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Competing Interests: Conflict-of-Interest Disclosures: Amy Abernethy reports personal fees from Verily/Alphabet, relationships with Georgiamune and EQRx, and personal investments in Iterative Health and One Health, outside the submitted work. Elliot Chaikof reports grants from the National Institutes of Health, outside the submitted work. George Q. Daley reports holding equity from Redona Therapeutics and from iTCells, outside the submitted work. Juan Enriquez reports investments with Excel Venture Management, outside the submitted work; investments in various life science technologies, including leading-edge brain technologies, and co-authoring a book on the impact of emerging brain technologies. Scott Gottlieb reports personal fees from Pfi zer, Inc, Illumina, Inc, Aetion, Tempus Labs, National Resilience, Inc, Cell-Carta, Parker Institute for Cancer Immunotherapy, Mount Sinai Health System, New Enterprise Associates, and American Enterprise Institute outside the submitted work. Sohail Tavazoie reports personal fees from Inspirna, outside the submitted work. Jacob S. Sherkow reports employment with the University of Illinois, grants from National Institutes of Health, and personal fees from Expert Consulting services, outside the submitted work.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12., Competing Interests: Competing interest statement: U.K.S., S.-J.L., M.E.S., B.R.B., K.R., M.Z., K.H.P., D.T.S., J.A.V.B., D.W.U., A.T.R., A.S.M., S.M., Z.W., M.S., K.K., J.P.M., E.P., D.C.G., R.M.P., S.A.T., and G.L.V. were employees of Warp Drive Bio, which has since become a wholly owned subsidiary of Revolution Medicines, Inc. G.L.V., R.D.K., and B.R.B. are minority shareholders in Revolution Medicines, Inc. R.D.K. is an employee of Lyell Immunopharma. U.K.S. is an employee of LifeMine Therapeutics. S.-J.L. is an employee of Beam Therapeutics. M.E.S. is an employee of C4 Therapeutics. B.R.B. is an employee of Inzen Therapeutics. K.R., D.T.S., K.K., J.P.M., and D.C.G. are employees of Ginko Bioworks. M.Z. is an employee of Kronos Bio. K.H.P. is an employee of A*STAR. A.S.M. is an employee of Agios Pharmaceuticals. S.M. is an employee of Morphic Therapeutic. Z.W. is an employee of Blueprint Medicines. M.S. is an employee of Bristol-Myers Squibb. E.P. is an employee of Amgen. S.A.T. is an employee of Kymera Therapeutics. G.L.V. is an employee of FOG Pharmaceuticals, Inc. and LifeMine Therapeutics., (Copyright © 2020 the Author(s). Published by PNAS.)

Curative therapies are most successful when cancer is diagnosed and treated at an early stage. We advocate that technological advances in next-generation sequencing of circulating, tumor-derived nucleic acids hold promise for addressing the challenge of developing safe and effective cancer screening tests., (Copyright © 2017. Published by Elsevier Inc.)

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.

von Hippel Lindau disease (VHL) is an autosomal dominant familial cancer syndrome linked to alteration of the VHL tumor suppressor gene. Affected patients are predisposed to develop pheochromocytomas and cystic and solid tumors of the kidney, CNS, pancreas, retina, and epididymis. However, organ involvement varies considerably among families and has been shown to correlate with the underlying germline alteration. Clinically, we observed a paradoxically lower prevalence of renal cell carcinoma (RCC) in patients with complete germline deletion of VHL. To determine if a relationship existed between the type of VHL deletion and disease, we retrospectively evaluated 123 patients from 55 families with large germline VHL deletions, including 42 intragenic partial deletions and 13 complete VHL deletions, by history and radiographic imaging. Each individual and family was scored for cystic or solid involvement of CNS, pancreas, and kidney, and for pheochromocytoma. Germline deletions were mapped using a combination of fluorescent in situ hybridization (FISH) and quantitative Southern and Southern blot analysis. An age-adjusted comparison demonstrated a higher prevalence of RCC in patients with partial germline VHL deletions relative to complete deletions (48.9 vs. 22.6%, p=0.007). This striking phenotypic dichotomy was not seen for cystic renal lesions or for CNS (p=0.22), pancreas (p=0.72), or pheochromocytoma (p=0.34). Deletion mapping revealed that development of RCC had an even greater correlation with retention of HSPC300 (C3orf10), located within the 30-kb region of chromosome 3p, immediately telomeric to VHL (52.3 vs. 18.9%, p <0.001), suggesting the presence of a neighboring gene or genes critical to the development and maintenance of RCC. Careful correlation of genotypic data with objective phenotypic measures will provide further insight into the mechanisms of tumor formation., (Copyright 2003 Wiley-Liss, Inc.)

Using current diagnostic criteria, primary mediastinal B cell lymphoma (PMBL) cannot be distinguished from other types of diffuse large B cell lymphoma (DLBCL) reliably. We used gene expression profiling to develop a more precise molecular diagnosis of PMBL. PMBL patients were considerably younger than other DLBCL patients, and their lymphomas frequently involved other thoracic structures but not extrathoracic sites typical of other DLBCLs. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients. Gene expression profiling strongly supported a relationship between PMBL and Hodgkin lymphoma: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in Hodgkin lymphoma cells. PDL2, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in Hodgkin lymphoma cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in Hodgkin lymphoma cell lines. The molecular diagnosis of PMBL should significantly aid in the development of therapies tailored to this clinically and pathogenetically distinctive subgroup of DLBCL.

A new collaborative model of research is needed to increase resources, to prioritize the R (ii) to increase the pace, reduce the overlap, and more systematically explore the elements of and delivery systems for vaccines; (iii) to use common standards for the prompt comparative testing of vaccine candidates; (iv) to expand resources for manufacturing vaccine candidates to speed their use in human trials; and (v) to increase the capacity for international clinical trials and to focus this effort toward quickly measuring the effectiveness of vaccine protection as prototype vaccine candidates are identified.

To identify potential molecular determinants of tumor biology and possible clinical outcomes, global gene-expression patterns were analyzed in the primary tumors of patients with metastatic renal cell cancer by using cDNA microarrays. We used grossly dissected tumor masses that included tumor, blood vessels, connective tissue, and infiltrating immune cells to obtain a gene-expression "profile" from each primary tumor. Two patterns of gene expression were found within this uniformly staged patient population, which correlated with a significant difference in overall survival between the two patient groups. Subsets of genes most significantly associated with survival were defined, and vascular cell adhesion molecule-1 (VCAM-1) was the gene most predictive for survival. Therefore, despite the complex biological nature of metastatic cancer, basic clinical behavior as defined by survival may be determined by the gene-expression patterns expressed within the compilation of primary gross tumor cells. We conclude that survival in patients with metastatic renal cell cancer can be correlated with the expression of various genes based solely on the expression profile in the primary kidney tumor.

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).

The heterodimeric Elongin BC complex has been shown to interact in vitro and in cells with a conserved BC-box motif found in an increasing number of proteins including RNA polymerase II elongation factor Elongin A, suppressor of cytokine signaling (SOCS)-box proteins, and the von Hippel-Lindau tumor suppressor protein. Recently, the Elongin BC complex was found to function as an adaptor that links these BC-box proteins to a module composed of Cullin family members Cul2 or Cul5 and RING-H2 finger protein Rbx1 to reconstitute a family of E3 ubiquitin ligases that activate ubiquitylation by the E2 ubiquitin-conjugating enzyme Ubc5. As part of our effort to understand the functions of Elongin BC-based ubiquitin ligases, we exploited a modified yeast two-hybrid screen to identify a mammalian BC-box protein similar in sequence to Saccharomyces cerevisiae Mediator subunit Med8p. In this report we demonstrate (i) that mammalian MED8 is a subunit of the mammalian Mediator complex and (ii) that MED8 can assemble with Elongins B and C, Cul2, and Rbx1 to reconstitute a ubiquitin ligase. Taken together, our findings are consistent with the model that MED8 could function to recruit ubiquitin ligase activity directly to the RNA polymerase II transcriptional machinery.

Background: The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival., Methods: Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index., Results: Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators., Conclusions: DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.

Renal cell carcinoma is associated with mutation of the von Hippel-Lindau (VHL) tumor suppressor gene. Cell lines derived from these tumors cannot exit the cell cycle when deprived of growth factors, and the ability to exit the cell cycle can be restored by the reintroduction of wild-type protein VHL (pVHL). Here, we report that cyclin D1 is overexpressed and remains inappropriately high in during contact inhibition in pVHL-deficient cell lines. In addition, hypoxia increased the expression of cyclin D1 specifically in pVHL-negative cell lines into which pVHL expression was restored. Hypoxic-induction of cyclin D1 was not observed in other pVHL-positive cell lines. This suggests a model whereby in some kidney cell types, pVHL may regulate a proliferative response to hypoxia, whereas the loss of pVHL leads to constitutively elevated cyclin D1 and abnormal proliferation under normal growth conditions.

The Cancer Molecular Analysis Project (CMAP) of the NCI is integrating diverse cancer research data to elucidate fundamental etiologic processes, enable development of novel therapeutic approaches, and facilitate the bridging of basic and clinical science.

Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

High-affinity iron uptake by a ferrous permease in the opportunistic pathogen Candida albicans is required for virulence. Here this iron uptake system has been characterized by investigating three distinct activities: an externally directed surface ferric reductase, a membrane-associated PPD (p-phenylenediamine) oxidase and a cellular ferrous iron transport activity. Copper was required for the PPD oxidase and ferrous transport activities. In contrast, copper was not required for iron uptake from siderophores. Addition of iron to the growth medium repressed ferric reductase and ferrous transport, indicating homeostatic regulation. To identify the genes involved, orthologous mutants of Saccharomyces cerevisiae were transformed with a genomic library of C. albicans. CFL95, a gene with sequence similarity to ferric reductases, restored reductase activity to the orthologous S. cerevisiae mutant. CaFTR2 and CaFTR1, genes with homology to ferrous permeases, conferred ferrous transport activity to the orthologous S. cerevisiae mutant. However, neither a genomic library nor CaFET99, a multicopper oxidase homologue and candidate gene for the PPD oxidase, complemented the S. cerevisiae mutant, possibly because of problems with targeting or assembly. Transcripts for CFL95, CaFTR1 and CaFET99 were strongly repressed by iron, whereas the CaFTR2 transcript was induced by iron. Deletion of the TUP1 regulator perturbed the homeostatic control of reductive iron uptake. Incidentally, iron starvation was noted to induce flavin production and this was misregulated in the absence of TUP1 control. The opposite regulation of two iron permease genes and the role of TUP1 indicate that the process of iron acquisition by C. albicans may be more complex and potentially more adaptable than by S. cerevisiae.

Deletions of 3p25, gains of chromosomes 7 and 10, and isochromosome 17q are known cytogenetic aberrations in sporadic renal cell carcinoma (RCC). In addition, a majority of RCCs have loss of heterozygosity (LOH) of the Von Hippel-Lindau (VHL) gene located at chromosome band 3p25. Patients who inherit a germline mutation of the VHL gene can develop multifocal RCCs and other solid tumors, including malignancies of the pancreas, adrenal medulla, and brain. VHL tumors follow the two-hit model of tumorigenesis, as LOH of VHL, a classic tumor suppressor gene, is the critical event in the development of the neoplastic phenotype. In an attempt to define the cytogenetic aberrations from early tumors to late RCC further, we applied spectral karyotyping (SKY) to 23 renal tumors harvested from 6 unrelated VHL patients undergoing surgery. Cysts and low-grade solid lesions were near-diploid and contained 1-2 reciprocal translocations, dicentric chromosomes, and/or isochromosomes. A variety of sole numerical aberrations included gains of chromosomes 1, 2, 4, 7, 10, 13, 21, and the X chromosome, although no tumors had sole numerical losses. Three patients shared a breakpoint at 2p21-22, and three others shared a dicentric chromosome 9 or an isochromosome 9q. In contrast to the near-diploidy of the low-grade lesions, a high-grade lesion and its nodal metastasis were markedly aneuploid, revealed loss of VHL by fluorescence in situ hybridization (FISH), and contained recurrent unbalanced translocations and losses of chromosome arms 2q, 3p, 4q, 9p, 14q, and 19p as demonstrated by comparative genomic hybridization (CGH). By combining SKY, CGH, and FISH of multiple tumors from the same VHL kidney, we have begun to identify chromosomal aberrations in the earliest stages of VHL-related renal cell tumors. Our current findings illustrate the cytogenetic heterogeneity of different VHL lesions from the same kidney, which supports the multiclonal origins of hereditary RCCs. Published 2001 Wiley-Liss, Inc.

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL(-/-) RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL(-/-) RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-alpha (TGF-alpha), another VHL-regulated growth factor. We show that TGF-alpha mRNA and protein are hypoxia-inducible in VHL(-/-) RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL(-/-) RCC cells, TGF-alpha can also act as a specific growth-stimulatory factor for VHL(-/-) RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL(-/-) RCC cells, such as vascular endothelial growth factor and TGF-beta1, which do not stimulate RCC cell proliferation. A TGF-alpha-specific antisense oligodeoxynucleotide blocked TGF-alpha production in VHL(-/-) RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL(-/-) RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-alpha stimulates proliferation. These results suggest that the generation of a TGF-alpha autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer syndrome predisposing to a variety of tumor types that include retinal hemangioblastomas, hemangioblastomas of the central nervous system, renal cell carcinomas, pancreatic cysts and tumors, pheochromocytomas, endolymphatic sac tumors, and epididymal cystadenomas [W. M. Linehan et al., J. Am. Med. Assoc., 273: 564-570, 1995; E. A. Maher and W. G. Kaelin, Jr., Medicine (Baltimore), 76: 381-391, 1997; W. M. Linehan and R. D. Klausner, In: B. Vogelstein and K. Kinzler (eds.), The Genetic Basis of Human Cancer, pp. 455-473, McGraw-Hill, 1998]. The VHL gene was localized to chromosome 3p25-26 and cloned [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. Germline mutations in the VHL gene have been detected in the majority of VHL kindreds. The reported frequency of detection of VHL germline mutations has varied from 39 to 80% (J. M. Whaley et al., Am. J. Hum. Genet., 55: 1092-1102, 1994; Clinical Research Group for Japan, Hum. Mol. Genet., 4: 2233-2237, 1995; F. Chen et al., Hum. Mutat., 5: 66-75, 1995; E. R. Maher et al., J. Med. Genet., 33: 328-332, 1996; B. Zbar, Cancer Surv., 25: 219-232, 1995). Recently a quantitative Southern blotting procedure was found to improve this frequency (C. Stolle et al., Hum. Mutat., 12: 417-423, 1998). In the present study, we report the use of fluorescence in situ hybridization (FISH) as a method to detect and characterize VHL germline deletions. We reexamined a group of VHL patients shown previously by single-strand conformation and sequencing analysis not to harbor point mutations in the VHL locus. We found constitutional deletions in 29 of 30 VHL patients in this group using cosmid and P1 probes that cover the VHL locus. We then tested six phenotypically normal offspring from four of these VHL families: two were found to carry the deletion and the other four were deletion-free. In addition, germline mosaicism of the VHL gene was identified in one family. In sum, FISH was found to be a simple and reliable method to detect VHL germline deletions and practically useful in cases where other methods of screening have failed to detect a VHL gene abnormality.

Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B-elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.

The Mammalian Gene Collection (MGC) project is a new effort by the NIH to generate full-length complementary DNA (cDNA) resources. This project will provide publicly accessible resources to the full research community. The MGC project entails the production of libraries, sequencing, and database and repository development, as well as the support of library construction, sequencing, and analytic technologies dedicated to the goal of obtaining a full set of human and other mammalian full-length (open reading frame) sequences and clones of expressed genes.

Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.

The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein-protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can be used to express elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel-Lindau tumor-suppressor gene product (pVHL). Only when pVHL and elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with elongins B and C. These results reveal a striking structural resemblance of pVHL/elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.

Mutation of the von Hippel-Lindau tumor suppressor gene (vhl) causes the von Hippel-Lindau cancer syndrome as well as sporadic renal clear cell carcinoma. To pursue our study of the intracellular localization of VHL protein in relation to its function, we fused VHL to the green fluorescent protein (GFP) to produce the VHL-GFP fusion protein. Like VHL, VHL-GFP binds to elongins B and C and Cullin-2 and regulates target gene product levels, including levels of vascular endothelial growth factor and glucose transporter 1. VHL-GFP localizes predominantly to the cytoplasm, with some detectable nuclear signal. Inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB) causes VHL to be redistributed to the nucleus. A cellular fusion assay was used to demonstrate that inhibition of transcription induces a decrease in the nuclear export rate of VHL. The dependence of transcription for trafficking is lost with a deletion of exon 2, a region with a mutation causing a splice defect in the VHL gene in sporadic renal clear cell carcinoma. Addition of a strong nuclear export signal (NES) derived from the Rev protein results in complete nuclear exclusion and abrogates the redistribution of VHL-GFP-NES into the nucleus upon inhibition of transcription. Leptomycin B, which inhibits NES-mediated nuclear export, reverts the distribution of VHL-GFP-NES to that of VHL-GFP and restores sensitivity to actinomycin D and DRB. Uncoupling of VHL-GFP trafficking to transcription either by an exon 2 deletion or fusion to NES abolishes VHL function. We suggest that VHL function requires not only nuclear or cytoplasmic localization, but also exon 2-mediated transcription-dependent trafficking between these two cellular compartments.

Although iron is an essential nutrient, it is also a potent cellular toxin, and the acquisition of iron is a highly regulated process in eukaryotes. In yeast, iron uptake is homeostatically regulated by the transcription factor encoded by AFT1. Expression of AFT1-1(up), a dominant mutant allele, results in inappropriately high rates of iron uptake, and AFT1-1(up) mutants grow slowly in the presence of high concentrations of iron. We present evidence that when Aft1-1(up) mutants are exposed to iron, they arrest the cell division cycle at the G1 regulatory point Start. This arrest is dependent on high-affinity iron uptake and does not require the activation of the DNA damage checkpoint governed by RAD9. The iron-induced arrest is bypassed by overexpression of a mutant G1 cyclin, cln3-2, and expression of the G1-specific cyclins Cln1 and Cln2 is reduced when yeast are exposed to increasing amounts of iron, which may account for the arrest. This reduction is not due to changes in transcription of CLN1 or CLN2, nor is it due to accelerated degradation of the protein. Instead, this reduction occurs at the level of Cln2 translation, a recently recognized locus of cell-cycle control in yeast.

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.

YIpDCE1 (Dual Constitutive Expression), a novel Saccharomyces cerevisiae integrating plasmid, constitutively expresses two genes under the control of separate phosphoglycerol kinase promoters. YIpDCE1 contains the complete ADE2 gene which can be used as a marker for selecting integrants at mutant ade2 loci commonly present in laboratory yeast strains. The YIpDCE1 plasmid can be inserted into the ade2-101 locus of the HF7c strain used in two hybrid screens. Thus it could be useful for analysis of two hybrid interactions that occur in the context of additional protein components (e.g. modifying enzymes such as kinases or phosphatases, or multimeric complexes consisting of three or four distinct protein components). YIpDCE1 has been used to create strains simultaneously overexpressing the permease (FTR1) and oxidase (FET3) components of the yeast high-affinity iron uptake system. This confers constitutive high-affinity iron uptake on the transformed strains, bypassing the normal regulatory mechanisms.

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes affected individuals to the human VHL cancer syndrome and is associated with sporadic renal cell carcinomas (RCC) and brain hemangioblastomas. VHL-negative 786-0 RCC cells are tumorigenic in nude mice which is suppressed by the reintroduction of VHL. Remarkably, this occurs without affecting the growth rate and cell cycle profile of these cells in culture. The 786-0 cell line, like many cancer cells, fails to exit the cell cycle upon serum withdrawal. Here, it is shown that reintroduction of the wild-type VHL gene restores the ability of VHL-negative RCC cancer cells to exit the cell cycle and enter G0/quiescence in low serum. Both VHL-positive and VHL-negative RCC cells exit the cell cycle by contact inhibition. The cyclin-dependent kinase inhibitor, p27, accumulates upon serum withdrawal, only in the presence of VHL, as a result of the stabilization of the protein. We propose that the loss of wild-type VHL gene results in a specific cellular defect in serum-dependent growth control, which may initiate tumor formation. This is corrected by the reintroduction of wild-type VHL, implicating VHL as the first tumor suppressor involved in the regulation of cell cycle exit, which is consistent with its gatekeeper function in the kidney.

The iron responsive element (IRE) is a approximately 30 nucleotide RNA hairpin that is located in the 5' untranslated region of all ferritin mRNAs and in the 3' untranslated region of all transferrin receptor mRNAs. The IREs are bound by two related IRE-binding proteins (IRPs) which help control intracellular levels of iron by regulating the expression of both ferritin and transferrin receptor genes. Multi-dimensional NMR and computational approaches were used to study the structure and dynamics of the IRE RNA in solution. The NMR data are consistent with formation of A-form helical stem regions, a one-base internal bulge and a Watson-Crick C.G base-pair between the first and fifth nucleotides in the loop. A superposition of refined structures indicates that the conserved C in the internal bulge, and three residues in the six-nucleotide hairpin loop are quite dynamic in this RNA. The structural roles of the stems, the loop and the bulge in the function of the IRE RNA and in possible interactions with the iron regulatory protein are discussed., (Copyright 1997 Academic Press Limited.)

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.

Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the ccc2Delta strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel-Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/-) mice appeared phenotypically normal, VHL -/- mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL -/- embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL -/- mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL -/- embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.