Ribosome-associated quality control mediates degradation of the premature translation termination product Orf1p of ODC antizyme mRNA.

Decoding of OAZ1 (Ornithine decarboxylase AntiZyme 1) mRNA, which harbours two open reading frames (ORF1 and ORF2) interrupted by a naturally occurring Premature Termination Codon (PTC), produces an 8 kDa truncated polypeptide termed Orf1p, unless the PTC is bypassed by +1 ribosomal frameshifting. In this study, we identified Orf1p as an endogenous ubiquitin-dependent substrate of the 26S proteasome both in yeast and mammalian cells. Surprisingly, we found that the ribosome-associated quality control factor Rqc1 and the ubiquitin ligase Ltn1 are critical for Orf1p degradation. In addition, the cytosolic protein quality control chaperone system Hsp70/Hsp90 and their corresponding co-chaperones Sse1, Fes1, Sti1 and Cpr7 are also required for Orf1p proteolysis. Our study finds that Orf1p, which is naturally synthesized as a result of a premature translation termination event, requires the coordinated role of both ribosome-associated and cytosolic protein quality control factors for its degradation.

Ccr4-Not complex subunits Ccr4, Caf1, and Not4 are novel proteolysis factors promoting the degradation of ubiquitin-dependent substrates by the 26S proteasome.

Degradation of short-lived and abnormal proteins is essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4, Caf1, and Not4 proteins are known components of the Ccr4-Not multimeric complex. Ccr4 and Caf1 have established roles in transcription, mRNA de-adenylation and RNA degradation etc., while Not4 was shown to have important roles in regulating translation and protein quality control pathways. Here we show that Ccr4, Caf1, and Not4 have a novel function at a post-ubiquitylation step in the UPS pathway by promoting ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4, CAF1, or NOT4 genes in Saccharomyces cerevisiae. Additionally, we show that Ccr4, Caf1, and Not4 bind to cellular ubiquitin conjugates, and that Ccr4 and Caf1 proteins interact with the proteasome. In contrast to Ccr4, Caf1, and Not4, other subunits of the Ccr4-Not complex are dispensable for UFD substrate degradation. From our findings we conclude that the Ccr4-Not complex subunits Ccr4, Caf1, and Not4 have a novel function outside of the canonical Ccr4-Not complex as a factor targeting ubiquitylated substrates for proteasomal degradation.

Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates.

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

Correction to: Analysis of Cotranslational Polyamine Sensing During Decoding of ODC Antizyme mRNA.

Correction to: Chapter 26 in: Rubén Alcázar and Antonio F. Tiburcio (eds.), Polyamines: Methods and Protocols, Methods in Molecular Biology, vol. 1694, https://doi.org/10.1007/978-1-4939-7398-9_26.

Analysis of Cotranslational Polyamine Sensing During Decoding of ODC Antizyme mRNA.

Polyamines are essential poly-cations with vital functions in all cellular systems. Their levels are controlled by intricate regulatory feedback mechanisms. Abnormally high levels of polyamines have been linked to cancer. A rate-limiting enzyme in the biosynthesis of polyamines in fungi and higher eukaryotes is ornithine-decarboxylase (ODC). Its levels are largely controlled posttranslationally via ubiquitin-independent degradation mediated by ODC antizyme (OAZ). The latter is a critical polyamine sensor in a feedback control mechanism that adjusts cellular polyamine levels. Here, we describe an approach employing quantitative western blot analyses that provides in vivo evidence for cotranslational polyamine-sensing by nascent OAZ in yeast. In addition, we describe an in vitro method to detect polyamine binding by antizyme.

Polyamines directly promote antizyme-mediated degradation of ornithine decarboxylase by the proteasome.

Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates.

Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA.

Polyamines are essential organic polycations with multiple cellular functions relevant for cell division, cancer and ageing. Regulation of polyamine synthesis is mainly achieved by controlling the activity of ornithine decarboxylase (ODC) through an unusual mechanism involving ODC antizyme, the binding of which disrupts homodimeric ODC and targets it for ubiquitin-independent degradation by the 26S proteasome. Whereas mammals express several antizyme genes, we have identified a single orthologue, termed OAZ1, in Saccharomyces cerevisiae. Similar to its mammalian counterparts, OAZ1 synthesis is induced with rising intracellular polyamine concentrations, which also inhibit ubiquitin-dependent degradation of the OAZ1 protein. Together, these mechanisms contribute to a homeostatic feedback regulation of polyamines. Antizyme synthesis involves a conserved +1 ribosomal frameshifting (RFS) event at an internal STOP codon during decoding of its messenger RNA. Here we used S. cerevisiae OAZ1 to dissect the enigmatic mechanism underlying polyamine regulation of RFS. In contrast with previous assumptions, we report here that the nascent antizyme polypeptide is the relevant polyamine sensor that operates in cis to negatively regulate upstream RFS on the polysomes, where its own mRNA is being translated. At low polyamine levels, the emerging antizyme polypeptide inhibits completion of its synthesis causing a ribosome pile-up on antizyme mRNA, whereas polyamine binding to nascent antizyme promotes completion of its synthesis. Thus, our study reveals a novel autoregulatory mechanism, in which binding of a small metabolite to a nascent sensor protein stimulates the latter's synthesis co-translationally.

The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron.

Ornithine decarboxylase (ODC), a homodimeric enzyme with a rate-limiting function in polyamine biosynthesis, is subject to a feedback control involving its selective proteolysis. Targeting of ODC monomers to the proteasome is mediated by ODC antizyme (OAZ), the expression of which is induced by high levels of polyamines. Here, we report our analysis of the N-terminal degron in Saccharomyces cerevisiae ODC and the mechanism of its antizyme-dependent targeting. This ∼45-residue domain of ODC [termed ODC degradation signal (ODS)] is essential for degradation of ODC. Extensive mutagenesis indicated that it is not a specific sequence within ODS that is important but, rather, its unstructured nature. Consistent with this conclusion, ODS could be functionally replaced by an unrelated unstructured domain. We show that increasing the distance of ODS to the rest of the ODC protein reduced the dependence on Oaz1 for targeting, indicating that exposure of ODS is critical for its function. Disruption of ODC dimers by introducing interface mutations, in contrast, was insufficient for targeting. Binding of Oaz1 to ODC monomers is thus required to activate ODS. Fusion of ODS to the N terminus of Ura3 was sufficient to convert it into a ubiquitin-independent substrate of the proteasome. By contrast, ODS failed to destabilize maltose-binding protein or dihydrofolate reductase, indicating that this degron only operates in an appropriate structural context that enables rapid unfolding.

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme.

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.