Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown.

Hershko, Avram; Ciechanover, Aaron; Rose, Irwin A.

doi:10.1016/s0021-9258(19)69833-9

Cited by 104 publications

(13 citation statements)

References 13 publications

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“…However, when purified ubiquitin was added in the presence of ATP, it promoted twofold the degradation only of 3H-casein with free amino groups and did not affect hydrolysis of carbamylated casein. The failure of ubiquitin to stimulate hydrolysis of the blocked substrate supports the earlier suggestion (12,13) that this polypeptide acts by conjugation to amino groups on the substrate and our assumption that with the carbamylated proteins this linkage reaction is not possible.…”

Section: Effects Of a Tp And Ubiquitin On Proteolysis In Fraction IIsupporting

confidence: 87%

“…One of these is ubiquitin (10), an 8,500-dalton polypeptide (11), which in the presence of ATP can be ligated to eamino groups of lysine residues of various cellular proteins (12). In this process, ATP-Mg ÷+ is required for the activation of the carboxyl glycine residue of ubiquitin to a form which can be linked by an isopeptide bond to lysines in proteins (13).…”

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confidence: 99%

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ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin.

Tanaka¹,

Waxman²,

Goldberg³

1983

The Journal of Cell Biology

154

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Protein degradation in rabbit reticulocytes is a nonlysosomal process requiring ATP. Recently, appreciable evidence has been presented that ATP is required for the covalent binding of the polypeptide ubiquitin to ~-amino groups on protein substrates. To test whether linkage of ubiquitin to substrates is required for ATP-dependent proteolysis, the amino groups of 3H-methyl-casein and denatured 12Sl-bovine serum albumin (BSA) were completely (93-99%) blocked by methylation, acetylation, carbamylation, or succinylation. In each case, the proteins lacking amino groups were still degraded by an ATP-stimulated process, although these various treatments altered absolute rates of proteolysis and reduced the magnitude of the ATP stimulation (two-to fourfold) below that seen measured with the unmodified substrates. When ubiquitin was removed by ion exchange chromatography, ATP still stimulated breakdown of casein and carbamylated casein twofold. The addition of ubiquitin in the presence of ATP caused a further twofold increase in the hydrolysis of unmodified casein but did not affect the degradation of casein lacking amino groups. Thus ubiquitin conjugation to substrates appears important in the breakdown of certain substrates (especially of BSA), but this reaction is not essential for ATP-stimulated proteolysis. The AlP-activated step that is independent of ubiquitin probably is also involved in the degradation of unblocked proteins, since both processes require Mg +÷ and ATP hydrolysis and are inhibited by heroin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway.Protein degradation within mammalian and bacterial cells requires metabolic energy (1, 2). The finding that ATP can stimulate proteolysis in cell-free extracts (2-4) has made possible appreciable progress in elucidating the basis for this energy requirement. In rabbit reticulocyte extracts, Etlinger and Goldberg (3) described a soluble, alkaline proteolytic system that is dependent on ATP. This nonlysosomal system appears responsible for the selective degradation of abnormal proteins as well as for the elimination of many normal proteins during reticulocyte maturation (5, 6). Hershko, Rose, and coworkers (7-10) have presented extensive evidence that multiple protein components are necessary for the stimulatory effect of ATP. One of these is ubiquitin (10), an 8,500-dalton polypeptide (11), which in the presence of ATP can be ligated to eamino groups of lysine residues of various cellular proteins (12). In this process, ATP-Mg ÷+ is required for the activation of the carboxyl glycine residue of ubiquitin to a form which can be linked by an isopeptide bond to lysines in proteins (13).In support of this model, Chin et al. (14) showed that the degree of attachment of ubiquitin to denatured hemoglobins correlated with their rates of degradation when these proteins were microinjected into HeLa ceils. Hershko et al. (2,9) have argued that the formation of ubiquitin-protein conjugates ...

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Section: Effects Of a Tp And Ubiquitin On Proteolysis In Fraction IIsupporting

confidence: 87%

mentioning

confidence: 99%

ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin.

Tanaka¹,

Waxman²,

Goldberg³

1983

The Journal of Cell Biology

154

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“…Pellet 3H and 32P radioactivities were determined after rinsing with cold 2% Cl3CCOOH as described . ubiquitin ultimately formed a thiol ester between a sulfhydryl group on the enzyme and the carboxyl terminus of ubiquitin (Hershko et al, 1981) strongly indicated a mixed-anhydride linkage between the phosphate of AMP and the carboxyl terminus of the polypeptide. That AMP-Ub is hydrolyzed rapidly at alkaline pH but slowly at acidic pH conforms to the expected chemical stability of an acyl phosphate anhydride .…”

Section: Resultsmentioning

confidence: 98%

Ubiquitin adenylate: structure and role in ubiquitin activation

Haas¹,

Warms²,

Rose³

1983

Biochemistry

134

156

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The acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.

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“…The ubiquitin-proteasome pathway (UPP) has been determined to regulate important cellular functions via the degradation or processing of targeted proteins. Ubiquitin is first activated by a ubiquitin-activating enzyme (E1) prior to conjugating to a substrate through the ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) following degradation by proteasome. − E1 activates the C-terminal glycine of ubiquitin to an adenylated intermediate and transfers it to a thiol site in the enzyme. , This high-energy intermediate of ubiquitin is further transferred to the thiol site of an E2 and finally to the targeted protein by combination with an E3 . Recent observations suggested that ubiquitination of a protein is not only the tag for proteasomal degradation but also a modification state of the signal mediators.…”

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confidence: 99%

Panepophenanthrin, from a Mushroom Strain, a Novel Inhibitor of the Ubiquitin-Activating Enzyme

et al. 2002

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Screening for inhibitors of the ubiquitin-proteasome pathway, considered to regulate important cellular events and linked to serious diseases as well, led to isolation of a new compound, panepophenanthrin, from the fermented broth of a mushroom strain, Panus rudis Fr. IFO 8994. This is the first inhibitor of the ubiquitin-activating enzyme, which is indispensable for the ubiquitin-proteasome pathway. The structure of panepophenanthrin was determined by NMR and X-ray crystallographic analyses as 1,3a,10-trihydroxy-10c-(3-hydroxy-3-methylbut-1-enyl)-5,5-dimethyl-1,2,3,3a,5,5a,8,9,10,10a,10b,10c-dodecahydro-4-oxa-2,3,8,9-diepoxyacephenanthrylen-7-one.

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Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown.

Cited by 104 publications

References 13 publications

ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin.

ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin.

Ubiquitin adenylate: structure and role in ubiquitin activation

Panepophenanthrin, from a Mushroom Strain, a Novel Inhibitor of the Ubiquitin-Activating Enzyme

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