FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast

免疫抑制剤の新しい作用メカニズムの解明 --FKBP12は真菌のイソロイシン生合成酵素を抑制する--. 京都大学プレスリリース. 2022-12-13.FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis

The copper transport-associated protein Ctr4 can form prion-like epigenetic determinants in Schizosaccharomyces pombe

Prions are protein-based infectious entities associated with fatal brain diseases in animals, but also modify a range of host-cell phenotypes in the budding yeast, Saccharomyces cerevisiae. Many questions remain about the evolution and biology of prions. Although several functionally distinct prion-forming proteins exist in S. cerevisiae, [HET-s] of Podospora anserina is the only other known fungal prion. Here we investigated prion-like, protein-based epigenetic transmission in the fission yeast Schizosaccharomyces pombe. We show that S. pombe cells can support the formation and maintenance of the prion form of the S. cerevisiae Sup35 translation factor [PSI+], and that the formation and propagation of these Sup35 aggregates is inhibited by guanidine hydrochloride, indicating commonalities in prion propagation machineries in these evolutionary diverged yeasts. A proteome-wide screen identified the Ctr4 copper transporter subunit as a putative prion with a predicted prion-like domain. Overexpression of the ctr4 gene resulted in large Ctr4 protein aggregates that were both detergent and proteinase-K resistant. Cells carrying such [CTR+] aggregates showed increased sensitivity to oxidative stress, and this phenotype could be transmitted to aggregate-free [ctr–] cells by transformation with [CTR+] cell extracts. Moreover, this [CTR+] phenotype was inherited in a non-Mendelian manner following mating with naïve [ctr–] cells, but intriguingly the [CTR+] phenotype was not eliminated by guanidine-hydrochloride treatment. Thus, Ctr4 exhibits multiple features diagnostic of other fungal prions and is the first example of a prion in fission yeast. These findings suggest that transmissible protein-based determinants of traits may be more widespread among fungi

The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation

Ubiquitination is a major post-translational modification of ribosomal proteins. The role of ubiquitination in the regulation of ribosome functions is still being elucidated. However, the importance of ribosome deubiquitination remains unclear. Here, we show that the cycle of ubiquitination and deubiquitination of the 40S ribosome subunit eS7 is important for efficient translation. eS7 ubiquitination at lysine 83 is required for efficient protein translation. We identified Otu2 and Ubp3 as the deubiquitinating enzymes for eS7. An otu2Δubp3Δ mutation caused a defect in protein synthesis. Ubp3 inhibited polyubiquitination of eS7 in polysomes to keep eS7 in a mono-ubiquitinated form, whereas Otu2 was specifically bound to the free 40S ribosome and promoted the dissociation of mRNAs from 40S ribosomes in the recycling step. Our results provide clues for understanding the molecular mechanism of the translation system via a ubiquitination-deubiquitination cycle

Overexpression profiling reveals cellular requirements in the context of genetic backgrounds and environments

Overexpression can help life adapt to stressful environments, making an examination of overexpressed genes valuable for understanding stress tolerance mechanisms. However, a systematic study of genes whose overexpression is functionally adaptive (GOFAs) under stress has yet to be conducted. We developed a new overexpression profiling method and systematically identified GOFAs in Saccharomyces cerevisiae under stress (heat, salt, and oxidative). Our results show that adaptive overexpression compensates for deficiencies and increases fitness under stress, like calcium under salt stress. We also investigated the impact of different genetic backgrounds on GOFAs, which varied among three S. cerevisiae strains reflecting differing calcium and potassium requirements for salt stress tolerance. Our study of a knockout collection also suggested that calcium prevents mitochondrial outbursts under salt stress. Mitochondria-enhancing GOFAs were only adaptive when adequate calcium was available and non-adaptive when calcium was deficient, supporting this idea. Our findings indicate that adaptive overexpression meets the cell's needs for maximizing the organism's adaptive capacity in the given environment and genetic context. Author summaryThe study aimed to investigate how overexpression of genes can aid organisms in adapting to stress. The researchers utilized a new method to identify genes in yeast that demonstrated functional adaptability when overexpressed under stress such as heat, salt, and oxidative stress. The results indicated that overexpressing specific genes, like calcium, during salt stress could counteract deficiencies and improve the organism's ability to withstand stress. The study also examined the effect of different genetic backgrounds on these genes and discovered that the impact differed among various yeast strains. Additionally, the study revealed that calcium could play a key role in adapting to salt stress by preventing mitochondrial outbursts. These findings suggest that overexpressing certain genes can help the organism maximize its adaptability to stress in a given environment and genetic context

Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking

AbstractThe membrane traffic and stability of the general amino acid permease Gap1 of Saccharomyces cerevisiae are under nitrogen control. Addition of a preferential nitrogen source such as ammonium to cells growing on a poor nitrogen source induces internalization of the permease and its subsequent degradation in the vacuole. This down-regulation requires ubiquitination of Gap1 through a process involving ubiquitin ligase Npi1/Rsp5, ubiquitin hydrolase Npi2/Doa4, and Bul1/2, two Npi1/Rsp5 interacting proteins. Here we report that yet another protein, Npi3, is involved in the regulation of Gap1 trafficking. We show that Npi3 is required for NH4+-induced down-regulation of Gap1, and particularly for efficient ubiquitination of the permease. Npi3 plays a pleiotropic role in permease down-regulation, since it is also involved in ubiquitination and stress-induced down-regulation of the uracil permease Fur4 and in glucose-induced degradation of hexose transporters Hxt6/7. We further provide evidence that Npi3 is required for direct vacuolar sorting of neosynthesized Gap1 permease as it occurs in npr1 mutant cells. NPI3 is identical to BRO1, a gene encoding a protein of unknown biochemical function and recently proposed to be involved in protein turnover. Npi3/Bro1 homologues include fungal proteins required for proteolytic cleavage of zinc finger proteins and the mouse Aip1 protein involved in apoptosis. We propose that proteins of the Npi3/Bro1 family, including homologues from higher species, may play a conserved role in ubiquitin-dependent control of membrane protein trafficking

Arrestin-Mediated Endocytosis of Yeast Plasma Membrane Transporters

Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters

Broad targeting of resistance to apoptosis in cancer

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer

Targeting of Sna3p to the Endosomal Pathway Depends on Its Interaction with Rsp5p and Multivesicular Body Sorting on Its Ubiquitylation

Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains

Small molecule inhibitors of CRM1

The transport through the nuclear pore complex is used by cancer cells to evade tumor-suppressive mechanisms. Several tumor-suppressors have been shown to be excluded from the cell nucleus in cancer cells by the nuclear export receptor CRM1 and abnormal expression of CRM1 is oncogenic. Inhibition of CRM1 has long been postulated as potential approach for the treatment of cancer and to overcome therapy resistance. Furthermore, the nuclear export of viral components mediated by the CRM1 is crucial in various stages of the viral lifecycle and assembly of many viruses from diverse families, including coronavirus. However, the first nuclear export inhibitors failed or never entered into clinical trials. More recently CRM1 reemerged as a cancer target and a successful proof of concept was achieved with the clinical approval of Selinexor. The chemical complexity of natural products is a promising perspective for the discovery of new nuclear export inhibitors with a favorable toxicity profile. Several screening campaigns have been performed and several natural product-based nuclear export inhibitors have been identified. With this review we give an overview over the role of CRM1-mediated nuclear export in cancer and the effort made to identify and develop nuclear export inhibitors in particular from natural sources.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research 1334 and by the Spanish Ministry of Science, Innovation and Universities through Grant RTI2018-094629-B-I00 to WL. BF was supported by FCT-SFRH/BPD/100434/2014 and Marie Curie Individual Fellowship project TRIBBLES (#748585). This work was also supported by two LPCC-NRS/Terry Fox grants (2016/2017; 2017/2018).info:eu-repo/semantics/publishedVersio

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein