Induction Of Proteolysis Research Articles

Isoflavin-β modifies muscle oxidative stress and prevents a thyrotoxicosis-induced loss of muscle mass in rats.

We sought to verify whether isoflavin-beta (Iso-β), a mixture of isoflavones with antioxidant properties, could prevent thyrotoxicosis-induced loss of muscle mass and the participation of oxidative stress (OS) in the mechanisms of this prevention. Two experimental periods of thyrotoxicosis induction were used in Wistar rats: 3 and 5 days to assess Iso-β effects before and after thyrotoxicosis-induced muscle wasting. After euthanasia, peritoneal fat and gastrocnemius muscle were collected, weighed, and muscle OS was assessed. Iso-β prevented the loss of gastrocnemius mass in thyrotoxic rats through the prevention of muscle OS generation during thyrotoxicosis, increasing muscle total antioxidant capacity and decreasing mitochondrial cytochrome c oxidase activity, lipid peroxidation, and protein carbonyl content. Iso-β decreased oxidative modification of proteins, which is known to exert a major role during proteolysis induction and is present in thyrotoxic myopathy, highlighting the potential action of Iso-β in this complication of the disease. Muscle Nerve 56: 975-981, 2017.

Induced maturation of human immunodeficiency virus.

HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion--termed maturation--that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

The PA influenza virus polymerase subunit is a phosphorylated protein.

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

Hyperthermia, unlike ionizing radiation and chemical oxidative stress, does not stimulate proteolysis in erythrocytes

1. 1. Oxidative stress by phenazine methosulfate stimulated proteolysis in erythrocytes. 2. 2. Gamma-irradiation of erythrocytes in the range of 50–1000 Gy also resulted in the induction of proteolysis. 3. 3. Though it has been suggested that hyperthermia imposes an oxidative stress on a cell, hyperthermic exposure of erythrocytes (30 min, 39–49°C) did not stimulate proteolysis during subsequent incubation of whole cells or hemolysates. 4. 4. Proteolytic degradation of spectrin was accelerated during incubation of membranes isolated from cells heated above 45°C but this effect seems to be due rather to thermal denaturation of spectrin than to oxidative modification of cellular proteins by hyperthermia.

Induction of proteolytic activity in serum by treatment with anionic detergents and organic solvents.

Using casein plates as a sensitive assay for proteolytic activity, it was observed that sodium-dodecyl sulfate (SDS) and other anionic detergents induce caseinolysis when mixed with sera and plasma. Caseinolysis was dependent on the presence of plasminogen in the fluids and could be blocked by inhibitors of serine proteases and antibody to plasminogen. Similarly, organic solvents such as isopropanol induced caseinolysis after mixing with plasma, but not normal serum. Isopropanol dissociated complexes of alpha 1-antitrypsin or alpha 2-macroglobulin with trypsin preformed in vitro. As both SDS and organic solvents are widely used in biochemical investigations of biological fluids, attention should be paid to the possible induction of proteolysis.

Differential Role of Fractionated Heparin in Antithrombin-III Proteolysis

AbstractCommercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively. Each fraction, in microgram quantities, was examined in the reaction of α-thrombin with a molar excess of 126I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21 %, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

Differential role of fractionated heparin in antithrombin-III proteolysis

Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.