We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.

Formin homology-2-domain containing protein 1 (FHOD1) regulates gene transcription, actin-cytoskeleton structure, and cell migration. To gain insight into the mechanisms by which FHOD1 mediates these diverse activities, a yeast-two-hybrid screen was performed to identify FHOD1-binding proteins. Three proteins specifically interacted with the carboxy-terminal two-thirds of FHOD1, which includes the FH1, FH2, and diaphanous activating domains (DAD). The newly identified FHOD1-binding proteins are protein kinase C binding protein 1 (PRKCBP1), cyclophilin B, and an isoform of WASP-interacting SH3-domain protein/diaphanous-interacting protein 1 (WISH/DIP1), named WISH-B. The proline-rich FH1 domain of FHOD1 was sufficient to interact with the central portion of PRKCP1 and full-length cyclophilin B. The FH1 domain also interacted with full-length WISH-B, but the extreme amino-terminus was sufficient to associate with WISH-B as well. WISH-B altered the solubility of FHOD1 in vitro and a truncation mutant containing the amino-terminal 227 residues of WISH-B disrupted FHOD1-induced stress fibers. WISH-B did not affect FHOD1-induced gene transcription through the serum response factor (SRF) recognition site on the skeletal alpha actin promoter (SkA). However, stabilization of F-actin prevented FHOD1 dependent activation of this promoter in presence of high, but not low serum concentrations. Thus, the identification of a new FHOD1-binding protein provides insight into the mechanisms by which FHOD1 regulates actin polymerization and transcription.

Dyggve-Melchior-Clausen syndrome (DMC), a severe autosomal recessive skeletal disorder with mental retardation, is caused by mutation of the gene encoding Dymeclin (DYM). Employing patient fibroblasts with mutations characterized at the genomic and, for the first time, transcript level, we identified profound disruption of Golgi organization as a pathogenic feature, resolved by transfection of heterologous wild-type Dymeclin. Collagen targeting appeared defective in DMC cells leading to near complete absence of cell surface collagen fibers. DMC cells have an elevated apoptotic index (P< 0.01) likely due to a stress response contingent upon Golgi-related trafficking defects. We performed spatiotemporal mapping of Dymeclin expression in zebrafish embryos and identified high levels of transcript in brain and cartilage during early development. Finally, in a chondrocyte cDNA library, we identified two novel secretion pathway proteins as Dymeclin interacting partners: GOLM1 and PPIB. Together these data identify the role of Dymeclin in secretory pathways essential to endochondral bone formation during early development.

We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.

We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.