Reduction In Proteolytic Activity Research Articles

Multifunctional Casein-Based Wound Dressing Capable of Monitoring and Moderating the Proteolytic Activity of Chronic Wounds.

Every 1.2 s, a diabetic foot ulcer is developed, and every 20 s, one amputation is carried out in diabetic patients. Monitoring and controlling protease activity have been considered as a strategy for more efficient management of diabetic and other chronic wounds. This study aimed to develop a casein-based dressing that, by its disappearance, provides information about the activity of proteases and simultaneously harnesses proteolytic activity. Casein films were fabricated by using an aqueous solution, and heat treatment was successfully deployed as a green and clean approach to confer hydrolytic stability. Our results showed that casein-based films' mechanical characteristics, water absorption, and proteolytic stability could be controlled by the length of the heat treatment, which proved to be a useful tool. An increase in the treatment duration from 30 min to 3 h led to toleration of 2.4 times higher stress, 2 times lower water uptake, and 3.4 times higher proteolytic stability at examined conditions. Selected casein-based structures responded to Bacillus sp. bacteria's protease (BSP) and human neutrophil elastase (HNE) as representatives of bacterial and nonbacterial proteases found in the wounds at 10 and 200 ng mL-1 levels, respectively. The hydrolysis was accompanied by a 36% reduction in proteolytic activity measured by using a casein-based universal protease activity assay. The released casein fragments could scavenge 90% of the examined radicals. In-vitro cell culture studies showed that the hydrolysates were not cytotoxic, and the casein-based film had a favorable interaction with fibroblast cells, indicating its potential as a scaffold in the case that proteolytic activity would not be to the extent that causes its rapid disintegration. In general, these findings hold promise for applying the developed casein-based structure for detecting proteolytic activity without the need for any equipment, kits, or expertise and, more importantly, in a highly economical manner. In the case that the proteolytic activity would not be severe, it could also serve as a substrate for cell adhesion and growth; this would aid in the healing process.

Reduced lysosomal activity and increased amyloid beta accumulation in silica-coated magnetic nanoparticles-treated microglia.

Nanoparticles have been used in neurological research in recent years because of their blood-brain barrier penetration activity. However, their potential neuronanotoxicity remains a concern. In particular, microglia, which are resident phagocytic cells, are mainly exposed to nanoparticles in the brain. We investigated the changes in lysosomal function in silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]-treated BV2 murine microglial cells. In addition, we analyzed amyloid beta (Aβ) accumulation and molecular changes through the integration of transcriptomics, proteomics, and metabolomics (triple-omics) analyses. Aβ accumulation significantly increased in the 0.1μg/μl MNPs@SiO2(RITC)-treated BV2 cells compared to the untreated control and 0.01μg/μl MNPs@SiO2(RITC)-treated BV2 cells. Moreover, the MNPs@SiO2(RITC)-treated BV2 cells showed lysosomal swelling, a dose-dependent reduction in proteolytic activity, and an increase in lysosomal swelling- and autophagy-related protein levels. Moreover, proteasome activity decreased in the MNPs@SiO2(RITC)-treated BV2 cells, followed by a concomitant reduction in intracellular adenosine triphosphate (ATP). By employing triple-omics and a machine learning algorithm, we generated an integrated single molecular network including reactive oxygen species (ROS), autophagy, lysosomal storage disease, and amyloidosis. In silico analysis of the single triple omics network predicted an increase in ROS, suppression of autophagy, and aggravation of lysosomal storage disease and amyloidosis in the MNPs@SiO2(RITC)-treated BV2 cells. Aβ accumulation and lysosomal swelling in the cells were alleviated by co-treatment with glutathione (GSH) and citrate. These findings suggest that MNPs@SiO2(RITC)-induced reduction in lysosomal activity and proteasomes can be recovered by GSH and citrate treatment. These results also highlight the relationship between nanotoxicity and Aβ accumulation.

Inhibition constant and stability of tripeptide inhibitors of gut trypsin-like enzyme of the soybean pest Anticarsia gemmatalis.

Insects overcome the action of natural protease inhibitors(PIs) due to evolutionary adaptations through endogenous proteolysis and reprogramming proteases. Insect adaptations complicate the formulation of IP-based crop protection products. However, small peptides designed based on the active site of enzymes have shown promising results that could change this scenario. GORE1 and GORE2 are designed tripeptides that reduce the survival of Anticarsia gemmatalis when ingested orally. In this article, the stability and ability of the peptides to bind trypsin-like enzymes of A. gemmatalis were evaluated by molecular dynamics (MD) simulations. The ability of the peptides to inhibit trypsin-like enzymes in vivo was compared with the SKTI protein by feeding A. gemmatalis larvae at different concentrations, followed by an inhibition persistence assay. During the MD simulation of enzyme-ligand complexes, both peptides showed a small variation of root-mean-square deviation and root-mean-square fluctuation, suggesting that these molecules reach equilibrium when forming a complex with the trypsin-like enzyme. Furthermore, both peptides form hydrogen bonds with substrate recognition sites of A. gemmatalis trypsin-like enzyme, with GORE2 having more interactions than GORE1. Larvae of A. gemmatalis exposed to the peptides and SKTI showed a similar reduction in proteolytic activity, but the persistence of inhibition of trypsin-like enzyme was longer in peptide-fed insects. Despite their size, the peptides exhibit important active and substrate binding site interactions, stability during complex formation, and steadiness effects in vivo. The results provide fundamental information for the development of mimetic molecules and help in decision-making for the selection of delivery methods for larger-scale experiments regarding similar molecules.

Cuminaldehyde exhibits potential antibiofilm activity against Pseudomonas aeruginosa involving reactive oxygen species (ROS) accumulation: a way forward towards sustainable biofilm management.

Pseudomonas aeruginosa often causes various acute and chronic infections in humans exploiting biofilm. Molecules interfering with microbial biofilm formation could be explored for the sustainable management of infections linked to biofilm. Towards this direction, the antimicrobial and antibiofilm activity of cuminaldehyde, an active ingredient of the essential oil of Cuminum cyminum was tested against Pseudomonas aeruginosa. In this regard, the minimum inhibitory concentration (MIC) of cuminaldehyde was found to be 150μg/mL against the test organism. Experiments such as crystal violet assay, estimation of total biofilm protein, fluorescence microscopy and measurement of extracellular polymeric substances (EPS) indicated that the sub-MIC doses (up to 60µg/mL) of cuminaldehyde demonstrated considerable antibiofilm activity without showing any antimicrobial activity to the test organism. Moreover, cuminaldehyde treatment resulted in substantial accumulation of cellular reactive oxygen species (ROS) that led to the inhibition of microbial biofilm formation. To this end, the exposure of ascorbic acid was found to restore the biofilm-forming ability of the cuminaldehyde-treated cells. Besides, a noticeable reduction in proteolytic activity was also observed when the organism was treated with cuminaldehyde. Taken together, the results demonstrated that cuminaldehyde could be used as a promising molecule to inhibit the biofilm formation of Pseudomonas aeruginosa.

APPLICATION OF GALVANOENTEROSORPTION IN MULTIMODAL TREATMENT OF PATIENTS WITH THERMAL BURNS

Abstract. Aim – to investigate the influence of enterosorption and its combination with intra-tissue electrophoresis (ITE) of antibacterial preparations on the proteolytic and fibrinolytic activity of blood plasma in patients with common thermal burns.
 Material and methods. Enterosorbtion detoxification by enterosgel was used in the multimodal treatment of 28 patients with thermal burns of II-IV stages. The drug was prescribed from 2-3 days after burn for 7-14 days, depending on the severity of the injury and manifestations of the intoxication syndrome. In 24 patients the treatment was complemented by galvanization of burn wounds with the preliminary introduction of antibacterial drugs. ITE began with 3-4 days after burn, taking into account the sensitivity of the microflora to antibiotics. The comparison group was made up of 30 patients with similar areas and depths of thermal burns receiving traditional treatment.
 Results and discussion. It was discovered that the inclusion of enterosorption in multimodal therapy of burns diminished the level of unlimited proteolysis, favoured the increase of the level of blood plasma fibrinolytic activity. Its combination with ITE of antibacterial agents (galvanoenterosorption) ensured a higher therapeutic effect. Common deep thermal burns are accompanied by a significant increase in proteolytic and fibrinolytic activity (FA) of blood. Inclusion of enterosorbtion in the integrated therapy of burns provides reduction of proteolytic activity and maintenance of a high level of FA of plasma, which reduces the risk of thromboembolic complications.
 Conclusion. Combined use of enterosorption and ITE of antibacterial agents (galvanoentersorption) with thermal burns provides a higher therapeutic effect.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

BackgroundOcriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases.ResultsThe present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations.ConclusionsThe results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

Nanoparticle Based Delivery of Protease Inhibitors to Cancer Cells.

Proteases are involved in a variety of processes associated with tumor development and progression. Because of their integral role in extracellular matrix and basal lamina degradation they play important roles in cancer cell migration, invasion, angiogenesis and metastasis. They are also involved in cancer cell signaling, the epithelial-mesenchymal transition, the antitumor immune response, cell de-differentiation and cancer stem cell remodeling. Their involvement in pro-tumorigenic processes makes them interesting targets for anticancer therapy. The most promising are matrix metalloproteases, cysteine cathepsins, the urokinase-type plasminogen activator system and proteasome; these constitute the focus of this review. Several inhibitors have been developed for reducing their activities that are in different phases of development, with some already in clinical use. However, systemic delivery of protease inhibitors can result in undesired reduction of proteolytic activity in normal tissues, leading to adverse effects and limited therapeutic efficacy. This caveat can be circumvented by nanoparticle delivery systems that direct protease inhibitors specifically to cancer cells. In this article we review the current state of nanoparticle delivery systems for delivering protease inhibitors to cancer cells.

Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.

Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145–166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co‐eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:666–676, 2017

ЛАБОРАТОРНА ХАРАКТЕРИСТИКА РІДКОГО ЕНЗИМНОГО МИЙНОГО ЗАСОБУ «ЕНЗИМИЙ» ДЛЯ САНОБРОБКИ У МОЛОЧНІЙ ПРОМИСЛОВОСТІ

We have developed a liquid enzyme detergent «Enzymyi» for sanitization of CIP–installations on milk plants that includes potassium hydroxide, complexones, proteolytic enzyme, stabilizers, and distilled water.The previous laboratory studies showed that the peak activity of «Enzymyi» on milk proteins was observed at as high as 65.8% at 60°C. Its activity decrease with the rise of temperature above 60 °C due to denaturation of enzymes since the heat causes changes in the primary structure and conformation of enzyme. It is also established that at pH solutions of 8.35 units proteolytic activity increased 1.7 times (P ≤ 0.01) compared to 7.2 pH units and reached its maximum activity 65.8 – 64%. Further increase of pH solutions from 8.35 units to 8.6 units contributed to gradual inhibition of enzymes resulting in reduction of proteolytic activity to 0. Proteolytic activity of the studied sample of enzyme detergent at water hardness of 0.357 mg–eq/l also reached the maximum of 60.5% and at increasing water hardness from 0.714 to 1.071 mg– eq/l PA decreased by 25.4% (P ≤ 0,05), i.e. with increasing water hardness PA reduced respectively. The article presents the study results as regards foam generating ability, foam stability and surface tension of solutions. The liquid detergent enzyme «Enzymyi» in concentrations of 0.03; 0.05; 0.07; 0.1; 0.3; 0.5; 0.7; 1% at the temperatures 20 °C and 60 °C was used for study. The article presents the study results as regards foam generating ability, foam stability and surface tension of solutions. The liquid detergent enzyme «Enzymyi» in concentrations of 0.03; 0.05; 0.07; 0.1; 0.3; 0.5; 0.7; 1% at the temperatures 20 °C and 60 °C was used for study.The results showed that «Enzymyi» practically doesn’t develop foam generating ability at the concentration from 0.03 to 1%. Foam stability does not exceed the standard, thus no more than 50% of the solution in 5 minutes that allows using the available composition for circulation washing of dairy equipment without its dismounting.

Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37kDa, and optimum activity was achieved at pH 5.5 and 55°C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45°C for 60min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.

Characteristics of the effect of cestodes parasitizing the fish intestine on the activity of the host proteinases

The activity and spectrum of proteinases in the intestines of host fishes change upon infestation with cestodes. Serine proteinases are found to make a greater contribution to the total proteolytic activity. The reduction of proteolytic activity is associated with adsorption of the enzymes of the host on the surface of cestodes, and the increase in the activity is caused by the injury of the intestinal mucosa by the attachment apparatuses of cestodes. The inhibition of proteainase activity indicates the possible participation of microbiota enzymes in protein hydrolyses.

ATIVIDADE PROTEOLÍTICA DE PROTEASE PRODUZIDA POR Pseudomonas fluorescens IB 2312 EM LEITE DESNATADO SUBMETIDO AO PROCESSO DE HOMOGENEIZAÇÃO À ALTA PRESSÃO

The presence of thermoresistant proteases produced by psychrotrophic microorganisms have been identified as a limiting factor of the UHT milk shelflife, causing undesirable changes in milk products. High pressure homogenization (HPH) processing is a non-thermal method of food preservation, able to promotes the microbiological safety and inactivation of some enzymes. Thus, this work assessed the proteolytic activity of protease produced by Pseudomonas fluorescens in skim milk subjected to high pressure homogenization process. The milk samples were added by the protease enzymatic extract (10% v/v) and subjected to pressures up to 300 MPa. The assays showed that pressures on the order of 300 MPa caused a 72.5% reduction in proteolytic activity. Therefore, the process at high pressures resulted in significant inactivation of this thermoresistent enzyme, which possibly favors the shelf-life extension of the UHT milk and also limits the yield and quality loss of cheeses due to undesirable sensory changes in flavor and texture caused by this enzyme.

The identification and biochemical properties of the catalytic specificity of a serine peptidase secreted by Aspergillus fumigatus Fresenius.

Aspergillus fumigatus is a saprophytic fungus as well as a so-called opportunist pathogen. Its biochemical potential and enzyme production justify intensive studies about biomolecules secreted by this microorganism. We describe the alkaline serine peptidase production, with optimum activity at 50°C and a pH of 7.5 and a reduction in proteolytic activity in the presence of the Al(+3) ions. When using intramolecularly quenched fluorogenic substrates, the highest catalytic efficiency was observed with the amino acid leucine on subsite S'(3) (60,000 mM(-1)s(-1)) and preference to non-polar amino acids on subsite S(3). In general, however, the peptidase shows non-specificity on other subsites studied. According to the biochemical characteristics, this peptidase may be an important biocatalyst for the hydrolysis of an enormous variety of proteins and can constitute an essential molecule for the saprophytic lifestyle or invasive action of the opportunistic pathogen. The peptidase described herein exhibits an estimated molecular mass of 33 kDa. Mass spectrometry analysis identified the sequence GAPWGLGSISHK displaying similarities to that of serine peptidase from Aspergillus fumigatus. These data may lead to a greater understanding of the advantageous biochemical potential, biotechnological interest, and trends of this fungus in spite of being an opportunist pathogen.

Proteolytic and milk-clotting activities of calf rennet processed by high pressure homogenization and the influence on the rheological behavior of the milk coagulation process

This work studied the effects of high pressure homogenization (HPH) on the performance of a commercial calf rennet. The enzyme solution was processed at 50, 100, 150 and 190 MPa and then stored for up to five days at 4 °C. The effects were evaluated on the proteolytic and milk-clotting activities of the enzyme and on the rheological behavior of the milk gels obtained using the processed enzyme. HPH caused a reduction in proteolytic activity with increase in pressure (up to 52% loss of activity at 190 MPa). At the first 48 h of storage, the processed enzyme maintained its activity while the non-processed sample showed a continuous reduction in activity. The results showed that HPH processing did not alter the milk-clotting activity and the rheological tests showed high G' values for the milk coagulated with the enzyme processed at 190 MPa (7% higher after 84 min of coagulation). Thus, HPH improves the milk coagulation by calf rennet and reduces the proteolytic activity of enzyme, which may improve the quality of fresh cheeses, especially after their storage. The changes caused by HPH in the calf rennet improved the cheese manufacturing process and the quality of the product (higher consistency of the milk gel and lower proteolysis during storage). Therefore, the HPH processing of calf rennet can improve its application in the dairy industry to obtain high quality fresh cheese and an extended shelf-life.

Chlorhexidine Inhibits the Proteolytic Activity of Root and Coronal Carious Dentin in vitro

The purpose of this study was to evaluate the effect of chlorhexidine on the proteolytic activity of carious coronal and root dentin collected from patients. Sound dentin from freshly extracted human teeth was used as a control. Dentin fragments were mixed with a synthetic substrate for proteolytic enzymes (N-benzoyl-DL-arginine-naphthylamide – BANA) and the suspensions mixed with either 0.12% chlorhexidine digluconate or distilled water. These mixtures were incubated for 18 h at 37°C, color was developed by the addition of 0.1% Fast Garnet and their optical density was recorded spectrophotometrically. BANA hydrolysis measured by the optical density of incubated specimens was detected in all tested groups, but was significantly higher for carious than for sound dentin (p < 0.05). The proteolytic activity was reduced for carious coronal and root dentin by chlorhexidine (p < 0.05; 50 and 30%, respectively). Chlorhexidine also reduced the proteolytic activity in sound root dentin (p < 0.05; 20%). Conversely, changes in the proteolytic activity of sound coronal dentin were not observed in the presence of chlorhexidine. The reduction in proteolytic activity by chlorhexidine was significantly higher in carious coronal dentin than in carious root dentin (p < 0.05). In conclusion, part of the effect of chlorhexidine in controlling caries progression in humans may be due to a decrease in the proteolytic activity of carious coronal and root dentin. Because of the prolonged incubation time in the present study, similar results may be obtained clinically with prolonged dentin exposure to chlorhexidine, e.g. chlorhexidine-containing varnishes.

076 A Clinical Study Investigating the Temporal Changes in Proteolytic Activity in Wounds Treated with Promogran*

The pathophysiology of wound repair is a delicately balanced, dynamic process involving both stimulatory and degradative factors to remove damaged tissue and synthesize new tissue. An imbalance of any of these factors may result in the formation of a chronic wound. Previous studies have indicated that the chronic wound environment is detrimental to cell and cytokine function, leading to delayed healing. The presence of a hostile chronic wound environment has been supported by many studies reporting elevated proteolytic activity in these wounds. However, it has not been determined if this elevation is due to an excess of a particular protease or a reduction in inhibitor levels. In this study we investigated the effect of PROMOGRANTM, a protease modulating matrix, on wound healing in patients with venous leg ulcers and examined the effect on the wound environment. Wound fluid samples were collected from a number of patients throughout the course of treatment and both protease and inhibitor levels were analyzed. Our results indicate that wounds which respond to PROMOGRANTM also exhibited an overall decrease in human neutrophil-derived elastase and MMP activity. An increase in the ratio of alpha-1-antitrypsin to human neutrophil-derived elastase was also observed suggesting a greater degree of regulation post therapy, and a return to a more acute-like wound environment. Wounds that did not respond to this treatment demonstrated a greatly reduced ratio, signifying a persistent excess of proteolytic activity and a continued delay in healing. It is unclear whether the observed biochemical changes in the wound environment are responsible for healing or merely symptomatic of the disease state. We have, however, shown that an overall reduction in proteolytic activity is concomitant with healing. This suggests that therapies that redress the balance of inhibitor to protease may be beneficial in treatment of chronic wounds.

Potential biological consequences of mRNA oxidation‐induced translation errors

Reactive oxygen species (ROS) are generated in living mammalian cells. This leads to oxidation of intracellular biomacromolecules. Much attention has been focused on the effects of DNA, protein, and lipid oxidation. However, since RNAs are less protected against ROS than nuclear DNAs, they are more susceptible to oxidation. We report the detection of elevated levels of oxidized nucleic acid bases, including 8-hydroxyguanine, using GC/MS methods, in polyA mRNA in HEK293 cells after mRNA synthesis was inhibited by ActinoymycinD for 8 h, the average half life of mRNA. When cells were either transfected with radiolabeled mRNA or pulse-labeled mRNA, the labeled mRNA was found to be extensively oxidized. These results suggest that under normal conditions, intracellular mRNAs are constantly subjected to oxidative modification. When HEK293 cells were transfected with a reporter mRNA followed by 10 h incubation, it was found that the reporter mRNA exhibited a time-dependent decrease in its translational efficiency. Concomitantly, there was an accumulation of full-length modified and short polypeptides derived from the transfected mRNA due to translation errors. The short polypeptides were derived, in part, from premature termination of the translation process. Both the modified and short polypeptides were highly susceptible to proteasome-catalyzed degradation. Thus, translation errors caused by mRNA oxidation may contribute, in part, to the reported high quantity of nascent polypeptides degraded and to the accumulation of protein aggregates observed in neurodegenerative diseases and in age-dependent changes in protein modification and thermostability, in conjunction with disease- and age-related reductions in proteolytic activity.

ID: 229 Rational design of cysteine mutants of PAI-1 with very long half-lives

Medical University of Ohio, Toledo, Ohio, USA Proteolytic activity initiated by uPA is commonly recognized as a critical factor in angiogenesis and metastasis. Many cancers overexpress uPA and the reduction of proteolytic activity has been proposed as a cancer treatment option. Indeed, uPA inhibitors have been shown to reduce angiogenesis and tumor growth. Thus, we want to identify novel inhibitors of uPA suitable for cancer treatment. We have chosen PAI-1, which inhibits the urokinase plasminogen activator. However, PAI-1 is not a stable molecule and converts itself into the latent form with a half-life in the range of t1/2~2 hours. Based on the known structure of active PAI-1, we have identified amino acids that can be substituted with a cysteine residue to produce disulfide bridges linking the top and bottom parts of strands A3 and A5 as well as sites within the helix D region in hopes of preventing a conversion into the latent PAI-1. We have created a total of seven cysteine mutants via point mutation (two to six point mutations) generating possible sites for a disulfide bridge formation at the top and bottom parts of A3 and A5, within the helix D region, or by a combination thereof. Desired mutations were introduced by PCR using appropriate primers. The mutant forms of PAI-1 containing the chitin binding intein tag were then purified using affinity chromatography wherein the intein tag is cleaved leaving the mutant PAI-1 protein. Cysteine mutations resulted in proteins with an extended half-life of PAI-1 from 2 to over 700 hours depending on the mutant. Novel PAI-1s were fully functional against uPA and showed activity in the in vitro model of angiogenesis e.g. inhibition of sprout formation. The mutant with the longest half-life (one that produced a disulfide bridge linking the top part of strands A3 and A5) was chosen for further study. VLHL PAI-1 expressed in the E. Coli vector produced a modest yield of 1 mg-purified protein from 1L of cell culture. Thus, we expressed it in the baculovirus vector, with an 6His purification tag that produced ~18 mg of PAI-1/1L. Two different forms were made: fully active VLHL PAI-1 and VLHLNS PAI-1 with an Arg369?Ala mutation in the P1 position, which will be used in future anticancer study as a negative control. VLHLNS PAI-1 mutation (Arg369?Ala) was introduced by PCR and gene was transferred into the baculovirus vector in the same way as VLHL PAI-1. We assume that the VLHLNS construct will remain in an active conformation as VLHL PAI-1 does, but will not have any inhibitory activity toward uPA. Our study suggests that both proteins are in an active conformation, however they convert into the latent form after treatment with reducing agents, which break the disulfide bridge. Fully active VLHL PAI-1 inhibits uPA as demonstrated by chromogenic assay (SPEC-TROZYME®) and forms the uPA/PAI-1 complex as shown on PAGE gel while VLHLNS PAI-1 does not. We believe that PAI-1s with extended half-lives are therapeutically desired in cancer treatment and Cys mutated PAI-1s could launch a new class of novel anti-cancer agents. Keywords: PAI-1, cysteine mutants. 170

Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence.

Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.