RESUMO
Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here, we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous Parkinson's disease-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38-binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.
Assuntos
Membranas Intracelulares , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Melanócitos , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Melanócitos/metabolismo , Mutação , Doença de Parkinson/metabolismo , Fosforilação , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Expressão Gênica , Domínios Proteicos , Ligação Proteica , Membranas Intracelulares/metabolismoRESUMO
The NEK1 kinase controls ciliogenesis, mitosis, and DNA repair, and NEK1 mutations cause human diseases including axial spondylometaphyseal dysplasia and amyotrophic lateral sclerosis. C21ORF2 mutations cause a similar pattern of human diseases, suggesting close functional links with NEK1 Here, we report that endogenous NEK1 and C21ORF2 form a tight complex in human cells. A C21ORF2 interaction domain "CID" at the C-terminus of NEK1 is necessary for its association with C21ORF2 in cells, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts an extended binding interface between a leucine-rich repeat domain in C21ORF2 and the NEK1-CID, and our model may explain why pathogenic mutations perturb the complex. We show that NEK1 mutations that inhibit kinase activity or weaken its association with C21ORF2 severely compromise ciliogenesis, and that C21ORF2, like NEK1 is required for homologous recombination. These data enhance our understanding of how the NEK1 kinase is regulated, and they shed light on NEK1-C21ORF2-associated diseases.
Assuntos
Reparo do DNA , Osteocondrodisplasias , Humanos , Mutação/genética , Quinase 1 Relacionada a NIMA/genética , Osteocondrodisplasias/genética , FosforilaçãoRESUMO
BACKGROUND: Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. METHODS: Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. RESULTS: Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. CONCLUSIONS: Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.
Assuntos
Centrossomo/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/patologia , FosforilaçãoRESUMO
This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the "Warburg" and "Crabtree" effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the "Warburg effect" and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the "Warburg effect", and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the "Warburg effect", i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as "multiple myeloma". Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.
Assuntos
Antineoplásicos/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Hexoquinase/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Piruvatos/uso terapêutico , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Mitocôndrias/metabolismo , Mieloma Múltiplo/tratamento farmacológicoRESUMO
In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicólise/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Piruvatos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Genoma Fúngico/genética , Glicólise/genética , Humanos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Via de Pentose Fosfato/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossínteseRESUMO
The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/patologia , Piruvatos/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/metabolismo , Benzamidas/farmacologia , Butionina Sulfoximina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mieloma Múltiplo/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Piruvatos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
We have investigated the antifungal activity of the pyruvic acid analogue: 3-bromopyruvate (3-BP). Growth inhibition by 3-BP of 110 strains of yeast-like and filamentous fungi was tested by standard spot tests or microdilution method. The human pathogen Cryptococcus neoformans exhibited a low Minimal Inhibitory Concentration (MIC) of 0.12-0.15 mM 3-BP. The high toxicity of 3-BP toward C. neoformans correlated with high intracellular accumulation of 3-BP and also with low levels of intracellular ATP and glutathione. Weak cytotoxicity towards mammalian cells and lack of resistance conferred by the PDR (Pleiotropic Drug Resistance) network in the yeast Saccharomyces cerevisiae, are other properties of 3-BP that makes it a novel promising anticryptococcal drug.
Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Piruvatos/farmacologia , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Anfotericina B/farmacologia , Transporte Biológico , Cryptococcus neoformans/patogenicidade , Fluconazol/farmacologia , Glutationa/metabolismo , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Fatores de TempoRESUMO
We have investigated the cytotoxicity in Saccharomyces cerevisiae of the novel antitumor agent 3-bromopyruvate (3-BP). 3-BP enters the yeast cells through the lactate/pyruvate H(+) symporter Jen1p and inhibits cell growth at minimal inhibitory concentration of 1.8 mM when grown on non-glucose conditions. It is not submitted to the efflux pumps conferring Pleiotropic Drug Resistance in yeast. Yeast growth is more sensitive to 3-BP than Gleevec (Imatinib methanesulfonate) which in contrast to 3-BP is submitted to the PDR network of efflux pumps. The sensitivity of yeast to 3-BP is increased considerably by mutations or chemical treatment by buthionine sulfoximine that decrease the intracellular concentration of glutathione.
Assuntos
Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/toxicidade , Piruvatos/farmacocinética , Piruvatos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Antineoplásicos Alquilantes/metabolismo , Butionina Sulfoximina/farmacologia , Glutationa/metabolismo , Testes de Sensibilidade Microbiana , Transportadores de Ácidos Monocarboxílicos/metabolismo , Piruvatos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismoRESUMO
Mechanisms of arsenic uptake and detoxification are present in all studied organisms. These mechanisms are considerably well described in unicellular organisms such as bacterium Escherichia coli and baker's yeast Saccharomyces cerevisiae, still leaving much to be revealed in multicellular organisms. Full identification of arsenic uptake and detoxification is of great importance. This knowledge can be very helpful in improving effectiveness of arsenic-containing drugs used in chemotherapy of parasitoses as well as in treatment of acute promielyocytic leukemia. Increased proficiency of bioremediation of arsenic-contaminated soils can be obtained by using plants hyperaccumulating arsenic. This kind of plants can be engineered by modulating expression levels of genes encoding arsenic transporters. The same technique may be used to decrease levels of accumulated arsenic in crops. The aim of this paper is to review current knowledge about systems of arsenic uptake in every studied organism--from bacteria to human.