Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.

Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.

Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae.

The stress response promoter element (STRE) confers increased transcription to a set of genes following environmental or metabolic stress in Saccharomyces cerevisiae. A lambda gt11 library was screened to isolate clones encoding STRE-binding proteins, and one such gene was identified as MSN2, which encoded a zinc-finger transcriptional activator. Disruption of the MSN2 gene abolished an STRE-binding activity in crude extracts as judged by both gel mobility-shift and Southwestern blot experiments, and overexpression of MSN2 intensified this binding activity. Northern blot analysis demonstrated that for the known or suspected STRE-regulated genes DDR2, CTT1, HSP12, and TPS2, transcript induction was impaired following heat shock or DNA damage treatment in the msn2-disrupted strain and was constitutively activated in a strain overexpressing MSN2. Furthermore, heat shock induction of a STRE-driven reporter gene was reduced more than 6-fold in the msn2 strain relative to wild-type cells. Taken together, these data indicate that Msn2p is the transcription factor that activates STRE-regulated genes in response to stress. Whereas nearly 85% of STRE-mediated heat shock induction was MSN2 dependent, there was significant MSN2-independent expression. We present evidence that the MSN2 homolog, MSN4, can partially replace MSN2 for transcriptional activation following stress. Moreover, our data provides evidence for the involvement of additional transcription factors in the yeast multistress response.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.

In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

HLH106, a Drosophila transcription factor with similarity to the vertebrate sterol responsive element binding protein.

We cloned a Drosophila homolog to the sterol responsive element binding proteins (SREBPs). In vertebrates, the SREBPs are regulated by a mechanism that involves cleavage of the protein that normally residues in the cellular membranes and translocation of the released transcription factor into the nucleus. Regulation of the Drosophila factor HLH106 apparently follows the same mechanism, and we find the full-length gene product in the membrane fraction and a shorter cross-reacting form in the nuclear fraction. This nuclear form, which may correspond to proteolytically activated HLH106, is abundant in the blood cell line mbn-2. The general domain structure of HLH106 is very similar to that in SREBP. HLH106 is expressed throughout development, and it is present at high levels in Drosophila cell lines. In contrast to the rat homolog, HLH106 transcripts are not more abundant in adipose tissue than in other tissues.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily.

We report the three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-1 is a member of the transforming growth factor beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in embryonic development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors.

Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.