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1.
Nat Commun ; 10(1): 4215, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527595

RESUMO

A major constraint for developing new anti-tuberculosis drugs is the limited number of validated targets that allow eradication of persistent infections. Here, we uncover a vulnerable component of Mycobacterium tuberculosis (Mtb) persistence metabolism, the aspartate pathway. Rapid death of threonine and homoserine auxotrophs points to a distinct susceptibility of Mtb to inhibition of this pathway. Combinatorial metabolomic and transcriptomic analysis reveals that inability to produce threonine leads to deregulation of aspartate kinase, causing flux imbalance and lysine and DAP accumulation. Mtb's adaptive response to this metabolic stress involves a relief valve-like mechanism combining lysine export and catabolism via aminoadipate. We present evidence that inhibition of the aspartate pathway at different branch-point enzymes leads to clearance of chronic infections. Together these findings demonstrate that the aspartate pathway in Mtb relies on a combination of metabolic control mechanisms, is required for persistence, and represents a target space for anti-tuberculosis drug development.


Assuntos
Ácido Aspártico/metabolismo , Mycobacterium tuberculosis/metabolismo , Antituberculosos/farmacologia , Humanos , Lisina/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Treonina/metabolismo , Tuberculose/microbiologia
2.
Amino Acids ; 51(9): 1337-1351, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31428912

RESUMO

The application of high concentrations of taurine induces long-lasting potentiation of synaptic responses and axon excitability. This phenomenon seems to require the contribution of a transport system with a low affinity for taurine. The prototypic taurine transporter TauT (SLC6A6) was discarded by experimental evidence obtained in TauT-KO mice. The purpose of the present study was to determine whether the proton-coupled amino acid transporter 1 (PAT1; SLC36A1) which is a transport system with low affinity and high capacity for a great variety of amino acids including taurine, contributes to the taurine-induced synaptic potentiation. In rat hippocampal slices, the application of several amino acids (L- and D-alanine, L-glutamine, ß-guanidinopropionic acid, glycine, L-histidine, L- and D-serine, sarcosine, L- and D-threonine) imitated the synaptic potentiation induced by taurine. The magnitude of the potentiation caused by some of these amino acids was even greater than that induced by taurine. By contrast, the application of other amino acids (L-arginine, betaine, L-leucine, L-methionine, L- and D-proline, and L-valine) did not induce potentiation. The behaviour of these different amino acids on synaptic potentiation is not compatible with a role of PAT1 in synaptic potentiation. There was a positive correlation between the accumulation of the different amino acids in the slice and the magnitude of synaptic potentiation induced by them. Some of the amino acids inducing synaptic potentiation, like taurine and L-threonine, also increased electrical resistance of the slice, whereas L-leucine did not modify this parameter. Modifications induced by either taurine or L-threonine in synaptic potentiation, slice resistance and amino acid accumulation were dependent on extracellular chloride concentration. These findings support the idea that the accumulation of amino acids throughout the action of transporters causes cell swelling enhancing the electrical resistance of the slice, which by itself could be sufficient to increase field synaptic potentials.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Hipocampo/fisiologia , Simportadores/metabolismo , Potenciais Sinápticos , Aminoácidos/química , Aminoácidos/farmacologia , Animais , Impedância Elétrica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Taurina/metabolismo , Taurina/farmacologia , Treonina/metabolismo , Treonina/farmacologia
3.
PLoS Pathog ; 15(8): e1007980, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31461506

RESUMO

Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses.


Assuntos
Replicação do DNA , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Treonina/metabolismo , Transcrição Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , DNA Viral/genética , Células HEK293 , Humanos , Mutação , Fosforilação , Ligação Proteica , Homologia de Sequência , Treonina/química , Treonina/genética , Proteínas Virais/química , Replicação Viral
4.
Artif Cells Nanomed Biotechnol ; 47(1): 2882-2890, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31307224

RESUMO

Background: Histone H1.5 has been considered as a novel cancer marker as its expression is associated with various human cancers. The objective of this study was to explore the effects of H1.5 phosphorylation in Ras-driven growth and migration of glioma cells. Methods: The plasmids for expression of wide-type of Ras or RasG12V/Y40C were transfected into A172 cells. The expression levels of phosphorylated AKT and H1.5T10ph were tested by Western blot. The effects of H1.5T10ph on A172 cells growth and migration were determined by MTT, soft-agar colony formation, and transwell assay. qRT-PCR and ChIP assay were utilized to assess the role of H1.5T10ph in the transcription of Ras downstream genes. Besides, qRT-PCR and Western blot analysis were carried out to reveal the enzymes which were responsible for phosphorylating H1.5. Results: H1.5T10ph was down-regulated by Ras mutation, which accompanied by the activation of AKT signaling. Ras-driven A172 cells growth and migration were inhibited when H1.5T10ph was overexpressed. Additionally, H1.5T10ph was able to regulate the transcription of Ras downstream genes, including CYR61, IGFBP3, WNT16B, NT5E, GDF15, and CARD16. Further experiments revealed that Ras-AKT signaling repressed H1.5T10ph expression through degradation of GSK3, and the degradation was dependent on MDM2 mediation. Conclusion: Ras-AKT signaling driven the growth and migration of glioma cells possibly through repressing the phosphorylation of H1.5 at threonine 10. Ras-AKT activation repressed H1.5T10ph through MDM2-dependent degradation of GSK3. The findings provide a better understanding of Ras's oncogenic functions which further suggest Ras as a therapeutic target for glioma.


Assuntos
Progressão da Doença , Glioma/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Histonas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Treonina/metabolismo , Proteínas ras/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Histonas/química , Humanos , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Transcrição Genética
5.
Nat Commun ; 10(1): 3325, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346171

RESUMO

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system.


Assuntos
Proteínas do Sistema Complemento/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/metabolismo , Glicina/metabolismo , Serina/metabolismo , Soro/química , Treonina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Ciclo do Ácido Cítrico , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Purinas/biossíntese
6.
Cancer Sci ; 110(9): 2941-2959, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31343810

RESUMO

A sensitive and specific diagnosis biomarker, in principle scalable to most cancer types, is needed to reduce the prevalent cancer mortality. Meanwhile, the investigation of diagnosis determinants of a biomarker will facilitate the interpretation of its screening results in clinic. Here we design a large-scale (1558 enrollments), multicenter (multiple hospitals), and cross-validation (two datasets) clinic study to validate plasma Hsp90α quantified by ELISA as a pan-cancer biomarker. ROC curve shows the optimum diagnostic cutoff is 69.19 ng/mL in discriminating various cancer patients from all controls (AUC 0.895, sensitivity 81.33% and specificity 81.65% in test cohort; AUC 0.893, sensitivity 81.72% and specificity 81.03% in validation cohort). Similar results are noted in detecting early-stage cancer patients. Plasma Hsp90α maintains also broad-spectrum for cancer subtypes, especially with 91.78% sensitivity and 91.96% specificity in patients with AFP-limited liver cancer. In addition, we demonstrate levels of plasma Hsp90α are determined by ADAM10 expression, which will affect Hsp90α content in exosomes. Furthermore, Western blotting and PRM-based quantitative proteomics identify that partial false ELISA-negative patients secret high levels of plasma Hsp90α. Mechanism analysis reveal that TGFß-PKCγ gene signature defines a distinct pool of hyperphosphorylated Hsp90α at Theronine residue. In clinic, a mechanistically relevant population of false ELISA-negative patients express also higher levels of PKCγ. In sum, plasma Hsp90α is a novel pan-cancer diagnosis biomarker, and cancer diagnosis with plasma Hsp90α is particularly effective in those patients with high expression of ADAM10, but may be insufficient to detect the patients with low ADAM10 and those with hyperphosphorylated Hsp90α.


Assuntos
Biomarcadores Tumorais/sangue , Proteínas de Choque Térmico HSP90/sangue , Neoplasias/diagnóstico , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Adolescente , Adulto , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Conjuntos de Dados como Assunto , Ensaio de Imunoadsorção Enzimática , Exossomos/metabolismo , Reações Falso-Negativas , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/patologia , Fosforilação , Estudos Prospectivos , Curva ROC , Treonina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
7.
Mol Pharmacol ; 96(2): 204-211, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31189667

RESUMO

The calcium-sensing receptor (CaS) is the principal controller of extracellular calcium (Ca2+ o) homeostasis and is inhibited in vitro and in vivo by protein kinase C (PKC)-mediated phosphorylation at CaST888 However, PKC inhibition enhances signaling even in CaSs lacking Thr-888, suggesting that an additional inhibitory site exists. An apparently equivalent PKC regulatory site in metabotropic glutamate receptor 5 (Ser-839) aligns not with CaST888 but instead with CaSS875, which was not previously considered to be a PKC site. CaSS875A (nonphosphorylatable) exhibited significantly enhanced Ca2+ o sensitivity of both intracellular Ca2+ mobilization and extracellular signal-regulated kinase 1/2 activation, whereas the phosphomimetic CaSS875D mutant exhibited a loss of function. The CaSS875A/T888A double mutant exhibited even greater Ca2+ o sensitivity than CaST888A alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaSS875A/T888A double mutation elicited maximal activation even under control conditions, but remained sensitive to negative allosteric modulation [N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine] or Ca2+ o removal. Therefore, we have now identified CaSS875 as the missing PKC phosphorylation site that, together with CaST888, shapes the CaS signaling that underpins Ca2+ o homeostasis. Together with the inactive form of the CaS extracellular domain, these sites attenuate Ca2+ o sensitivity to attain appropriate physiologic Ca2+ o sensing. SIGNIFICANCE STATEMENT: Serine-875 represents the missing inhibitory PKC phosphorlyation site in CaS that in tandem with Thr-888 controls receptor activity.


Assuntos
Mutação , Proteína Quinase C/metabolismo , Receptores de Detecção de Cálcio/química , Serina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Células HEK293 , Humanos , Fosforilação , Domínios Proteicos , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Treonina/metabolismo
8.
Elife ; 82019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31124786

RESUMO

Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the -3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling.


Assuntos
Quinases Relacionadas a NIMA/metabolismo , Transdução de Sinais , Especificidade por Substrato , Humanos , Quinases Relacionadas a NIMA/química , Fosforilação , Serina/metabolismo , Treonina/metabolismo
9.
Eur J Pharmacol ; 855: 137-148, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059711

RESUMO

Selective pharmacologic targeting of cell cycle regulators is a potent anti-cancer therapeutic strategy. Here, we show that caspase-3-mediated p21 cleavage involves p53 independent of triptolide (TPL)-induced S phase arrest in human type 1 nasopharyngeal carcinoma (NPC) cells. Coimmunoprecipitation studies demonstrated that TPL causes S phase cell cycle arrest by suppressing the formation of cyclin A-phosphor (p)-cyclin-dependent kinas 2 (CDK2) (Thr 39) complexes. Ectopic expression of constitutively active protein kinase B1 (Akt1) blocks the induction of S phase arrest and the suppression of cyclin A expression and CDK2 Thr 39 phosphorylation by TPL. Expression of the phosphomimetic mutant CDK2 (T39E) rescues the cells from TPL-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TPL-triggered S phase arrest. Treatment with TPL induces an increase in the formation of complexes between unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and p85α in the plasma membrane. Decreased microRNA (miR)-144 expression and increased PTEN expression after TPL treatment were demonstrated, and TPL-enhanced p85α-PTEN complexes and inhibitory effects on Akt (Ser 473) phosphorylation and S phase arrest were suppressed by ectopic PTEN short hairpin RNA or miR-144 expression. Knockdown of endogenous miR-144 by miR-144 Trap upregulated PTEN expression and accordingly enhanced p85α-PTEN complex formation and S phase arrest. Collectively, the effect of TPL on S phase arrest in human NPC cells is likely to enhance the p85α-PTEN interaction in the plasma membrane by suppressing miR-144 expression, resulting in the attenuation of cyclin A-p-CDK2 (Thr 39) complex formation via Akt inactivation.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Diterpenos/farmacologia , Regulação para Baixo/efeitos dos fármacos , MicroRNAs/genética , Neoplasias Nasofaríngeas/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fenantrenos/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Compostos de Epóxi/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Treonina/metabolismo , Quinases Ativadas por p21/metabolismo
10.
Nat Struct Mol Biol ; 26(6): 450-459, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133701

RESUMO

Stalled translation produces incomplete, ribosome-tethered polypeptides that the ribosome-associated quality control (RQC) pathway targets for degradation via the E3 ubiquitin ligase Ltn1. During this process, the protein Rqc2 and the large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins can aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae. We found that CAT tails enhance the efficiency of Ltn1 in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails to ubiquitylate those stalled polypeptides or becomes limiting, CAT tails act as degrons, marking proteins for proteasomal degradation off the ribosome. Thus, CAT tails functionalize the carboxy termini of stalled polypeptides to drive their degradation on and off the ribosome.


Assuntos
Peptídeos/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alanina/química , Alanina/metabolismo , Peptídeos/química , Biossíntese de Proteínas , Proteólise , Proteínas de Ligação a RNA/metabolismo , Especificidade por Substrato , Treonina/química , Treonina/metabolismo
11.
Planta ; 250(2): 589-601, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31134341

RESUMO

MAIN CONCLUSION: The absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.


Assuntos
Genoma de Cloroplastos/genética , Genoma de Planta/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Tabaco/genética , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Fosforilação , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteômica , Treonina/metabolismo , Tabaco/fisiologia
12.
Reprod Domest Anim ; 54(8): 1085-1094, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145488

RESUMO

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.


Assuntos
Acrossomo/fisiologia , Progesterona/farmacologia , Suínos , Treonina/metabolismo , Acrossomo/efeitos dos fármacos , Animais , Exocitose , Regulação da Expressão Gênica , Masculino , Fosforilação , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Treonina/química
13.
Plant Sci ; 283: 195-201, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128689

RESUMO

Methionine and threonine are two essential amino acids whose low levels limit the nutritional quality of seeds. The current objective was to define factors that regulate and might increase their levels in seeds. Feeding experiments carried out on receptacles of developing tobacco (Nicotiana tabacum) capsules showed that 1 mM of S-methylmethionine increased the level of methionine to contents similar to 2.5 mM of homoserine, an intermediate metabolite of the aspartate family of amino acids. The latter also increased the level of threonine. Based on these findings, we generated tobacco seeds that expressed a combination of bacterial feedback-insensitive aspartate kinase (bAK), which was previously reported to have a high level of threonine/methionine, and feedback-insensitive cystathionine γ-synthase (CGS), the regulatory enzyme of the methionine biosynthesis pathway. Plants expressing this latter gene previously showed having higher levels of methionine. The results of total amino acids analysis showed that the level of threonine was highest in the bAK line, which has moderate levels of methionine and lysine, while the highest level of methionine was found in seeds expressing both heterologous genes. The results suggest that the level of threonine in tobacco seeds is limited by the substrate, while that of methionine is limited also by the activity of CGS.


Assuntos
Carbono-Oxigênio Liases/metabolismo , Metionina/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Treonina/metabolismo , Tabaco/metabolismo , Aminoácidos/metabolismo , Homosserina/metabolismo , Redes e Vias Metabólicas , Plantas Geneticamente Modificadas , Sementes/enzimologia , Tabaco/enzimologia
14.
Nat Cell Biol ; 21(4): 420-429, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936473

RESUMO

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.


Assuntos
Dopamina/metabolismo , Olho/irrigação sanguínea , Luz , Proteínas de Membrana/metabolismo , Opsinas/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Endotélio Vascular/metabolismo , Olho/enzimologia , Olho/crescimento & desenvolvimento , Olho/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Opsinas/genética , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Treonina/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/fisiologia , Corpo Vítreo/metabolismo
15.
Nat Commun ; 10(1): 1676, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976006

RESUMO

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.


Assuntos
Divisão Celular/fisiologia , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Cristalografia por Raios X , Ciclina A/isolamento & purificação , Quinase 2 Dependente de Ciclina/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/isolamento & purificação , Proteínas de Fusão bcr-abl/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo , Tirosina/metabolismo , Quinases da Família src/isolamento & purificação , Quinases da Família src/metabolismo
16.
Nat Commun ; 10(1): 1689, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976076

RESUMO

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.


Assuntos
Inflamassomos/metabolismo , Melanoma Experimental/patologia , Mitocôndrias/fisiologia , Piroptose/fisiologia , Receptores Estrogênicos/metabolismo , Neoplasias Cutâneas/patologia , Animais , Caspase 3/metabolismo , Citocromos c/metabolismo , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Mutação , Fosforilação/fisiologia , Cultura Primária de Células , Domínios Proteicos/genética , Receptores Estrogênicos/genética , Treonina/metabolismo
17.
Biochemistry ; 58(16): 2125-2132, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30912640

RESUMO

Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.


Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Bactérias/metabolismo , Peptídeos Cíclicos/metabolismo , Prochloron/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Ciclização , Cisteína/química , Cisteína/metabolismo , Difosfatos/metabolismo , Modelos Químicos , Estrutura Molecular , Peptídeos Cíclicos/química , Prochloron/fisiologia , Treonina/química , Treonina/metabolismo , Urocordados/microbiologia
18.
Poult Sci ; 98(8): 3233-3239, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30778573

RESUMO

This study aimed to find the digestible lysine (d.Lys), digestible sulfur amino acids (d.SAA), and digestible threonine (d.Thr) requirements to optimize body weight gain (BWG) and feed conversion ratio (FCR) via adaptive neuro-fuzzy inference systems (ANFIS) using either the Genetic algorithm (ANFIS-GA) or Particle Swarm Optimization algorithm (ANFIS-PSO) in Cobb-500 chicks from 1 to 10 d of age. The range of amino acids was 90 to 115% of the recommendations for male Cobb-500 chicks. The estimated dietary d.Lys, d.SAA, and d.Thr requirements by ANFIS-GA and ANFIS-PSO to optimize BWG were the same and were 12.10, 8.98, and 7.89 g/kg, respectively. The optimum BWG predicted by ANFIS-GA and ANFIS-PSO were 270 and 266 g, respectively for the 1 to 10 d period. The estimated dietary requirements of d.Lys, d.SAA, and d.Thr to minimize FCR at 0.995 by ANFIS-GA were 12.10, 8.98, and 7.89 g/kg, respectively. Although the estimated d.Lys and d.SAA requirements by ANFIS-PSO and ANFIS-GA were identical, the predicted d.Thr requirement by ANFIS-PSO was 0.01 g/kg higher than by ANFIS-GA to minimize FCR at 0.963. Comparison of goodness of fit in term of root mean square error revealed that the ANFIS-GA prediction was more accurate than ANFIS-PSO. This study demonstrates that the hybrid methodology of ANFIS-GA is as an effective and accurate approach to modeling and optimizing nutrient requirements.


Assuntos
Aminoácidos/metabolismo , Galinhas/fisiologia , Necessidades Nutricionais , Algoritmos , Aminoácidos Sulfúricos/metabolismo , Ração Animal/análise , Animais , Dieta/veterinária , Digestão , Lisina/metabolismo , Masculino , Modelos Teóricos , Treonina/metabolismo , Ganho de Peso
19.
Immunity ; 50(3): 576-590.e6, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30770249

RESUMO

Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.


Assuntos
Apoptose/fisiologia , Inflamação/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Imunidade Inata/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Serina/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo
20.
Nat Chem Biol ; 15(2): 123-131, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30598543

RESUMO

Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of Y1S2P3T4S5P6S7 heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3' regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases place phospho-Thr4 marks to regulate expression of targeted genes.


Assuntos
Proteínas Quinases/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/fisiologia , Sequência de Aminoácidos , Caseína Quinase I/metabolismo , Fosforilação , Filogenia , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Transcrição Genética
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