Proteasome control of [URE3] prion propagation by degradation of anti-prion proteins Cur1 and Btn2 in Saccharomyces cerevisiae

[URE3] is a prion of the nitrogen catabolism controller, Ure2p, and [PSI+] is a prion of the translation termination factor Sup35p in S. cerevisiae. Btn2p cures [URE3] by sequestration of Ure2p amyloid filaments. Cur1p, paralogous to Btn2p, also cures [URE3], but by a different (unknown) mechanism. We find that an array of mutations impairing proteasome assembly or MG132 inhibition of proteasome activity result in loss of [URE3]. In proportion to their prion—curing effects, each mutation affecting proteasomes elevates the cellular concentration of the antiprion proteins Btn2 and Cur1. Of > 4600 proteins detected by SILAC, Btn2p was easily the most overexpressed in a pre9Δ (α3 core subunit) strain. Indeed, deletion of BTN2 and CUR1 prevents the prion—curing effects of proteasome impairment. Surprisingly, the 15 most unstable yeast proteins are not increased in pre9Δ cells suggesting altered proteasome specificity rather than simple inactivation. Hsp42, a chaperone that cooperates with Btn2 and Cur1 in curing [URE3], is also necessary for the curing produced by proteasome defects, although Hsp42p levels are not substantially altered by a proteasome defect. We find that pre9Δ and proteasome chaperone mutants that most efficiently lose [URE3], do not destabilize [PSI+] or alter cellular levels of Sup35p. A tof2 mutation or deletion likewise destabilizes [URE3], and elevates Btn2p, suggesting that Tof2p deficiency inactivates proteasomes. We suggest that when proteasomes are saturated with denatured/misfolded proteins, their reduced degradation of Btn2p and Cur1p automatically upregulates these aggregate-handling systems to assist in the clean-up.

The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.

Normal levels of ribosome-associated chaperones cure two groups of [PSI+] prion variants

The yeast prion [PSI+] is a self-propagating amyloid of the translation termination factor, Sup35p. For known pathogenic prions, such as [PSI+], a single protein can form an array of different amyloid structures (prion variants) each stably inherited and with differing biological properties. The ribosome-associated chaperones, Ssb1/2p (Hsp70s), and RAC (Zuo1p (Hsp40) and Ssz1p (Hsp70)), enhance de novo protein folding by protecting nascent polypeptide chains from misfolding and maintain translational fidelity by involvement in translation termination. Ssb1/2p and RAC chaperones were previously found to inhibit [PSI+] prion generation. We find that most [PSI+] variants arising in the absence of each chaperone were cured by restoring normal levels of that protein. [PSI+] variants hypersensitive to Ssb1/2p have distinguishable biological properties from those hypersensitive to Zuo1p or Ssz1p. The elevated [PSI+] generation frequency in each deletion strain is not due to an altered [PIN+], another prion that primes [PSI+] generation. [PSI+] prion generation/propagation may be inhibited by Ssb1/2/RAC chaperones by ensuring proper folding of nascent Sup35p, thus preventing its joining amyloid fibers. Alternatively, the effect of RAC/Ssb mutations on translation termination and the absence of an effect on the [URE3] prion suggest an effect on the mature Sup35p such that it does not readily join amyloid filaments. Ssz1p is degraded inzuo1Δ [psi-] cells, but not if the cells carry any of several [PSI+] variants. Our results imply that prions arise more frequently than had been thought but the cell has evolved exquisite antiprion systems that rapidly eliminate most variants.

Nonsense Mutations in the Yeast SUP35 Gene Affect the [PSI+] Prion Propagation

The essential SUP35 gene encodes yeast translation termination factor eRF3. Previously, we isolated nonsense mutations sup35-n and proposed that the viability of such mutants can be explained by readthrough of the premature stop codon. Such mutations, as well as the prion [PSI+], can appear in natural yeast populations, and their combinations may have different effects on the cells. Here, we analyze the effects of the compatibility of sup35-n mutations with the [PSI+] prion in haploid and diploid cells. We demonstrated that sup35-n mutations are incompatible with the [PSI+] prion, leading to lethality of sup35-n [PSI+] haploid cells. In diploid cells the compatibility of [PSI+] with sup35-n depends on how the corresponding diploid was obtained. Nonsense mutations sup35-21, sup35-74, and sup35-218 are compatible with the [PSI+] prion in diploid strains, but affect [PSI+] properties and lead to the formation of new prion variant. The only mutation that could replace the SUP35 wild-type allele in both haploid and diploid [PSI+] strains, sup35-240, led to the prion loss. Possibly, short Sup351–55 protein, produced from the sup35-240 allele, is included in Sup35 aggregates and destabilize them. Alternatively, single molecules of Sup351–55 can stick to aggregate ends, and thus interrupt the fibril growth. Thus, we can conclude that sup35-240 mutation prevents [PSI+] propagation and can be considered as a new pnm mutation.

A Novel Cereblon E3 Ligase Modulator Eradicates Acute Myeloid Leukemia Stem Cells through Degradation of Translation Termination Factor GSPT1

Acute myeloid leukemia (AML) is a clonal malignant disease initiated and propagated by leukemia stem cells (LSCs). Both LSCs and normal hematopoietic stem cells (HSCs) share many biological properties including self-renewal and quiescence. One such shared property that we have recently established involves the pro-survival features of proteostatic stress signaling. Stem cells have reduced protein translation initiation due to scarcity of the eIF2α translation initiation complex (van Galen et al Nature 2014; Cell Reports 2018). This in turn, increases the activity of activating transcription factor 4 (ATF4) uniquely in HSCs and LSCs. In homeostasis, this level of ATF4 facilitates stem cell persistence and survival, but upon stronger stress activation stem cell apoptosis ensues. This mechanism predicts that agonists of the integrated stress response (ISR) could provide a novel therapeutic approach to eradicate LSCs. Here we report that the novel cereblon E3 ligase modulator (CELMoD) CC-90009, which causes degradation of the translation termination factor G1 to S phase transition protein 1 (GSPT1) and downstream activation of ISR, is potent against primary AML both in vitro and in vivo, and reduces self-renewing LSCs in preclinical xenograft models for human AML. We first carried out in vitro assays to evaluate the effect of CC-90009 on primary AML samples. We found that CC-90009 degraded GSPT1 in primary AML cells and induced leukemic cell apoptosis in 24 hours. Leukemic colony forming progenitors were also reduced by CC-90009 in a dose-dependent manner. We next tested the efficacy of CC-90009 against primary AML samples in xenografts in NOD/SCID mice. Leukemia cells were transplanted intrafemorally 21 days prior to CC-90009 treatment. Mice were treated with vehicle or CC-90009 at 2.5mg/kg BID for 4 weeks. Heterogeneous responses to the CC-90009 treatment were observed. Of 35 AML samples tested, 16 were highly responsive to CC-90009 with >75% reduction of AML engraftment, 10 showed moderate response between 45% and 75% reductions, and 9 showed reductions of <25%. AML is clinically characterized by accumulation of blasts that are impaired for differentiation and maturation. We observed that, in addition to the reduction of total AML graft, CC-90009 also induced myeloid differentiation of AML blasts in the CC-90009 responders, as evidenced by increases in late myeloid cell surface markers (CD14, CD15 and CD11b) and reductions of the immature marker CD34. To determine the efficacy of CC-90009 against AML cases at high risk of relapse following standard induction chemotherapy, we assessed CC-90009 efficacy vs. the status of an expression-based 17-gene leukemia stem cell score (the LSC17 score) that was recently implemented for rapid risk stratification of AML patients (Ng et al, Nature 2016). LSC17-high patients are predicted to have poor treatment response and poor clinical outcome. We found that, while 8 out of 9 poor responders to CC-90009 had high LSC17 scores, 20 out of 28 samples that had high LSC17 scores responded well to CC-90009, indicating that the drug is able to target high risk cases. Serial transplantation utilizing limiting dilution analysis showed that CC-90009 targeted self-renewing LSCs. Our data established that a new CELMoD CC-90009 has anti-proliferative effects on human primary AML cells and self-renewing LSCs evaluated in xenograft assays. These observations provide important implications for CC-90009 in its clinical development as a new therapeutic agent to treat AML patients with high risk disease when treated with standard of care therapies. Currently, a phase I study evaluating CC-90009 in relapsed or refractory AML is ongoing (CC-90009-AML-001; NCT02848001). Disclosures Jin: Trillium Therapeutics: Other: licensing agreement. Ng:Celgene: Research Funding. Wang:Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; NanoString: Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation. Minden:Trillium Therapetuics: Other: licensing agreement. Fan:Celgene Corporation: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership.

The Pub1 and Upf1 Proteins Act in Concert to Protect Yeast from Toxicity of the [PSI+] Prion

The [PSI+] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI+] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI+], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI+] formation, while Sla2 can prevent [PSI+] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI+] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI+] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI+] detoxification system.