Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Effect of alkyl side chain variation on the electron-transfer activity of ubiquinone derivatives

The effect of the alkyl side chain of the ubiquinone molecule on the electron-transfer activity of ubiquinone in mitochondrial succinate-cytochrome c reductase is studied by using synthetic ubiquinone derivatives that possess the basic ubiquinone structure of 2,3-dimethoxy-5-methyl-1,4-benzoquinone with different alkyl side chains at the 6-position. The alkyl side chains vary in chain length, degree of saturation, and location of double bonds. When a ubiquinone derivative is used as an electron acceptor for succinate-ubiquinone reductase, an alkyl side chain of six carbons is needed to obtain the maximum activity. However, when it serves as an electron donor for ubiquinol-cytochrome c reductase or as a mediator in succinate-cytochrome c reductase, an alkyl side chain of 10 carbons gives maximal efficiency. Introduction of one or two isolated double bonds into the alkyl side chain of the ubiquinone molecule has little effect on electron-transfer activity. However, a conjugated double bond system in the alkyl side chain drastically reduces electron-transfer efficiency. The effect of the conjugated double bond system on the electron-transferring efficiency of ubiquinone depends on its location in the alkyl side chain. When location is far from the benzoquinone ring, the effect is minimal. These observations together with the results obtained from photoaffinity-labeling studies lead us to conclude that flexibility in the portion of the alkyl side chain immediately adjacent to the benzoquinone ring is required for the electron-transfer activity of ubiquinone.     

ACE/ACE2 Ratio and MMP-9 Activity as Potential Biomarkers in Tuberculous Pleural Effusions

Objective: Pleural effusion is common problem, but the rapid and reliable diagnosis for specific pathogenic effusions are lacking. This study aimed to identify the diagnosis based on clinical variables to differentiate pleural tuberculous exudates from other pleural effusions. We also investigated the role of renin-angiotensin system (RAS) and matrix metalloproteinase (MMPs) in the pathogenesis of pleural exudates.Experimental design: The major components in RAS and extracellular matrix metabolism, including angiotensin converting enzyme (ACE), ACE2, MMP-2 and MMP-9 activities, were measured and compared in the patients with transudative (n = 45) and exudative (n = 80) effusions. The exudative effusions were come from the patients with tuberculosis (n = 20), pneumonia (n = 32), and adenocarcinoma (n = 28).Results: Increased ACE and equivalent ACE2 activities, resulting in a significantly increased ACE/ACE2 ratio in exudates, were detected compared to these values in transudates. MMP-9 activity in exudates was significantly higher than that in transudates. The significant correlation between ACE and ACE2 activity that was found in transudates was not found in exudates. Advanced analyses showed significantly increased ACE and MMP-9 activities, and decreased ACE2 activity in tuberculous pleural effusions compared with those in pneumonia and adenocarcinoma effusions. The results indicate that increased ACE and MMP-9 activities found in the exudates were mainly contributed from a higher level of both enzyme activities in the tuberculous pleural effusions.Conclusion: Interplay between ACE and ACE2, essential functions in the RAS, and abnormal regulation of MMP-9 probably play a pivotal role in the development of exudative effusions. Moreover, the ACE/ACE2 ratio combined with MMP-9 activity in pleural fluid may be potential biomarkers for diagnosing tuberculous pleurisy.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.