Analysis of peptidase activities of a cathepsin B-like (TcoCBc1) from Trypanosoma congolense.

International audienceThe substrate specificity of TcoCBc1 was evaluated using two internally quenched fluorescent peptide libraries with randomized sequences designed to detect carboxydipeptidase (Abz-GXXZXK(Dnp)-OH) and endopeptidase (Abz-GXXZXXQ-EDDnp) activities at acidic and neutral pHs, respectively. All the data obtained with TcoCBc1 were compared with those of human cathepsin B, including the pH profiles of the hydrolytic reactions. The most relevant observation is the preference of TcoCBc1 for substrates with a pair of acidic amino acids at positions P(2) and P(1) for its carboxydipeptidase activity and the well acceptance for E and D at P(1) position for endopeptidase activity. These peculiar preferences for negatively charged groups of TcoCBc1 and its requirements for carboxydipeptidase activity were also observed on Abz labeled analogues of bradykinin (Abz-RPPG(↓)FSAFR-OH, Abz-RPPG(↓)FS(↓)AF-OH, Abz-RPPG(↓)DE(↓)AF-OH) and angiotensin I (Abz-DR(↓)VYIHAFHL-OH), where (↓) indicates the cleavage site. TcoCBc1 was modeled based on the atomic coordinates of the cathepsin B from Trypanosoma brucei and the positively charged environment in TcoCBc1 catalytic site contrasts with the negatively charged environment in human cathepsin B. The preferences of S1 and S2 subsites of TcoCBc1 for acidic amino acids have to be taken into consideration for future studies of physiological roles of TcoCBc1 as for instance in apoptotic processes of Trypanosoma congolense

Yellow fever virus NS2B/NS3 protease: Hydrolytic Properties and Substrate Specificity

Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P'1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the k(cat)/K-m of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. the catalytic significance of this homogeneity - a unique feature among worldwide prominent flavivirus - was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on k(cat)/K-m. the parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates. (C) 2011 Elsevier Inc. All rights reserved

Phenotypic and mutational spectrum of ROR2-related Robinow syndrome.

Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as the main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome