Selective Degradation of Abnormal Proteins in Mammalian Tissue Culture Cells

Capecchi, Mario R.; Capecchi, N. E.; Hughes, Stephen H.; Wahl, Geoffrey M.

doi:10.1073/pnas.71.12.4732

Cited by 85 publications

(22 citation statements)

References 17 publications

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“…This hypothesis may not completely explain the discrepancy, since no correlation has been found between enzyme heat stability in vitro and enzyme degradation rates in vivo in a series of cell lines with mutant hypoxanthine-guanine phosphoribosyltransferase (51). Molecules of mutant hypoxanthine-guanine phosphoribosyltransferase are degraded much faster than the normal enzyme in mammalian cells (51). Therefore, the level of mutant enzyme specific activity of a particular tissue may be dependent upon the relative balance of synthesis and degradation of the mutant protein.…”

Section: Discussionmentioning

confidence: 68%

“…A discrepency between erythrocyte and leukocyte activity in one study has been attributed to enzyme lability in the absence of protein synthesis in the former (50). This hypothesis may not completely explain the discrepancy, since no correlation has been found between enzyme heat stability in vitro and enzyme degradation rates in vivo in a series of cell lines with mutant hypoxanthine-guanine phosphoribosyltransferase (51). Molecules of mutant hypoxanthine-guanine phosphoribosyltransferase are degraded much faster than the normal enzyme in mammalian cells (51).…”

Section: Discussionmentioning

confidence: 83%

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Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Fox¹,

Dwosh²,

Marchant³

et al. 1975

J. Clin. Invest.

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ABSTRA CT The mutation in a young gouty male with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase has been evaluated. The serum uric acid was 11.8 mg/100 ml, and the urinary uric acid excretion was 1,279 mg/24 h. Erythrocyte hypoxanthine-guanine phosphoribosyltransferase was 34.2 nmol/h/mg, adenine phosphoribosyltransferase was 36.5 nmol/h/mg and phosphoribosylpyrophosphate was 2.6 AM. Hypoxanthine-guanine phosphoribosyltransferase from peripheral leukocytes and cultured diploid skin fibroblasts was within the normal range, but enzyme activity in rectal mucosa was below the normal range.Initial velocity studies of the normal enzyme and the mutant enzyme from erythrocytes with the substrates hypoxanthine, guanine, or phosphoribosylpyrophosphate showed that the Michaelis constants were similar. Product inhibition studies distinguished the mutant enzyme from the normal enzyme. Hyperbolic kinetics with increasing phosphoribosylpyrophosphate were converted to sigmoid kinetics by 0.2 mM GMP with the mutant enzyme but not with the normal enzyme.The mutant erythrocyte hypoxanthine-guanine phosphoribosyltransferase was inactivated normally at 80'C and had a normal half-life in the peripheral circulation. The mol wt of 48,000 was similar to the normal enzyme mol wt of 47,000. With isoelectric focusing, the mutant erythrocyte enzyme had two major peaks with isoelectric pH's of 5.50 and 5.70, in contrast to the isoelectric pH's of 5.76, 5.82, and 6.02 of the normal isoAn abstract of this work was published in Clin. Res. 22: 389a, 1974. Dr.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 83%

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Fox¹,

Dwosh²,

Marchant³

et al. 1975

J. Clin. Invest.

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“…All of the mutations that caused a loss of hypoxanthine-guanine phosphoribosyltransferase activity in cultured cells also decreased the half-life of the mutant proteins (6). It seems likely that many proteins (both host and viral) have been selected to be folded into compact structures that are resistant to degradation by PRs.…”

Section: Discussionmentioning

confidence: 99%

Mutations in the Thumb Allow Human Immunodeficiency Virus Type 1 Reverse Transcriptase To Be Cleaved by Protease in Virions

Dunn

McWilliams

Das

et al. 2009

J Virol

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Although human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been extensively studied, there are still significant questions about the effects of mutations on the maturation and stability of RT. We show here that a significant fraction (>80%) of the single point mutations we generated in the thumb subdomain of HIV-1 (RT) affect the stability of RT in virions. Fragments of the unstable mutant RTs can be detected in Western blots of virion proteins; however, the degree of degradation varies. The titers of the mutants whose virions contain degraded RTs are reduced. Some, but not all, of the unstable RT thumb subdomain mutants we analyzed have a temperature-sensitive phenotype. A preliminary survey of mutations in other subdomains of RT shows that some of these mutations also destabilize RT. The stability of the RT mutants is enhanced by the addition of a protease inhibitor, suggesting that the viral protease plays an important role in the degradation of the mutant RTs. These results confirm and extend earlier reports of mutations that affect the stability of RT in virions. The data suggest that the stability of a mutant RT in virions could be a major factor in determining the virus titer and, by extension, viral fitness, which could affect whether a mutation in RT is acceptable to the virus.

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“…This suggests that if breakdown is occurring, it happens rapidly and nearly completely. This process must be much faster than the reported degradation of a missense mutant cellular enzyme (9).…”

Section: Methodsmentioning

confidence: 99%

Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

Summers¹,

Wagner²,

Summers³

1975

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in the viral gene coding for the thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) induced by herpes simplex virus have been obtained by selection of virus resistant to bromoodeoxyuridine when grown in thymidine-kinase-deficient LMTK-mouse cells. Proteins labeled after infection of Vero (monkey) cells with herpes simplex virus were analyzed by gel electrophoresis and one protein of about 40,000 daltons was consistently altered in a number of thymidine-kinase-deficient mutants. Many viral mutants lacked this peptide and one class of these mutants induced the synthesis of new shorter peptides. Revertant virus could be selected which simultaneously regained the ability to induce thymidine kinase activity, regained the intact thymidine kinase peptide, and lost the ability to synthesize the shorter peptide fragment. These mutants comprise a class of animal virus mutants which have the properties expected of peptide chain termination mutants.The use of mutants which are sensitive to translational suppression has greatly advanced the study of bacteria and bacterial viruses (1). With the exception of fungi (2, 3), such mutations have not been demonstrated in eukaryotic systems. Two approaches to obtain such mutants are immediately apparent. First is to obtain a cell which has a suppressor gene and then to select mutants which are suppressible. Second, one can obtain potentially suppressible mutations and then use these to search for suppressor mutations. It is this latter course which we have taken, and we report here on the first part of the problem, namely the isolation and characterization of mutants which have the properties expected of peptide chain termination, potentially suppressible mutations.We have studied mutants in the thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.75) (TK) gene of herpes simplex virus (HSV). This system was chosen for several reasons. The enzyme thymidine kinase is not a required viral function, so defective mutants can be grown even in the absence of suppressor activity. TK-deficient mutants can be selected on the basis of their resistance to the lethal effects of 5-bromo-deoxyuridine (BrdUrd) (4). In addition, a sensitive and simple enzyme assay is available (5). The recent identification of the TK polypeptide by gel electrophoresis of immunoprecipitated, labeled infected cell proteins was also highly encouraging (6). The results of our studies show that a single viral-induced polypeptide is affected by mutation to BrdUrd resistance with simultaneous loss of TK activity, that revertants to BrdUrd sensitivity regain the affected polypeptide and TK activity, and that, in some cases, a shorter peptide is observed which appears in the mutants and is absent in the revertants. The simplest interpretation of these results is in terms of classical peptide chain termination mutants. Selection of Revertants. In order to select revertants of HSV-TK which had regained the ability to induce TK, certain of the virus mutants were grown in LMTK-cells...

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Selective Degradation of Abnormal Proteins in Mammalian Tissue Culture Cells

Cited by 85 publications

References 17 publications

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Mutations in the Thumb Allow Human Immunodeficiency Virus Type 1 Reverse Transcriptase To Be Cleaved by Protease in Virions

Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

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