No abstract

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc 2 155, making it a useful tool for mycobacterial genetics.The genus Mycobacterium comprises a wide range of species, from the pathogenic slow growers, such as the causative agents of tuberculosis and leprosy, to fast-growing mycobacteria found in the environment, such as Mycobacterium fortuitum. Traditionally, mycobacterial genetics has not been developed because of the pathogenicity and slow growth rate of Mycobacterium tuberculosis. The global increase in the incidence of tuberculosis, however, has been accompanied by an increase in molecular studies of mycobacteria, and although genetic systems are steadily being developed, there is still a need for more tools in this area.Several plasmids have been detected in mycobacteria from both environmental organisms and opportunistic pathogens (4,5,17). They are especially common in Mycobacterium avium complex isolates recovered from infected humans with pulmonary infections or AIDS and in Mycobacterium scrofulaceum and the M. fortuitum complex of both clinical and environmental isolates (3,10). Of all of the plasmids isolated, the most extensively studied is the M. fortuitum plasmid pAL5000 (14,21,25). This plasmid has been used as the basis for several other mycobacterial vectors (6,8,9). The other two mycobacterial plasmids whose replication regions have been defined and sequenced are the M. scrofulaceum plasmid pMSC262 (20) and the M. avium plasmid pLR7 (2). These two replicons were found to show homology in the replication region. Related plasmids have been detected throughout the mycobacteria by hybridization studies with pLR7 as a probe (5).Several insertion sequences have been isolated from mycobacteria (18), but only one composite transposon, Tn610, has been isolated. This transposon was isolated from M. fortuitum and contains a sulfonamide-resistant determinant and two copies of insertion sequence IS6100 which show homologies with the IS6 family elements. This is the only mycobacterial mobile element whose mechanism of transposition is a replicative process leading to the formation of cointegrates by duplication of one IS element in a direct orientation. Tn611 is a kanamycin-resistant derivative of Tn610 (16). Hybridization of transposition events shows ...

Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.

Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.