A ubiquitin-based tagging system for controlled modulation of protein stability

Stack, Jeffrey H.; Whitney, Michael; Rodems, Steven; Pollok, Brian A.

doi:10.1038/82422

Cited by 85 publications

(78 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, little is known about the suitability of linear chains as degradation signals in vivo. Noncleavable tandem arrays of 2-8 ubiquitin units were shown to confer half-lives of less than 10 min to their fusion partners in reticulocyte lysates and cell culture (35,36). When overexpressed in yeast, they effectively inhibit the proteasomal degradation of short-lived proteins (37).…”

Section: Discussionmentioning

confidence: 99%

Distinct consequences of posttranslational modification by linear versus K63-linked polyubiquitin chains

Zhao

Ulrich²

2010

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

View full text Add to dashboard Cite

Polyubiquitin chains mediate a variety of biological processes, ranging from proteasomal targeting to inflammatory signaling and DNA repair. Their functional diversity is in part due to their ability to adopt distinct conformations, depending on how the ubiquitin moieties within the chain are linked. We have used the eukaryotic replication clamp PCNA, a natural target of lysine (K)63-linked polyubiquitylation, as a model substrate to directly compare the consequences of modification by different types of polyubiquitin chains. We show here that K63-polyubiquitylated PCNA is not subject to proteasomal degradation. In contrast, linear, noncleavable ubiquitin chains do not promote DNA damage tolerance, but function as general degradation signals. We find that a linear tetraubiquitin chain is sufficient to afford proteasomal targeting through the Cdc48-Npl4-Ufd1 complex without further modification. Although a minimum chain length of four is required for degradation, a longer chain does not further reduce the half-life of the respective substrate protein. Our results suggest that the cellular machinery responsible for recognition of ubiquitylated substrates can make subtle distinctions between highly similar forms of the polyubiquitin signal.

show abstract

Section: Discussionmentioning

confidence: 99%

Distinct consequences of posttranslational modification by linear versus K63-linked polyubiquitin chains

Zhao

Ulrich²

2010

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

View full text Add to dashboard Cite

show abstract

“…The activity of the ubiquitin proteasome was monitored with a model substrate as previously described (54). Briefly, Jurkat cells expressing 2XUb-␤-lactamase reporter were plated in 96-well plates at a density of 1.5 ϫ 10 6 cells/ml and incubated with the various concentrations of compounds.…”

Section: Methodsmentioning

confidence: 99%

Rescue of ΔF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Goor

Straley²,

Cao³

et al. 2006

American Journal of Physiology-Lung Cellular and Molecular Phys

Self Cite

464

556

View full text Add to dashboard Cite

show abstract

“…A plasmid conferring neomycin (G418) resistance, pUbBla3X NS2͞3-3A, was transfected into Jurkat cells. The cytomegalovirus promoter-driven ORF of the plasmid encodes a 91-kDa protein, ubiquitin-ubiquitinubiquitin-NS2͞3-BLA, with the C termini of the three ubiquitin domains rendered noncleaveable to ubiquitin C-terminal hydrolases (40). The ubiquitin C-terminal sequence here is Arg-LeuArg-Gly-Val.…”

Section: Methodsmentioning

confidence: 99%

Host cell factor requirement for hepatitis C virus enzyme maturation

Waxman¹,

Whitney²,

Pollok³

et al. 2001

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

Self Cite

View full text Add to dashboard Cite

The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2͞3 protease of hepatitis C virus. The cleavage activity of NS2͞3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2͞3. Furthermore, these HSP90 inhibitors prevent NS2͞3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2͞3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone͞folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.A n estimated 170 million people are infected with hepatitis C virus (HCV) worldwide. The infection is usually persistent, and after an asymptomatic period often lasting years, many patients develop chronic liver disease, including cirrhosis and hepatocellular carcinoma (1, 2). The preferred existing treatment with IFN-␣ is successful in only a small fraction of cases, so an urgent need exists for the development of therapeutically effective inhibitors of HCV replication. With the precedent of numerous successful antiviral therapies based on viral enzyme inhibition, the enzymes encoded by HCV present particularly attractive targets. The recombinant HCV enzymes, NS5B RNA polymerase, NS3 protease, and NS3 helicase have been purified, their enzymatic properties are being defined in vitro, and their potential for inhibition is being explored by many laboratories (3-7).In contrast, the HCV protease activity responsible for cleavage between NS2 and the NS3 proteins, known as NS2͞3 protease, has been reported only in cell lysate translation systems and transfection experiments, despite having been described initially several years ago (8-10). Thus, NS2͞3 cleavage as a target for inhibition has been particularly refractory to detailed analysis, so that the definition of the critical components of this activity is needed. Based on mutational analyses, the protein region essential for NS2͞3 cleavage activity has been approximately mapped to amino acids 898-1207 of the HCV ORF. This region of the HCV polyprotein includes the N-terminal portion of NS3 that is known to be sufficient for NS3 protease activity, residues 1027-1207. Because of the overlap of sequence essential for the two distinct protease activities, it is worthwhile to emphasize that the NS3 protease activity can be totally eliminated through mutagenesis of the NS3 active site Ser-1165 without affecting NS2͞3 cleavage activity (10).Additional features of NS2͞3 cleavage activity known at this time include identification of Cys-993 and His-952 as essential residues (8-13). The only known cleavage site sequence, ArgLeu-Leu2Ala-Pro-Ile, is conserved across HCV strains, and the catalytic mechanism of NS2͞3 cleavage is unknown, but the enzyme is sp...

show abstract

A ubiquitin-based tagging system for controlled modulation of protein stability

Cited by 85 publications

References 25 publications

Distinct consequences of posttranslational modification by linear versus K63-linked polyubiquitin chains

Distinct consequences of posttranslational modification by linear versus K63-linked polyubiquitin chains

Rescue of ΔF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Host cell factor requirement for hepatitis C virus enzyme maturation

Contact Info

Product

Resources

About