Expression of von Hippel-Lindau protein in normal and pathological human tissues.

To examine the localization of von Hippel-Lindau (VHL) protein in human tissues, we produced four novel monoclonal antibodies against human VHL protein. Western blot analysis revealed that two of these antibodies recognized the epitope in amino acid sequence 60-89 of the VHL protein and the others recognized sequences 54-60 and 189-213. In a wild-type VHL gene-transfected cell line, immunocytochemistry and immunoelectron microscopy demonstrated the intracytoplasmic localization of VHL protein, particularly in mitotic cells. In normal human tissues, VHL protein was detected immunohistochemically in epithelial cells covering the body surface and the alimentary, respiratory, and genitourinary tracts; in secretory epithelial cells of exocrine and endocrine organs; in parenchymal cells of visceral organs; in cardiomyocytes; in neurons in nervous tissue; in lymphocytes in lymphoid tissue; and in macrophages. In pathological specimens, VHL protein was expressed in VHL-related tumor, as well as in endothelial cells, fibroblasts, and pericytes, all of which are involved in active angiogenesis. These findings suggest that these monoclonal antibodies can be useful for various immunological assays and that the VHL protein plays fundamental roles in physiological and pathological situations, especially in neovascularization.

Cellular proteins that bind the von Hippel-Lindau disease gene product: mapping of binding domains and the effect of missense mutations.

The von Hippel-Lindau disease (VHL) gene is a novel tumor suppressor gene that plays a role in the pathogenesis of renal cell carcinomas and hemangioblastomas of the central nervous system. To begin an evaluation of the biological functions of the VHL gene product (pVHL), we prepared bacterial fusion protein between glutathione S-transferase and wild-type or mutant pVHLs. The fusion proteins were used to identify cellular proteins that bind to pVHL in vitro. Monkey kidney cells transfected with wild-type or mutant VHL cDNAs were used to identify cellular proteins that bind to pVHL in vivo. Wild-type pVHL consistently bound two cellular proteins with apparent molecular masses of 10 and 14 kilodaltons that were designated p10 and p14, respectively. Mapping studies with a panel of VHL deletion mutant proteins demonstrated that p10 and p14 bound to a 32-amino acid peptide located in the carboxy terminal portion of pVHL. Missense mutation located within this 32-amino acid peptide abrogated the ability of the VHL protein to bind p10 and p14. Of 67 VHL families with identified germline mutations, 42 families had mutations predicted to affect the p10/p14-binding region. Maintenance of the integrity of the p10/p14-binding region appears to be essential for cellular growth regulation by pVHL.

Release of the sigma subunit from Escherichia coli RNA polymerase transcription complexes is dependent on the promoter sequence.

The sigma subunit of bacterial RNA polymerase is required for the specific initiation of transcription at promoter sites. However, sigma is released from the transcription complex shortly after transcription is initiated, and elongation proceeds in the absence of sigma. In order to study the position of sigma release, we have developed a method to quantify the photoaffinity labeling produced by an aryl azide positioned at the leading (5'-) end of nascent RNA, as a function of the transcript length [Stackhouse, T.M., & Meares, C.F. (1988) Biochemistry 27, 3038-3045]. Here we compare photoaffinity labeling of transcription complexes containing three natural bacteriophage promoters (lambda PR, lambda PL, and T7 A1) and two recombinant constructs, A1/PR (T7 A1 promoter with the lambda PR transcribed region) and PR/A1 (lambda PR promoter with the T7 A1 transcribed region). Significant photoaffinity labeling of the sigma subunit was observed only on the templates containing the lambda PR promoter region, regardless of the sequence of the transcribed region. These results indicate the molecular interactions responsible for the position of sigma release from the transcription complex mainly involve the nucleotide sequence of the promoter region--rather than the transcribed region--of the DNA template. Further studies on transcription complexes containing the A1/PR and the PR/A1 templates were performed, using polyclonal antibodies against the holoenzyme or against the sigma subunit. These experiments corroborate the promoter dependence of sigma release. They also show a correlation between the release of sigma and stable binding of the transcript by the transcription complex.

Photoaffinity labeling of Escherichia coli RNA polymerase/poly[d(A-T)] transcription complexes by nascent RNA.

To elucidate the molecular interactions during transcription by Escherichia coli RNA polymerase, we have performed a quantitative analysis of the photoaffinity labeling produced by an aryl azide positioned at the leading (5') end of the nascent RNA. Macromolecular contacts on the path of RNA across the transcription complex containing the template poly[d(A-T)] are observed as a function of the length of the transcript. Quantitative analysis provides the percent yield of photoaffinity labeling in the transcription complex by each length of RNA. Significant yields are observed for DNA, the beta/beta' subunits (analyzed together), and the sigma subunit. The alpha subunit is not labeled under these experimental conditions. The DNA template is labeled by the leading ends of RNA molecules 5-18 bases long, with yields ranging from 1% to 6%. Photoaffinity labeling of poly[d(A-T)] is also observed for many transcript lengths longer than 18 nucleotides, but the yields are too low to quantitate. Labeling of the beta/beta' subunits occurs with approximately 50% yields for transcripts of lengths greater than or equal to 12 nucleotides; low but significant labeling yields of 1-8% by shorter RNAs (3-10 nucleotides) are observed. Labeling of the sigma subunit is detectable for transcripts from 7 to more than 19 nucleotides long; quantitative measurements were possible up to the 19-mer. The RNAs most likely to be photoattached to the sigma subunit are 9-12 nucleotides long, with a maximum photoaffinity labeling yield of 15% by the decanucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Folding of homologous proteins: conservation of the folding mechanism of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids.

The equilibrium and kinetic properties for the urea-induced unfolding of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids were compared to determine the role of protein folding in evolution. The parent proteins differ at 40 positions in the sequence of 268 amino acids, and the hybrids differ by up to 15 amino acids from the Escherichia coli alpha subunit. The results show that all the proteins follow the same folding mechanism and are consistent with a previously proposed hypothesis [Hollecker, M., & Creighton, T. E. (1983) J. Mol. Biol. 168, 409; Krebs, H., Schmid, F. X., & Jaenicke, R. (1983) J. Mol. Biol. 169, 619] that the folding mechanisms are conserved in homologous proteins. Analysis of the kinetic data suggests that the 15 positions at which the parent proteins differ in the amino folding unit, residues 1-188, do not play a role in a rate-limiting step in folding that has been previously identified as the association of the amino and carboxyl folding units [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T. S., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965]. One or more of the 25 positions at which the parent proteins differ in the carboxyl folding unit, residues 189-268, do appear to play a role in this same rate-limiting step.