclassical class I genes in the control of iron absorption. This gene system is characterized by multiple copies of two head-to-head genes encoded on opposite strands and driven by one common regulatory motif. This regulatory motif has a striking homology to the promoter region of the beta-globin gene, a gene obviously involved in iron metabolism and hence termed beta-globin analogous promoter (betaGAP). Upstream of the betaGAP sequence are nonclassical class I genes. At least one of these nonclassical class I genes, Q2, is expressed in the gastrointestinal tract, the primary site of iron absorption. Also expressed in the gastrointestinal tract and downstream of the betaGAP motif is a second set of putative genes, termed Hephaestus (HEPH). Based on these observations, we hypothesized that the genes that seem to be controlled by the betaGAP regulatory motifs would be responsible for the control of Fe absorption. As a test of this hypothesis, we predicted that mice which have altered expression of class I gene products, the beta2-microglobulin knockout mice, [beta2m(-/-)], would develop Fe overload. This prediction was confirmed, and these results indicate beta2m-associated proteins are involved in the control of intestinal Fe absorption.

The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells.

The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.

Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex.

cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. We have identified a new Sch. pombe gene, orp1+, that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homologue. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man.