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1.
Cell ; 187(14): 3602-3618.e20, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38823389

RESUMO

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.


Assuntos
Neoplasias , Nucleotídeos de Purina , Purinas , Animais , Camundongos , Purinas/metabolismo , Purinas/biossíntese , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotídeos de Purina/metabolismo , Humanos , Inosina/metabolismo , Hipoxantina/metabolismo , Camundongos Endogâmicos C57BL , Adenina/metabolismo , Linhagem Celular Tumoral , Feminino
2.
Microb Cell Fact ; 23(1): 66, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402195

RESUMO

BACKGROUND: Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (PPEPCK) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins. RESULTS: PPEPCK-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from PPEPCK. The traditional, methanol-inducible alcohol oxidase 1 promoter (PAOX1) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100-120 A600 units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from PPEPCK that is comparable or even higher than that from PAOX1. CONCLUSIONS: We have designed an induction medium for recombinant protein production from K. phaffii PPEPCK in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from PPEPCK equaled and even surpassed those from PAOX1. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii PPEPCK in bioreactors.


Assuntos
Metanol , Saccharomycetales , Metanol/metabolismo , Glutamato de Sódio/farmacologia , Glutamato de Sódio/metabolismo , Proteínas Recombinantes , Glutamatos/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Etanol/metabolismo , Pichia/genética , Pichia/metabolismo
3.
Yeast ; 39(5): 337-347, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35384037

RESUMO

The yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbours a unique glutamate utilization pathway in which the cytosolic enzymes glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the sequential conversion of glutamate to α-ketoglutarate, oxaloacetate and phosphoenolpyruvate respectively. GDH2 and PEPCK are essential for glutamate catabolism. Their synthesis is induced by autophagy during carbon starvation and are essential for cell survival. Here, we demonstrate that GDH2 and PEPCK reciprocally regulate each other's protein levels during glutamate catabolism such that GDH2 is downregulated in Δpepck and PEPCK is downregulated in Δgdh2. We further demonstrate that sequential conversion of glutamate to α-ketoglutarate and oxaloacetate by GDH2 and AAT2, respectively, is essential for PEPCK synthesis in cells metabolizing glutamate. Our studies indicate that translation of GDH2 mRNA is induced by glutamate while oxaloacetate derived from glutamate is likely to be the inducer of PEPCK mRNA translation during glutamate catabolism. Thus, GDH2- and PEPCK-catalyzed reactions are essential for ATP generation and gluconeogenesis respectively during carbon starvation and glutamate catabolism in K. phaffii. We conclude that K. phaffii harbours a unique translational regulatory circuit in which substrates of GDH2 and PEPCK act as inducers of their synthesis, a phenomenon not reported in any yeast species.


Assuntos
Glutamato Desidrogenase , Ácidos Cetoglutáricos , Carbono/metabolismo , Regulação Fúngica da Expressão Gênica , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Glutamatos/metabolismo , Oxaloacetatos , Fosfoenolpiruvato , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Saccharomycetales , Leveduras/metabolismo
4.
Biochem Biophys Res Commun ; 581: 25-30, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34653675

RESUMO

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.


Assuntos
Carbono/deficiência , Proteínas Fúngicas/genética , Desidrogenase de Glutamato (NADP+)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , RNA Mensageiro/genética , Saccharomycetales/efeitos dos fármacos , Aminoácidos/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia , Carbono/farmacologia , Proteínas Fúngicas/agonistas , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Desidrogenase de Glutamato (NADP+)/biossíntese , Metabolismo/genética , Viabilidade Microbiana , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/agonistas , RNA Mensageiro/biossíntese , Saccharomycetales/enzimologia , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento
5.
J Biol Chem ; 293(43): 16647-16660, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30185617

RESUMO

Rtg1p and Rtg3p are two basic helix-loop-helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Pichia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Citosol/metabolismo , Proteínas Fúngicas/genética , Mitocôndrias/metabolismo , Pichia/genética , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica
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