Proteasomes Begin Ornithine Decarboxylase Digestion at the C Terminus

Zhang, Mingsheng; MacDonald, Alasdair A.; Hoyt, M. Andrew; Coffino, Philip

doi:10.1074/jbc.m314043200

Cited by 55 publications

(50 citation statements)

References 41 publications

(57 reference statements)

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“…Is that also true of eukaryotic proteasomes? Studies from our laboratory (5,6) and by others (7)(8)(9)(10) are consistent with this conclusion, but studies using purified proteasomes and substrates of well-defined structure have been limited and have not determined the kinetic parameters associated with proteasome action.…”

supporting

confidence: 71%

“…Vertebrate forms of ODC have a small conserved degradation tag (22) (37 amino acids in mouse and human) at its C terminus (cODC). Degradation is strictly unidirectional and begins at the tag (5). Fusing this tag to other proteins promotes their rapid turnover by the eukaryotic proteasome (17), including those of mammals, plants, and fungi.…”

Section: Design and Structure Of Substrates Used In These Studies-mentioning

confidence: 99%

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Dependence of Proteasome Processing Rate on Substrate Unfolding

Henderson

Erales

Hoyt

et al. 2011

Journal of Biological Chemistry

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Protein degradation by eukaryotic proteasomes is a multistep process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steadystate time for degradation ranged from ϳ5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be ratelimiting for degradation.To degrade folded proteins, chambered protease complexes unfold substrates in an energy-dependent manner that requires two components (1). The first, a regulatory complex, recognizes substrates and initiates their processing. The second, an associated proteolytic complex, contains sites at which peptide bonds are hydrolyzed. These sites are present in a closed chamber sequestered from the general cellular environment. The proteolytic chamber is accessed through a pore that excludes folded protein domains but accommodates an unfolded or unstructured polypeptide (2, 3). Regulatory and catalytic complexes must thus collaborate to degrade native proteins: the regulatory complex actively unfolds substrates containing structured domains and translocates them into the catalytic complex. Substrate unfolding and translocation by bacterial ATP-dependent proteases has been extensively studied. It was found that substrate unfolding can be rate-limiting for degradation and that increased mechanical stability of substrates prolongs degradation (4). Is that also true of eukaryotic proteasomes? Studies from our laboratory (5, 6) and by others (7-10) are consistent with this conclusion, but studies using purified proteasomes and substrates of well-defined structure have been limited and have not determined the kinetic parameters associated with proteasome action.There are several requirements for rigorously performing such an analysis. Homogeneous substrates must be available that differ in their resistance to unfolding, but are otherwise identical in structure and proteasomal interaction. Most substrates are designated for destruction by the conjugation of ubiquitin chains, which provide a high affinity tag for proteasome association, but some substrates utilize alternate tags to designate degradation (11,12). Capture of ubiquitin chains and their...

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supporting

confidence: 71%

Section: Design and Structure Of Substrates Used In These Studies-mentioning

confidence: 99%

Dependence of Proteasome Processing Rate on Substrate Unfolding

Henderson

Erales

Hoyt

et al. 2011

Journal of Biological Chemistry

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“…Even modest reductions in mammalian ODC activity can lead to marked resistance to tumor development (1,2). ODC is turned over rapidly, and this, in part, is because of it being targeted to the 26S proteasome without ubiquitination by the protein antizyme, its key regulator (3,4). Antizyme itself is negatively regulated by antizyme inhibitor, a homolog of ODC with a higher affinity for antizyme that has lost the ability to decarboxylate ornithine (5).…”

mentioning

confidence: 99%

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

Ivanov

Loughran

Atkins

2008

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

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In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes.antizyme ͉ AUU ͉ non-AUG ͉ polyamines ͉ upstream ORF

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“…Antizyme increases the degradation of ODC by enhancing its interaction with the proteasome but does not increase the rate of proteasomal processing. It was also shown that proteasomes actually start degradation of ODC at the COOH terminus (24). Therefore, rapid degradation of mammalian ODC requires a degradation domain that contains a PEST motif located at the COOH terminus of the protein.…”

mentioning

confidence: 99%

Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

Hsieh¹,

Chen²,

Wang³

et al. 2007

J Nucl Med

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Herpes simplex virus type 1 thymidine kinase (HSV1-TK) is a widely used reporter for in vivo noninvasive monitoring of therapeutic gene expression, immune cell trafficking, and proteinprotein interactions in various animal systems. However, the stability of HSV1-TK limits its application in studies that require rapid turnover of the reporter. The purpose of this study was to create a destabilized HSV1-TK as a transcription reporter that allows for dynamic studies of short-time-scale gene expression events. Methods: A destabilized HSV1-TK was created by targeting inactivating mutations in the nuclear localization signal of HSV1-TK and fusing the degradation domain of mouse ornithine decarboxylase to the C-terminal end. The protein or enzyme stability was determined by Western blot analysis and HSV1-TK enzyme activity assay, respectively. The proteasome inhibition assay was used to test whether the rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasomedependent manner. The suitability of destabilized HSV1-TK as a transcription reporter was tested by linking it to a tetracyclineturnoff-expressing system. The dynamic transcriptional events mediating a series of doxycycline inductions were monitored by destabilized HSV1-TK or by native HSV1-TK and were determined by an in vitro HSV1-TK enzyme activity assay and in vivo small-animal PET imaging. Results: The destabilized HSV1-TK, unlike wild-type HSV1-TK, was unstable in the presence of cycloheximide and had a short half-life of protein and enzyme activity. The rapid turnover of the destabilized HSV1-TK was processed in a 26S proteasome-dependent manner. Furthermore, the destabilized HSV1-TK had low cytotoxicity when it was highly expressed in living cells. The results of dynamic gene expression studies in vitro and in vivo showed that the destabilized HSV1-TK is an optimal reporter for monitoring short-time-scale dynamic transcriptional events mediating a series of doxycycline inductions, whereas the wild-type HSV1-TK is not optimal to achieve this purpose. Conclusion: The use of destabilized HSV1-TK as a transcription reporter together with a molecular probe, which has a short physical and biologic half-life, allows more direct monitoring of transcription induction and easier monitoring of its coincidence with other biochemical changes.

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Proteasomes Begin Ornithine Decarboxylase Digestion at the C Terminus

Cited by 55 publications

References 41 publications

Dependence of Proteasome Processing Rate on Substrate Unfolding

Dependence of Proteasome Processing Rate on Substrate Unfolding

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

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