Serine proteinases in the turnover of the cartilage extracellular matrix in the joint: implications for therapeutics

Cartilage destruction is a key characteristic of arthritic disease, a process now widely established to be mediated by metzincins such as MMPs. Despite showing promise in preclinical trials during the 1990s, MMP inhibitors for the blockade of extracellular matrix turnover in the treatment of cancer and arthritis failed clinically, primarily due to poor selectivity for target MMPs. In recent years, roles for serine proteinases in the proteolytic cascades leading to cartilage destruction have become increasingly apparent, renewing interest in the potential for new therapeutic strategies that utilize pharmacological inhibitors against this class of proteinases. Herein, we describe key serine proteinases with likely importance in arthritic disease and highlight recent advances in this field

Induced mutations in the<em> TI1</em> gene encoding a major double-headed protease inhibitor in <em>Pisum sativum</em> L. can reduce significantly the inhibition of target enzymes

International audienceTrypsin / chymotrypsin inhibitors in the seeds of many legume crop species are regarded as antinutritional proteins often leading to a requirement for heat-treatment of seed products prior to their use in feed. A TILLING resource developed in Pisum sativum L. (pea) was exploited to identify mutants in the major seed-expressed trypsin / chymotrypsin inhibitor gene, TI1, where the inhibition of either or both of the target enzymes may be reduced. Three lines with missense mutations in TI1, predicted to affect activity through alteration of (a) a conserved cysteine residue, (b) the P1′ serine within the active site of the chymotrypsin inhibitory domain or (c) overall charge of the carboxy-terminal domain involved in protein dimerization, were identified. The mutants were back-crossed to the parent cultivar, Cameor, to generate mutant and wild type segregant lines, where the effects of the mutations on overall activity, isoform charge and the association properties of the encoded proteins could be examined and compared. The data show that one mutation (C77Y) leads to a significant reduction in both trypsin and chymotrypsin inhibitory activity, associated with the loss of activity of two TI1 isoforms. The second and third classes of TI1 mutations showed lesser effects on overall inhibitory activity, despite their likely impact on protein structure. The resource provides potential for providing novel germplasm with improved properties for feed and food uses

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates

Clinical manifestations of intermediate allele carriers in Huntington disease

Objective: There is controversy about the clinical consequences of intermediate alleles (IAs) in Huntington disease (HD). The main objective of this study was to establish the clinical manifestations of IA carriers for a prospective, international, European HD registry. Methods: We assessed a cohort of participants at risk with <36 CAG repeats of the huntingtin (HTT) gene. Outcome measures were the Unified Huntington's Disease Rating Scale (UHDRS) motor, cognitive, and behavior domains, Total Functional Capacity (TFC), and quality of life (Short Form-36 [SF-36]). This cohort was subdivided into IA carriers (27-35 CAG) and controls (<27 CAG) and younger vs older participants. IA carriers and controls were compared for sociodemographic, environmental, and outcome measures. We used regression analysis to estimate the association of age and CAG repeats on the UHDRS scores. Results: Of 12,190 participants, 657 (5.38%) with <36 CAG repeats were identified: 76 IA carriers (11.56%) and 581 controls (88.44%). After correcting for multiple comparisons, at baseline, we found no significant differences between IA carriers and controls for total UHDRS motor, SF-36, behavioral, cognitive, or TFC scores. However, older participants with IAs had higher chorea scores compared to controls (p 0.001). Linear regression analysis showed that aging was the most contributing factor to increased UHDRS motor scores (p 0.002). On the other hand, 1-year follow-up data analysis showed IA carriers had greater cognitive decline compared to controls (p 0.002). Conclusions: Although aging worsened the UHDRS scores independently of the genetic status, IAs might confer a late-onset abnormal motor and cognitive phenotype. These results might have important implications for genetic counseling. ClinicalTrials.gov identifier: NCT01590589

Optimization of adsorptive removal of α-toluic acid by CaO2 nanoparticles using response surface methodology

The present work addresses the optimization of process parameters for adsorptive removal of α-toluic acid by calcium peroxide (CaO2) nanoparticles using response surface methodology (RSM). CaO2 nanoparticles were synthesized by chemical precipitation method and confirmed by Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) analysis which shows the CaO2 nanoparticles size range of 5–15 nm. A series of batch adsorption experiments were performed using CaO2 nanoparticles to remove α-toluic acid from the aqueous solution. Further, an experimental based central composite design (CCD) was developed to study the interactive effect of CaO2 adsorbent dosage, initial concentration of α-toluic acid, and contact time on α-toluic acid removal efficiency (response) and optimization of the process. Analysis of variance (ANOVA) was performed to determine the significance of the individual and the interactive effects of variables on the response. The model predicted response showed a good agreement with the experimental response, and the coefficient of determination, (R2) was 0.92. Among the variables, the interactive effect of adsorbent dosage and the initial α-toluic acid concentration was found to have more influence on the response than the contact time. Numerical optimization of process by RSM showed the optimal adsorbent dosage, initial concentration of α-toluic acid, and contact time as 0.03 g, 7.06 g/L, and 34 min respectively. The predicted removal efficiency was 99.50%. The experiments performed under these conditions showed α-toluic acid removal efficiency up to 98.05%, which confirmed the adequacy of the model prediction