Effects of Manipulation of Proteases on Myofibril Protein Degradation of Tilapia Muscle in vitro and in vivo

The recovery of approximately 40% of the total liver protein during the first day after partial hepatectomy was shown to be due to the near cessation of protein breakdown rather than to an increase in protein synthesis. The decrease in degradation of total protein was less if rats were adrenalectomized or protein-depleted prior to partial hepatectomy. The effect of these treatments originally suggested that changes in free amino acid levels in liver might be related to the rate of protein degradation. However, no correlation was found between levels of total free amino acids and rates of breakdown. Measurements of individual amino acids during liver regeneration suggested that levels of free methionine and phenylalanine, amino acids that have been found to lower rates of protein degradation in vitro, are not correlated with rates of breakdown in vivo. The difference between the fractional rate of ornithine aminotransferase degradation (0.68/day and 0.28/day in sham-hepatectomized and partially hepatectomized rats, respectively) was sufficient to account for the higher level of this protein 3 days after surgery in the latter group.

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Assay Systems for Profiling Deubiquitinating Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21165638 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5638

Author(s):

Jinhong Cho ◽

Jinyoung Park ◽

Eunice EunKyeong Kim ◽

Eun Joo Song

Keyword(s):

Protein Degradation ◽

Protein Interactions ◽

Live Cells ◽

Deubiquitinating Enzyme ◽

Protein Protein Interactions ◽

Deubiquitinating Enzymes ◽

Cell Lysates ◽

Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

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Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs

Blood ◽

10.1182/blood.2019000789 ◽

2019 ◽

Vol 134 (2) ◽

pp. 160-170 ◽

Cited By ~ 10

Author(s):

Adam S. Sperling ◽

Michael Burgess ◽

Hasmik Keshishian ◽

Jessica A. Gasser ◽

Shruti Bhatt ◽

...

Keyword(s):

Protein Degradation ◽

Ubiquitin Ligase ◽

Degradation Kinetics ◽

Hematologic Malignancies ◽

Clinical Activity ◽

Substrate Affinity ◽

Cell Type Specificity ◽

Drug Dependent

Abstract Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

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Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.

The Journal of Cell Biology ◽

10.1083/jcb.132.3.291 ◽

1996 ◽

Vol 132 (3) ◽

pp. 291-298 ◽

Cited By ~ 282

Author(s):

A A McCracken ◽

J L Brodsky

Keyword(s):

Protein Degradation ◽

Atp Hydrolysis ◽

Genetically Engineered ◽

Heat Inactivation ◽

Protein Factor ◽

Transport Vesicles ◽

Alpha Factor ◽

Processed Form

To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radio-labeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of p alpha F was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.

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Evidence for Separable Functions of Srp1p, the Yeast Homolog of Importin α (Karyopherin α): Role for Srp1p and Sts1p in Protein Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.20.16.6062-6073.2000 ◽

2000 ◽

Vol 20 (16) ◽

pp. 6062-6073 ◽

Cited By ~ 43

Author(s):

Michelle M. Tabb ◽

Prasad Tongaonkar ◽

Loan Vu ◽

Masayasu Nomura

Keyword(s):

Protein Degradation ◽

Nuclear Localization ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Localization Function ◽

Importin Α ◽

Yeast Homolog ◽

Two Hybrid

ABSTRACT Srp1p (importin α) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. Thesrp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of thesrp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressingsrp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-β-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not insrp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 insrp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

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Insulin and Analogue Effects on Protein Degradation in Different Cell Types

Journal of Biological Chemistry ◽

10.1074/jbc.m007988200 ◽

2000 ◽

Vol 276 (15) ◽

pp. 11552-11558 ◽

Cited By ~ 19

Author(s):

Janet Fawcett ◽

Frederick G. Hamel ◽

Robert G. Bennett ◽

Zoltan Vajo ◽

William C. Duckworth

Keyword(s):

Protein Degradation ◽

Hepg2 Cells ◽

Cell Types ◽

Major Effect ◽

Cellular Protein ◽

Experimental Conditions ◽

L6 Cells ◽

Different Cell Types

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activityin vitroandin vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (LysB28,ProB29-insulin (LysPro), AspB10-insulin (B10), and GluB4,GlnB16,PheB17-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [3H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC50of 4.6 × 10−9m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC50= 4.2 × 10−11and 1.6 × 10−10m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.

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Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0755 ◽

2006 ◽

Vol 17 (2) ◽

pp. 789-798 ◽

Cited By ~ 34

Author(s):

Taeyoon Kim ◽

Y. Peng Loh

Keyword(s):

Protein Degradation ◽

Secretory Granule ◽

Endocrine Cells ◽

Underlying Mechanism ◽

Genetic Profiling ◽

Regulated Secretion ◽

Granule Protein ◽

Protease Nexin

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

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The effects of calcium ions, ionophore A23187 and inhibition of energy metabolism on protein degradation in the rat diaphragm and epitrochlearis muscles in vitro

Biochemical Journal ◽

10.1042/bj1900593 ◽

1980 ◽

Vol 190 (3) ◽

pp. 593-603 ◽

Cited By ~ 29

Author(s):

P H Sugden

Keyword(s):

Energy Metabolism ◽

Protein Degradation ◽

Glycogen Phosphorylase ◽

Activity Ratio ◽

Ionophore A23187 ◽

Rat Diaphragm ◽

Phosphorylase Activity ◽

Glycogen Phosphorylase Activity

1. The effects of external Ca2+, EGTA, ionophore A23187, CN-, dinitrophenol and iodoacetamide on the rate of protein degradation in the rat diaphragm and epitrochlearis muscles in vitro were investigated. 2. External Ca2+ increased protein degradation when compared with external EGTA. Protein degradation was further increased by Ca2+ + ionophore A23187. 3. EGTA and ionophore A23187 decreased ATP and phosphocreatine concentrations and the ATP/ADP ratio. 4. CN-, dinitrophenol and iodoacetamide decreased protein degradation, presumably by interfering with energy metabolism. 5. The effects of EGTA may be caused by disturbances in energy metabolism. The effects of ionophore A23187 cannot be readily explained by disturbances in energy metabolism. 6. Incubation of diaphragms with Ca2+ causes a rapid increase in whole-tissue Ca content. This is further stimulated by ionophore A23187. The uptake of Ca2+ may be, at least in part, into the cytoplasm because an increase in the glycogen phosphorylase activity ratio is observed. 7. A Ca2+-activated proteinase is present in rat heart and diaphragm. This enzyme may mediate in part the effects of Ca2+ described above. The apparent KA of this enzyme for Ca2+ is about 0.25 mM. 8. Because effects of ionophore A23187 cause a large increase in whole-tissue Ca content and because the Ca2+-activated proteinase has a relatively low affinity for Ca2+, it is felt that the effects of Ca2+ upon muscle proteolysis are unlikely to be of importance in steady-state protein turnover in vivo. The mechanism may, however, be important in breakdown of necrotic tissue in the living animal.

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Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats

Biochemical Journal ◽

10.1042/bj2220395 ◽

1984 ◽

Vol 222 (2) ◽

pp. 395-400 ◽

Cited By ~ 34

Author(s):

V R Preedy ◽

D M Smith ◽

N F Kearney ◽

P H Sugden

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Protein Turnover ◽

Synthesis Rate ◽

Protein Synthesis Rate ◽

Ventricular Muscle ◽

Degradation Rates ◽

Perfused Hearts

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.

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Protein metabolism in lung: use of isolated perfused lung to study protein degradation

Journal of Applied Physiology ◽

10.1152/jappl.1979.47.1.72 ◽

1979 ◽

Vol 47 (1) ◽

pp. 72-78 ◽

Cited By ~ 8

Author(s):

M. J. Chiang ◽

P. Whitney ◽

D. Massaro

Keyword(s):

Protein Degradation ◽

Protein Metabolism ◽

Lung Weight ◽

Isolated Perfused Lung ◽

Constant Rate ◽

Perfused Lung

This study investigates the use of the isolated perfused lung to study protein degradation. Proteins were labeled in vivo for 10 min or for 5 h using L-[U-14C]phenylalanine. When prelabeled lungs were perfused in vitro virtually all of the acid-soluble and acid-insoluble radioactivity in the tissue and perfusate remained as phenylalanine. Protein degradation was measured as the accumulation of free [14C]phenylalanine in ther perfusate; during the time this accumulated the amount of intracellular free phenylalanine and the free phenylalanine space remained constant. Proteins labeled during 10 min had a constant rate of degradation between 45 and 90 min of perfusion (about 11%.h-1); those labeled during 5 h had a constant rate of degradation for 90 (about 3%.h-1). The percent dry lung weight did not change during the perfusion. We conclude that measurable rates of proteolysis of “rapid” and “slowly” turning over proteins can be obtained while the lung is virtually free of edema. This system should allow studies on the modulation of proteolysis in intact lung under defined conditions.

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