Yeast homologs of human MCUR1 regulate mitochondrial proline metabolism.
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ABSTRACT:
SUBMITTER: Zulkifli M
PROVIDER: S-EPMC7519068 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MCUR1 is an evolutionarily conserved mitochondrial protein that has been shown to act as a scaffold factor for the assembly of the mammalian mitochondrial calcium uniporter (MCU). Surprisingly, many fungi that do not have MCU contain MCUR1 homologs, suggesting these proteins possess alternate functions. Herein we characterized two Saccharomyces cerevisiae homologs of MCUR1- Put6 and Put7 as regulators of mitochondrial proline metabolism. Put6 and Put7 are tethered to the inner mitochondrial membrane in a large hetero-oligomeric complex. The abundance and assembly of this complex are regulated by proline and the loss of this complex results in unregulated proline uptake in the mitochondria, leading to redox imbalance in the cell. Yeast cells lacking either Put6 or Put7 exhibit a pronounced defect in proline utilization, which can be corrected by the heterologous expression of human MCUR1. Our work uncovers an unexpected role of evolutionarily conserved MCUR1 homologs in mitochondrial proline metabolism.
2020-09-30 | PXD015476 | Pride
Project description:Ca(2+) flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca(2+) signals and activation of cell death pathways. Mitochondrial Ca(2+) uptake occurs at regions of close apposition with intracellular Ca(2+) release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca(2+) selective ion channel (MiCa; ref. ) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca(2+) uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca(2+) uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca(2+) uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca(2+) uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca(2+) uptake and maintenance of normal cellular bioenergetics.
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Project description:The "last resort" pathway results in ubiquitylation and degradation of RNA polymerase II in response to transcription stress and is governed by factors such as Def1 in yeast. Here, we show that the SMY2 gene acts as a multi-copy suppressor of DEF1 deletion and functions at multiple steps of the last resort pathway. We also provide genetic and biochemical evidence from disparate cellular processes that Smy2 works more broadly as a hitherto overlooked regulator of Cdc48 function. Similarly, the Smy2 homologs GIGYF1 and -2 affect the transcription stress response in human cells and regulate the function of the Cdc48 homolog VCP/p97, presently being explored as a target for cancer therapy. Indeed, we show that the apoptosis-inducing effect of VCP inhibitors NMS-873 and CB-5083 is GIGYF1/2 dependent.
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Project description:The human tumor suppressor neurofibromin contains a cysteine and serine-rich domain/Ras-GTPase activating protein domain (CSRD/RasGAP) and a C-terminal domain (CTD). Domain studies of neurofibromin suggest it has other functions in addition to being a RasGAP, but the mechanisms underlying its tumor suppressor activity are not well understood. The budding yeast Saccharomyces cerevisiae is a good model system for studying neurofibromin function because it possesses Ira1 and Ira2, which are homologous to human neurofibromin in both sequence and function. We found that overexpression of CTD or a neurofibromin CTD-homologous domain (CHD) of Ira1/2 in budding yeast delayed degradation of the securin protein Pds1, whereas overexpression of CSRD/RasGAP did not affect Pds1 degradation. We also found that when CTD or CHD was overexpressed, the number of cells in metaphase was higher than in the control. These results demonstrate that CTD and CHD function in the metaphase to anaphase transition. In addition, ?ira1?ira2 cells bypassed mitotic arrest in response to spindle damage, indicating that Ira1 and Ira2 may be involved in the spindle assembly checkpoint (SAC). However, ?ira1?ira2?mad2 cells are more sensitive to spindle damage than ?mad2 or ?ira1?ira2 cells are, suggesting that Ira1/2 and Mad2 function in different pathways. Overexpression of CTD but not CSRD/RasGAP partially rescued the hypersensitivity of ?ira1?ira2?mad2 cells to microtubule-destabilizing drugs, indicating a role for CTD in the SAC pathway. Taken together, independently of RasGAP activity, the C-terminal domains of neurofibromin, Ira1, and Ira2 regulate the metaphase to anaphase transition in a Mad2-independent fashion.
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Project description:Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.
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Project description:During the mitochondrial permeability transition, a large channel in the inner mitochondrial membrane opens, leading to the loss of multiple mitochondrial solutes and cell death. Key triggers include excessive reactive oxygen species and mitochondrial calcium overload, factors implicated in neuronal and cardiac pathophysiology. Examining the differential behavior of mitochondrial Ca(2+) overload in Drosophila versus human cells allowed us to identify a gene, MCUR1, which, when expressed in Drosophila cells, conferred permeability transition sensitive to electrophoretic Ca(2+) uptake. Conversely, inhibiting MCUR1 in mammalian cells increased the Ca(2+) threshold for inducing permeability transition. The effect was specific to the permeability transition induced by Ca(2+), and such resistance to overload translated into improved cell survival. Thus, MCUR1 expression regulates the Ca(2+) threshold required for permeability transition.
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Project description:Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of ?1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).
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Project description:Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells' proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells' survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.
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Project description:In many plants and microorganisms, intracellular proline has a protective role against various stresses, including heat-shock, oxidation and osmolarity. Environmental stresses induce cellular senescence in a variety of eukaryotes. Here we showed that intracellular proline regulates the replicative lifespan in the budding yeast Saccharomyces cerevisiae. Deletion of the proline oxidase gene PUT1 and expression of the γ-glutamate kinase mutant gene PRO1-I150T that is less sensitive to feedback inhibition accumulated proline and extended the replicative lifespan of yeast cells. Inversely, disruption of the proline biosynthetic genes PRO1, PRO2, and CAR2 decreased stationary proline level and shortened the lifespan of yeast cells. Quadruple disruption of the proline transporter genes unexpectedly did not change intracellular proline levels and replicative lifespan. Overexpression of the stress-responsive transcription activator gene MSN2 reduced intracellular proline levels by inducing the expression of PUT1, resulting in a short lifespan. Thus, the intracellular proline levels at stationary phase was positively correlated with the replicative lifespan. Furthermore, multivariate analysis of amino acids in yeast mutants deficient in proline metabolism showed characteristic metabolic profiles coincident with longevity: acidic and basic amino acids and branched-chain amino acids positively contributed to the replicative lifespan. These results allude to proline metabolism having a physiological role in maintaining the lifespan of yeast cells.
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Project description:Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.
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