Molecular cloning and characterization of Aspergillus nidulans cyclophilin B.
Cyclophilins are an evolutionarily conserved family of proteins which serve as the intracellular receptors for the immunosuppressive drug cyclosporin A. Here we report the characterization of the first cyclophilin cloned from the filamentous fungus Aspergillus nidulans (CYPB). Sequence analysis of the cypB gene predicts an encoded protein with highest homology to the murine cyclophilin B protein. The sequence similarity includes an N-terminal sequence predicted to target the protein to the endoplasmic reticulum (ER) as well as a C-terminal sequence predicted to retain the mature protein in the ER. The bacterially expressed hexa-histidine tagged protein displays peptidyl-prolyl isomerase activity which is inhibited by cyclosporin A. In the presence of cyclosporin A, the expressed protein also inhibits purified calcineurin. When the endogenous cypB gene was disrupted and placed under the control of the regulatable alcohol dehydrogenase promoter, the strain demonstrated no detectable growth phenotype under conditions which induce or repress cypB transcription. Induction or repression of the cypB gene also did not effect sensitivity of A. nidulans to cyclosporin A. cypB mRNA levels were significantly elevated under severe heat shock conditions, indicating a possible role for the A. nidulans cyclophilin B protein during growth in high stress environments.
Joseph
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JD
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Heitman
J
J
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AR
D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001233:Aspergillus nidulans / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D019703:Calcineurin / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D021983:Cyclophilins; D016572:Cyclosporine / Q000494:pharmacology; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D020104:Immunophilins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000494:pharmacology
D000935:Antifungal Agents; D003470:Culture Media; D004271:DNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C081786:cyclophilin B; D013912:Threonine; D016572:Cyclosporine; D019703:Calcineurin; D021983:Cyclophilins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase; D005947:Glucose; D005990:Glycerol