RESUMO
Inosine 5' monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report the identification of two additional missense variants in IMPDH2 from affected individuals and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation.
Assuntos
IMP Desidrogenase , Purinas , Humanos , Regulação Alostérica , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Mutação , Guanosina TrifosfatoRESUMO
Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 µM. The enzyme then binds NADPH with a dissociation constant of 140 µM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.
Assuntos
Proteínas de Bactérias/genética , FMN Redutase/genética , Regulação Bacteriana da Expressão Gênica , Oxirredutases/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , FMN Redutase/metabolismo , Oxirredutases/metabolismo , Staphylococcus aureus/enzimologiaRESUMO
The Wnt1-Cre transgenic mouse line is extensively used in the study of the development of the neural crest and its derivatives and the midbrain. The Wnt1 gene has important developmental roles in formation of the midbrain-hindbrain boundary, regulation of midbrain size, and neurogenesis of ventral midbrain dopaminergic (mDA) neurons. Here, we report that Wnt1-Cre transgenic mice exhibit phenotypes in multiple aspects of midbrain development. Significant expansion of the midbrain and increased proliferation in the developing inferior colliculus is associated with ectopic expression of Wnt1. Marked elevation of Wnt1 expression in the ventral midbrain is correlated with disruption of the differentiation program of ventral mDA neurons. We find that these phenotypes can be attributed to ectopic expression of Wnt1 from the Wnt1-Cre transgene leading to the ectopic activation of canonical Wnt/ß-catenin signaling. Since these caveats could complicate the utility of Wnt1-Cre in some developmental circumstances, we report a new Wnt1-Cre2 transgenic mouse line that can serve the same purposes as the original without the associated phenotypic complications. These studies reveal an important caveat to a widely-used reagent, provide an improved version of this reagent, and indicate that the original Wnt1-Cre transgenic mouse line may be useful as a gain of function model for interrogating Wnt signaling mechanisms in multiple aspects of midbrain development.
Assuntos
Integrases/metabolismo , Mesencéfalo/embriologia , Neurogênese/fisiologia , Fenótipo , Transdução de Sinais/fisiologia , Proteína Wnt1/metabolismo , Animais , Western Blotting , Bromodesoxiuridina , Primers do DNA/genética , Imunofluorescência , Genótipo , Hibridização In Situ , Integrases/genética , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Crista Neural/metabolismo , Crista Neural/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Regeneration of lost tissue requires biosynthesis of metabolites needed for cell proliferation and growth. Among these are the critical purine nucleotides ATP and GTP. The abundance and balance of these purines is regulated by inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyzes the committing step of GTP synthesis. IMPDH2 assembles into filaments that resist allosteric inhibition under conditions of high GTP demand. Here we asked whether IMPDH2 is required in the highly proliferative context of regeneration, and whether its assembly into filaments takes place in regenerating tissue. We find that inhibition of IMPDH2 leads to impaired tail regeneration and reduced cell proliferation in the tadpole Xenopus tropicalis. We find that both endogenous and fluorescent fusions of IMPDH2 robustly assemble into filaments throughout the tadpole tail, and that the regenerating tail creates a sensitized condition for filament formation. These findings clarify the role of purine biosynthesis in regeneration and reveal that IMPDH2 enzyme filament formation is a biologically relevant mechanism of regulation in vertebrate regeneration.
RESUMO
Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.
Assuntos
Citoesqueleto , Guanosina Trifosfato , IMP Desidrogenase , Retina , Animais , Bovinos , Guanosina Trifosfato/biossíntese , Nucleotídeos , Fosforilação , Retina/enzimologia , IMP Desidrogenase/metabolismoRESUMO
The down-regulation or cellular depletion of protein kinase C (PKC) attendant to prolonged activation by phorbol esters is a widely described property of this key family of signaling enzymes. However, neither the mechanism of down-regulation nor whether this mechanism occurs following stimulation by physiological agonists is known. Here we show that the peptidyl-prolyl isomerase Pin1 provides a timer for the lifetime of conventional PKC isozymes, converting the enzymes into a species that can be dephosphorylated and ubiquitinated following activation induced by either phorbol esters or natural agonists. The regulation by Pin1 requires both the catalytic activity of the isomerase and the presence of a Pro immediately following the phosphorylated Thr of the turn motif phosphorylation site, one of two C-terminal sites that is phosphorylated during the maturation of PKC isozymes. Furthermore, the second C-terminal phosphorylation site, the hydrophobic motif, docks Pin1 to PKC. Our data are consistent with a model in which Pin1 binds the hydrophobic motif of conventional PKC isozymes to catalyze the isomerization of the phospho-Thr-Pro peptide bond at the turn motif, thus converting these PKC isozymes into species that can be efficiently down-regulated following activation.
Assuntos
Peptidilprolil Isomerase/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Bombesina/farmacologia , Células COS , Chlorocebus aethiops , Regulação para Baixo/fisiologia , Fibroblastos/citologia , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Neurotransmissores/farmacologia , Peptidilprolil Isomerase/genética , Proteína Quinase C beta , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Especificidade por Substrato/fisiologia , Ubiquitinação/fisiologiaRESUMO
Regulatory T cells (Tregs) have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to activate Akt efficiently, however, remains unknown. We show that PH-domain leucine-rich-repeat protein phosphatase (PHLPP), which dephosphorylates Akt, is upregulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than those of conventional T cells, and knockdown of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1(-/-) mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1(-/-) mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence, to our knowledge, for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.
Assuntos
Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD4/genética , Antígenos CD4/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/genética , Fosforilação , Reação em Cadeia da Polimerase , Interferência de RNA , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) differentially attenuates Akt, PKC, and ERK1/2 signaling, thereby controlling the duration and amplitude of responses evoked by these kinases. PHLPP1 is expressed in the mammalian central clock, the suprachiasmatic nucleus, where it oscillates in a circadian fashion. To explore the role of PHLPP1 in vivo, we have generated mice with a targeted deletion of the PHLPP1 gene. Here we show that PHLPP1-null mice, although displaying normal circadian rhythmicity, have a drastically impaired capacity to stabilize the circadian period after light-induced resetting, producing a large phase shift after light resetting. Our findings reveal that PHLPP1 exerts a previously unappreciated role in circadian control, governing the consolidation of circadian periodicity after resetting.
Assuntos
Ritmo Circadiano/efeitos da radiação , Luz , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Regulação da Expressão Gênica/efeitos da radiação , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Fosfoproteínas Fosfatases/deficiência , Proteínas tau/metabolismoRESUMO
Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands.
RESUMO
Inosine 5' monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report identification of two additional affected individuals with missense variants in IMPDH2 and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation.
RESUMO
Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.
Assuntos
Hiperalgesia/enzimologia , Hiperalgesia/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Medula Espinal/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Androstadienos/farmacologia , Androstadienos/uso terapêutico , Animais , Carragenina/efeitos adversos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Formaldeído/efeitos adversos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/complicações , Masculino , Proteínas do Tecido Nervoso/metabolismo , Medição da Dor , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/metabolismo , Sirolimo/farmacologia , Medula Espinal/patologia , Estatísticas não Paramétricas , Substância P/farmacologia , Fatores de Tempo , WortmaninaRESUMO
Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Domínios PDZ/fisiologia , Proteína Quinase C-alfa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Astrócitos/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Proteína 1 Homóloga a Discs-Large , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Domínios PDZ/genética , Fosforilação , Ligação Proteica , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/genética , RNA Interferente PequenoRESUMO
Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types, they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial, skeletal, and neurological anomalies. Heterozygous females are more severely affected than hemizygous males, a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation, no direct evidence for this has been demonstrated. Here, by generating hiPSCs from CFNS patients, we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells, thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients.
Assuntos
Anormalidades Craniofaciais/genética , Efrina-B1/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mosaicismo , Células Neuroepiteliais/metabolismo , Diferenciação Celular/genética , Autorrenovação Celular/genética , Reprogramação Celular , Cromossomos Humanos X , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Predisposição Genética para Doença , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/citologia , Inativação do Cromossomo XRESUMO
Cell segregation is the process by which cells self-organize to establish developmental boundaries, an essential step in tissue formation. Cell segregation is a common outcome of Eph/ephrin signaling, but the mechanisms remain unclear. In craniofrontonasal syndrome, X-linked mosaicism for ephrin-B1 expression has been hypothesized to lead to aberrant Eph/ephrin-mediated cell segregation. Here, we use mouse genetics to exploit mosaicism to study cell segregation in the mammalian embryo and integrate live-cell imaging to examine the underlying cellular and molecular mechanisms. Our data demonstrate that dramatic ephrin-B1-mediated cell segregation occurs in the early neuroepithelium. In contrast to the paradigm that repulsive bidirectional signaling drives cell segregation, unidirectional EphB kinase signaling leads to cell sorting by the Rho kinase-dependent generation of a cortical actin differential between ephrin-B1- and EphB-expressing cells. These results define mechanisms of Eph/ephrin-mediated cell segregation, implicating unidirectional regulation of cortical actomyosin contractility as a key effector of this fundamental process.
Assuntos
Actomiosina/metabolismo , Diferenciação Celular , Efrina-B1/metabolismo , Células Neuroepiteliais/citologia , Receptores da Família Eph/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Contagem de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Células Neuroepiteliais/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The recently discovered pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP) family is emerging as a central component in suppressing cell survival pathways. Originally discovered in a rational search for a phosphatase that directly dephosphorylates and inactivates Akt, PHLPP is now known to potently suppress cell survival both by inhibiting proliferative pathways and by promoting apoptotic pathways. In the first instance, PHLPP directly dephosphorylates a conserved regulatory site (termed the hydrophobic motif) on Akt, protein kinase C and S6 kinase, thereby terminating signalling by these pro-survival kinases. In the second instance, PHLPP dephosphorylates and thus activates the pro-apoptotic kinase Mst1, thereby promoting apoptosis. PHLPP is deleted in a large number of cancers and the genetic deletion of one isozyme in a PTEN (phosphatase and tensin homologue located on chromosome 1) +/- (or heterozygous) prostate cancer model results in increased tumourigenesis, underscoring the role of PHLPP as a tumour suppressor. This review summarizes the targets and cellular actions of PHLPP, with emphasis on its role as a tumour suppressor in the oncogenic phosphoinositide 3-kinase (PI3K)/Akt signalling cascade.
Assuntos
Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sobrevivência Celular , Humanos , Masculino , Mutação , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Hyperactivation of the PI 3-kinase/AKT pathway is a driving force of many cancers. Here we identify the AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes neoplasia and, on partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation as a condition for full-blown disease. Surprisingly, the codeletion of PTEN and PHLPP1 in patient samples is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data establish a conceptual framework for progression of PTEN mutant prostate cancer to life-threatening disease.
Assuntos
Mutação , Proteínas Nucleares/fisiologia , PTEN Fosfo-Hidrolase/genética , Fosfoproteínas Fosfatases/fisiologia , Neoplasias da Próstata/patologia , Progressão da Doença , Humanos , Masculino , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismoRESUMO
Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.