Peptidyl-prolyl cis/trans isomerases (PPIases) are a conserved group of enzymes that catalyse the conversion between cis and trans conformations of proline imidic peptide bonds. These enzymes play critical roles in regulatory mechanisms of cellular function and pathophysiology of disease. There are three different classes of PPIases and increasing interest in the development of specific PPIase inhibitors. Cyclosporine A, FK506, rapamycin and juglone are known PPIase inhibitors. Herein, we review recent advances in elucidating the role and regulation of the PPIase family in vascular disease. We focus on peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1), an important member of the PPIase family that plays a role in cell cycle progression, gene expression, cell signalling and cell proliferation. In addition, Pin1 may be involved in atherosclerosis. The unique role of Pin1 as a molecular switch that impacts on multiple downstream pathways necessitates the evaluation of a highly specific Pin1 inhibitor to aid in potential therapeutic drug discovery.
SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.
FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confirm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specificity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with kcat/KM of 4.5 + 0.13 × 105 M−1 s−1, while the kcat/KM of active site mutant of D55A is 0.81 + 0.05 × 105 M−1 s−1. These values were considered comparable to kcat/KM obtained from the protease-coupling assay. Interestingly, the substrate specificities of Pk-FKBP35 obtained from both methods are also similar, with the preference of Pk-FKBP35 towards Xaa at P1 position was Leu>Phe>Lys>Trp>Val>Ile>His>Asp>Ala>Gln>Glu. Altogether, we proposed that protease-free and protease-coupling assays arereliable for Pk-FKBP35.
The purpose of this study was to investigate the association between the peptidyl-propyl-cis/trans isomerase 1 (PIN1) -842(G > C) and -667(T > C) polymorphic variants and breast cancer risk among Malaysian ethnic groups namely the Malays, Chinese and Indians, as well as clinico-pathological characteristics of the patients.
Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.