Family C1 cysteine proteases: Biological diversity or redundancy?

Recent progress in the identification and partial characterization of novel genes encoding cysteine proteases of the papain family has considerably increased our knowledge of this family of enzymes. Kinetic data available to date for this large family indicate relatively broad, overlapping specificities for most enzymes, thus inspiring a growing conviction that they may exhibit functional redundancy. This is also supported in part by phenotypes of cathepsin knockout mice and suggests that several proteases can substitute for each other to degrade or process a given substrate. On the other hand, specific functions of one particular protease have also been documented. In addition, differences in cellular distribution and intracellular localization may contribute to defining specific functional roles for some of these proteases

In vitro effects of 0 to 120 Grays of irradiation on bone viability and release of growth factors

Background: High dose radiation therapy is commonly used in maxillofacial surgeries to treat a number of head and neck tumors. Despite its widespread use, little information is available regarding the effects of irradiation on bone cell viability and release of growth factors following dose-dependent irradiation. Methods: Bone samples were collected from porcine mandibular cortical bone and irradiated at doses of 0, 7.5, 15, 30, 60 and 120 Grays. Thereafter, cell viability was quantified, and the release of growth factors including TGFβ1, BMP2, VEGF, IL1β and RANKL were investigated over time. Results: It was observed that at only 7.5Gy of irradiation, over 85 % of cells were non-vital and by 60 Gy, all cells underwent apoptosis. Furthermore, over a 7-fold decrease in VEGF and a 2-fold decrease in TGFβ1 were observed following irradiation at all tested doses. Little change was observed for BMP2 and IL1β whereas RANKL was significantly increased for all irradiated samples. Conclusions: These results demonstrate the pronounced effects of irradiation on bone-cell vitality and subsequent release of growth factors. Interestingly, the largest observed change in gene expression was the 7-fold decrease in VEGF protein following irradiation. Future research aimed at improving our understanding of bone following irradiation is necessary to further improve future clinical treatments

Essential Role for Cathepsin S in MHC Class II–Associated Invariant Chain Processing and Peptide Loading

AbstractDestruction of Ii by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complexes to the cell surface. The cysteine protease cathepsin S is highly expressed in spleen, lymphocytes, monocytes, and other class II–positive cells, and is inducible with interferon-γ. Specific inhibition of cathepsin S in B lymphoblastoid cells prevented complete proteolysis of Ii, resulting in accumulation of a class II–associated 13 kDa Ii fragment in vivo. Consequently, the formation of SDS-stable complexes was markedly reduced. Purified cathepsin S, but not cathepsin B, H, or D, specifically digested Ii from αβIi trimers, generating αβ–CLIP complexes capable of binding exogenously added peptide in vitro. Thus, cathepsin S is essential in B cells for effective Ii proteolysis necessary to render class II molecules competent for binding peptides

Development and Characterization of a Eukaryotic Expression System for Human Type II Procollagen

Background Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Results Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Conclusions Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation

Functional expression of human cathepsin S in Saccharomyces cerevisiae. Purification and characterization of the recombinant enzyme.

A cDNA encoding the human lysosomal cysteine proteinase cathepsin S precursor has been expressed in yeast using the pVT100-U expression vector containing the alpha-factor promoter. The procathepsin S gene was expressed either as a fusion protein with the pre-region or with the prepro-region of the yeast alpha-factor precursor gene. Following in vitro processing both constructs gave an identical active mature enzyme with a molecular weight of 24,000. After prolonged cultivation of the cells the recombinant protein is also found as an active proteinase in the culture supernatant. The precursor can be activated in vitro at pH 4.5 and 40 degrees C under reducing conditions. The in vitro activated enzyme has a 6-amino acid NH2-terminal extension when compared with the native bovine enzyme. The purified enzyme displays a bell-shaped pH activity profile with a pH optimum of 6.5 and pK values of 4.5 and 7.8. The isoelectric point of the recombinant human cathepsin S is between 8.3 and 8.6 and about 1.5 pH units higher than for the bovine enzyme. The kinetic data for several synthetic substrates and inhibitors reveal a preference for smaller amino acid residues in the binding subsites S2 and S3 of cathepsin S. Like the bovine enzyme, the recombinant human cathepsin S is characterized by a broader range of pH stability (pH 5-7.5) than cathepsins B and L

In vitro antiatherogenicity of extracts from Halimeda incrassata seaweed: antioxidant activity and smooth muscle cell migration studies

Aim: The aim of this work was to evaluate the in vitro atheroprotective potential of the seaweed Halimeda incrassata in smooth muscle cell migration and lipoprotein oxidation in relation to its antioxidant activity. Material and methods: Antioxidant activity was determinate by DPPH• radical scavenging assay and ORAC method. The inhibitory effect of the aqueous extract on LDL oxidation mediated by Cu2+ ions was determinate by TBARS and conjugated diene quantification. The effect of the seaweed aqueous extract on smooth muscle cell migration was evaluated in MOVAS-1 mouse aortic smooth muscle cell. Results: The inhibitory effect of the aqueous extract on lipoprotein oxidation mediated by Cu2+ was demonstrated. Seaweed extract caused dose-dependent inhibition of TBARS (IC50 = 0.8 mg/mL) and conjugated dienes formation. The seaweed had a high antioxidant activity in the assays performed. The activity could be related to the phenolic content of Halimeda incrassata. Conclusions: In summary, the results of this study represent a further step in the characterization of the atheroprotective action of Halimeda incrassata and indicate the seaweed could be used for a nutraceutical and/or phytoterapeutic application.Objetivos: El objetivo de este trabajo fue evaluar el potencial ateroprotector in vitro del alga Halimeda incrassata en la migración de células de músculo liso de ratón y la oxidación de lipoproteínas en relación con su actividad antioxidante. Material y métodos: La actividad antioxidante fue determinada mediante los métodos de inhibición de radicales DPPH y la Capacidad antioxidante total (ORAC). La actividad inhibitoria de la oxidación de LDL mediada por iones Cu2+ se determinó por la cuantificación de TBARS y dienos conjugados. El efecto del extracto acuoso sobre la migración de las células de músculo liso se evaluó en la línea de células de músculo liso aórtica de ratón MOVAS-1. Resultados: Se demostró el efecto inhibidor del extracto sobre la oxidación de LDL mediada por Cu2+. El extracto del alga causa inhibición dosis-dependiente de la formación de TBARS (IC50 = 0,8 mg/mL) y dienos conjugados. Las algas tuvieron una alta actividad antioxidante en los ensayos realizados y podría estar relacionada con el contenido de compuestos fenólicos. Conclusiones: Los resultados de este trabajo representan un paso más en la caracterización de la acción ateroprotectora de Halimeda incrassata y evidencian sus posibles aplicaciones como nutracéutico y/o fitofármaco.The research was funded by IFS grant F/4897-1. Partial funding was also provided by CIHR grant MOP24447, the Canadian Research Chair award (D.B.) and a personal grant from GSEP, offered by the Canadian Bureau for International Education (A.C) and CNPq- (Brasil)

In vitro effects of 0 to 120 Grays of irradiation on bone viability and release of growth factors.

BACKGROUND High dose radiation therapy is commonly used in maxillofacial surgeries to treat a number of head and neck tumors. Despite its widespread use, little information is available regarding the effects of irradiation on bone cell viability and release of growth factors following dose-dependent irradiation. METHODS Bone samples were collected from porcine mandibular cortical bone and irradiated at doses of 0, 7.5, 15, 30, 60 and 120 Grays. Thereafter, cell viability was quantified, and the release of growth factors including TGFβ1, BMP2, VEGF, IL1β and RANKL were investigated over time. RESULTS It was observed that at only 7.5Gy of irradiation, over 85 % of cells were non-vital and by 60 Gy, all cells underwent apoptosis. Furthermore, over a 7-fold decrease in VEGF and a 2-fold decrease in TGFβ1 were observed following irradiation at all tested doses. Little change was observed for BMP2 and IL1β whereas RANKL was significantly increased for all irradiated samples. CONCLUSIONS These results demonstrate the pronounced effects of irradiation on bone-cell vitality and subsequent release of growth factors. Interestingly, the largest observed change in gene expression was the 7-fold decrease in VEGF protein following irradiation. Future research aimed at improving our understanding of bone following irradiation is necessary to further improve future clinical treatments

Chemoenzymatic synthesis of organoselenium(IV) compounds and their evaluation as cysteine protease inhibitors

A series of organoselenium dihalides (organoselenanes) was synthesized from organoselenides using a chemoenzymatic approach. The organoselenanes have variations in their stereochemistry and in the halogen atom bonded to the selenium atom. Because of the unique selenium-thiol chemistry displayed by several organoselenium compounds, the organoselenanes were evaluated as new potential inhibitors of cysteine proteases (cathepsins S and V). By the analysis of the second-order rate constants of the inhibition of cathepsin S and V, it was possible to conclude that organoselenanes inhibited the cathepsin S faster than cathepsin V. It was observed higher inhibitory potencies for the dibromo organoselenanes derivatives than the dichloro analogues. In addition, the present data suggest the use of hypervalent selenium compounds as novel motifs for cysteine proteases inhibitors.Uma série de organosselenanas foi sintetizada utilizando-se uma metodologia quimio-enzimática. Estas organosselenanas apresentam variações na estereoquímica e no halogênio ligado ao átomo de selênio. Devido à reação característica envolvendo compostos de selênio e tióis, estas organosselenanas foram avaliadas como inibidores de cisteíno proteases (catepsinas V e S). As constantes de inibição de segunda-ordem mostraram que a catepsina S é inibida mais rapidamente do que a catepsina V. Pode-se observar que as organosselenanas dibromadas são inibidores mais potentes do que as dicloradas. Desta forma, os resultados obtidos mostram que compostos hipervalentes de selênio podem ser aplicados como inibidores de cisteíno proteases

Taxane-containing induction chemotherapy followed by definitive chemoradiotherapy: Outcome in patients with locally advanced head and neck cancer

Background: Induction chemotherapy followed by definitive chemoradiotherapy is an intensified treatment approach for locally advanced squamous cell carcinoma of the head and neck (HNSCC) that might be associated with high rates of toxicity. Materials and methods: The data of 40consecutive patients who underwent induction chemotherapy with docetaxel-containing regimens followed by intensity-modulated radiotherapy (IMRT) and concomitant systemic therapy for unresectable locally advanced HNSCC were retrospectively analyzed. Primary objectives were RT-related acute and late toxicity. Secondary objectives were response to induction chemotherapy, locoregional recurrence-free survival (LRRFS), overall survival (OS), and influencing factors for LRRFS and OS. Results: The median follow-up for surviving patients was 21months (range, 2-53months). Patients received a median of three cycles of induction chemotherapy followed by IMRT to 72Gy. Three patients died during induction chemotherapy and one during chemoradiotherapy. Acute RT-related toxicity was of grade 3 and 4 in 72 and 3 % of patients, respectively, mainly dysphagia and dermatitis. Late RT-related toxicity was mainly xerostomia and bone/cartilage necrosis and was of grade 3 and 4 in 15 % of patients. One- and 2-year LRRFS and OS were 72 and 49 % and 77 and 71 %, respectively. Conclusion: Induction chemotherapy followed by chemoradiotherapy using IMRT was associated with a high rate of severe acute and late RT-related toxicities in this selected patient cohort. Four patients were lost because of fatal complications. Induction chemotherapy did not compromise the delivery of full-dose RT; however, the use of three cycles of concomitant cisplatin was impaire