Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.The FK506-binding protein FKBP65 is a member of the FK506-binding protein (FKBP) 2 class of immunophilins. Immunophilins are intracellular receptors of two immunosuppressant drugs cyclosporine A and FK506, and these receptors were divided into two classes in accordance with their binding ability of the immunosuppressant drugs. Proteins that bind to cyclosporine A are called the cyclophilins. FKBPs bind FK506 and are highly conserved and found in bacteria, yeast, and many tissues of higher eukaryotes. Almost every cellular compartment contains a member of this protein family. The FKBP class includes FKBP9, FKBP12, FKBP13, FKBP25, FKBP52, FKBP54, FKBP60, and FKBP65. All these proteins contain the binding motif for FK506. Both protein families have peptidylprolyl cistrans-isomerase (PPIase) activity.FKBP65 was first identified in mouse NIH3T3 fibroblasts (1), and it consists of four basic FKBP13 domains. It was originally thought that FKBP65 interacts with c-Raf-1 (2), in analogy to the function of FKBP52 in stabilizing the glucocorticoid receptor (3) or FKBP12 in stabilizing the ryanodine (4) or the inositol 1,4,5-triphosphate receptor (5). However, it was shown later that FKBP65 is a luminal rough endoplasmic reticulum (rER)-resident protein that co-localized with tropoelastin (6). It was suggested that the PPIase activity of FKBP65 is important for the folding of the proline-rich tropoelastin (7).The biosynthesis of collagens involves a large number of post-translational modifications in which many different rERresident proteins are involved (8). After the translocation of the growing polypeptide chains of procollagens into the rER, proline residues become 4-hydroxylated by prolyl 4-hydroxlase. 4-Hydroxylation of proline residues increases the stability of the triple helix and is a key element in the folding of the triple helix. Prolyl 4-hydroxylase requires an unfolded chain as a substrate. The chain selection and association for triple helix formation is determined by the C-terminal propeptides in fibrillar collagens. Premature association between procollagen chains is thought to be prevented by chaperones such as PDI, BiP/ GRP78, GRP94, and HSP47 and collagen modifying enzymes until the biosynthesis of the individual chain is completed. Additional modifications are the 3-hydroxylation of proline residues by the P3H1/CRTAP/cyclophilin B complex, the hydroxylation of lysine residues by lysyl hydroxylases and glycosylation. The chains are then selected, and trimers are for...

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the ␣ chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cistrans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1⅐CRTAP⅐CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1⅐CRTAP⅐CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1⅐CRTAP⅐CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

Background: C-terminal binding protein 1 (CtBP1) assembles into a tetrameric transcriptional co-repressor but how it directs gene expression is not clear. Results: CtBP1 requires NAD(H) for transition into multimers. Its biochemical activities are separable from transcriptional repression. Conclusion: Tryptophan 318 permits CtBP1 to first dimerize and then tetramerize after the binding of NAD(H). Significance: Clarification of how CtBP1 tetramerizes will permit development of CtBP inhibitors to target oncogenesis.

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The launch of the eagle-i Consortium, a collaborative network for sharing information about research resources, such as protocols and reagents, provides a vivid demonstration of the challenges that researchers, libraries and institutions face in making their data available to others.