Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.978
Filtrar
1.
Plant Sci ; 313: 111063, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34763857

RESUMO

Kiwifruit is known as 'the king of vitamin C' because of the high content of ascorbic acid (AsA) in the fruit. Deciphering the regulatory network and identification of the key regulators mediating AsA biosynthesis is vital for fruit nutrition and quality improvement. To date, however, the key transcription factors regulating AsA metabolism during kiwifruit developmental and ripening processes remains largely unknown. Here, we generated a putative transcriptional regulatory network mediating ascorbate metabolism by transcriptome co-expression analysis. Further studies identified an ethylene response factor AcERF91 from this regulatory network, which is highly co-expressed with a GDP-galactose phosphorylase encoding gene (AcGGP3) during fruit developmental and ripening processes. Through dual-luciferase reporter and yeast one-hybrid assays, it was shown that AcERF91 is able to bind and directly activate the activity of the AcGGP3 promoter. Furthermore, transient expression of AcERF91 in kiwifruit fruits resulted in a significant increase in AsA content and AcGGP3 transcript level, indicating a positive role of AcERF91 in controlling AsA accumulation via regulation of the expression of AcGGP3. Overall, our results provide a new insight into the regulation of AsA metabolism in kiwifruit.


Assuntos
Actinidia/genética , Actinidia/metabolismo , Ácido Ascórbico/metabolismo , Etilenos/metabolismo , Galactose/metabolismo , Guanosina Difosfato/metabolismo , Fosforilases/metabolismo , Ácido Ascórbico/genética , China , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Frutas/genética , Frutas/metabolismo , Galactose/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Guanosina Difosfato/genética , Fosforilases/genética
2.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445383

RESUMO

As crucial signal transducers, G-proteins and G-protein-coupled receptors (GPCRs) have attracted increasing attention in the field of signal transduction. Research on G-proteins and GPCRs has mainly focused on animals, while research on plants is relatively rare. The mode of action of G-proteins is quite different from that in animals. The G-protein α (Gα) subunit is the most essential member of the G-protein signal cycle in animals and plants. The G-protein is activated when Gα releases GDP and binds to GTP, and the relationships with the GPCR and the downstream signal are also achieved by Gα coupling. It is important to study the role of Gα in the signaling pathway to explore the regulatory mechanism of G-proteins. The existence of a self-activated Gα in plants makes it unnecessary for the canonical GPCR to activate the G-protein by exchanging GDP with GTP. However, putative GPCRs have been found and proven to play important roles in G-protein signal transduction. The unique mode of action of G-proteins and the function of putative GPCRs in plants suggest that the same definition used in animal research cannot be used to study uncanonical GPCRs in plants. This review focuses on the different functions of the Gα and the mode of action between plants and animals as well as the functions of the uncanonical GPCR. This review employs a new perspective to define uncanonical GPCRs in plants and emphasizes the role of uncanonical GPCRs and Gα subunits in plant stress resistance and agricultural production.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Plantas/metabolismo , Estresse Fisiológico , Animais , Regulação da Expressão Gênica de Plantas , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Desenvolvimento Vegetal , Proteínas de Plantas/metabolismo , Transdução de Sinais
3.
Nat Commun ; 12(1): 3440, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103529

RESUMO

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.


Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Estresse Fisiológico , Fosfatos Açúcares/metabolismo , Regulação Alostérica , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/ultraestrutura , Evolução Molecular , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Leucoencefalopatias/patologia , Ligantes , Metaboloma , Modelos Moleculares , Mutação/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Fosfatos Açúcares/química
4.
Methods Mol Biol ; 2262: 259-267, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977482

RESUMO

Small GTPases cycle between active GTP bound and inactive GDP bound forms in live cells. They act as molecular switches and regulate diverse cellular processes at different times and locations in the cell. Spatiotemporal visualization of their activity provides important insights into dynamics of cellular signaling. Conformational sensors for GTPase activity (COSGAs) are based on the conserved GTPase fold and have been used as a versatile approach for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.


Assuntos
Técnicas Biossensoriais/métodos , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Proteínas ras/química , Proteínas ras/metabolismo , Humanos , Microscopia Confocal , Conformação Proteica , Transdução de Sinais
5.
Sci Rep ; 11(1): 10523, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006972

RESUMO

Proteasome inhibitors (PIs) represent the gold standard in the treatment of multiple myeloma. Among PIs, Bortezomib (BTZ) is frequently used as first line therapy, but peripheral neuropathy (PN), occurring approximately in 50% of patients, impairs their life, representing a dose-limiting toxicity. Carfilzomib (CFZ), a second-generation PI, induces a significantly less severe PN. We investigated possible BTZ and CFZ off-targets able to explain their different neurotoxicity profiles. In order to identify the possible PIs off-targets we used the SPILLO-PBSS software that performs a structure-based in silico screening on a proteome-wide scale. Among the top-ranked off-targets of BTZ identified by SPILLO-PBSS we focused on tubulin which, by contrast, did not turn out to be an off-target of CFZ. We tested the hypothesis that the direct interaction between BTZ and microtubules would inhibit the tubulin alfa GTPase activity, thus reducing the microtubule catastrophe and consequently furthering the microtubules polymerization. This hypothesis was validated in a cell-free model, since BTZ (but not CFZ) reduces the concentration of the free phosphate released during GTP hydrolysis. Moreover, NMR binding studies clearly demonstrated that BTZ, unlike CFZ, is able to interact with both tubulin dimers and polymerized form. Our data suggest that different BTZ and CFZ neurotoxicity profiles are independent from their proteasome inhibition, as demonstrated in adult mice dorsal root ganglia primary sensory neurons, and, first, we demonstrate, in a cell free model, that BTZ is able to directly bind and perturb microtubules.


Assuntos
Antineoplásicos/toxicidade , Bortezomib/toxicidade , Oligopeptídeos/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Inibidores de Proteassoma/toxicidade , Tubulina (Proteína)/metabolismo , Animais , Biopolímeros/metabolismo , Linhagem Celular , Simulação por Computador , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Ligação Proteica
6.
J Mol Biol ; 433(15): 167061, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34023403

RESUMO

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.


Assuntos
4-Hidroxicumarinas/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Mycobacterium tuberculosis/metabolismo , 4-Hidroxicumarinas/química , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice
7.
J Mol Biol ; 433(15): 167046, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-33971210

RESUMO

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.


Assuntos
Archaea/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Archaea/química , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Mutação , Fatores de Alongamento de Peptídeos/genética , Conformação Proteica , Domínios Proteicos
8.
Plant Physiol ; 185(4): 1574-1594, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33793952

RESUMO

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Ácido Ascórbico/biossíntese , Galactose/metabolismo , Guanosina Difosfato/metabolismo , Fosforilases/metabolismo , Tabaco/genética , Tabaco/metabolismo , Ácido Ascórbico/genética , Galactose/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Guanosina Difosfato/genética , Mutação , Fosforilases/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
9.
Med Sci (Paris) ; 37(4): 372-378, 2021 Apr.
Artigo em Francês | MEDLINE | ID: mdl-33908855

RESUMO

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane.


Assuntos
Proliferação de Células , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Estrutura Quaternária de Proteína , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Proto-Oncogênicas/química , Proteína Enriquecida em Homólogo de Ras do Encéfalo/química , Proteína Regulatória Associada a mTOR/química , Serina-Treonina Quinases TOR/química , Proteínas Supressoras de Tumor/química
10.
Biochemistry ; 60(19): 1552-1563, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33900734

RESUMO

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-d-glycero-α-d-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is d-glycero-l-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-d-glycero-4-keto-ß-l-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-d-glycero-ß-l-gluco-heptose. The three-dimensional crystal structure of Cj1430 was determined to a resolution of 1.85 Å in the presence of bound GDP-d-glycero-ß-l-gluco-heptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 Å. Its fold places it into the short-chain dehydrogenase/reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.


Assuntos
Campylobacter jejuni/metabolismo , Heptoses/biossíntese , Proteínas de Bactérias/química , Campylobacter jejuni/patogenicidade , Guanosina Difosfato/metabolismo , Heptoses/química , Heptoses/metabolismo , Ácidos Cetoglutáricos/metabolismo , Monossacarídeos/metabolismo , Oxirredutases/metabolismo , Polissacarídeos/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/metabolismo
11.
RNA ; 27(6): 665-675, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33758037

RESUMO

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNAHis, producing mature tRNAHis In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G-1 Little attention has been paid to the structural requirements of incoming GTP in 3'-5' nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson-Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5'-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5'-terminal modification of RNAs.


Assuntos
Guanosina Trifosfato/metabolismo , Nucleotidiltransferases/metabolismo , Biotinilação , Candida albicans/enzimologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Humanos , Methanosarcina/enzimologia , Mitocôndrias/enzimologia , Modelos Moleculares , Nucleotídeos/metabolismo , Nucleotidiltransferases/química , RNA de Transferência de Histidina/metabolismo
12.
Nat Struct Mol Biol ; 28(3): 258-267, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33633398

RESUMO

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.


Assuntos
Microscopia Crioeletrônica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/ultraestrutura , Bicamadas Lipídicas , Nanoestruturas/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestrutura , Regulação Alostérica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Guanosina Difosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Micelas , Modelos Moleculares , Neurotensina/química , Neurotensina/metabolismo , Conformação Proteica , Receptores de Neurotensina/química , Transdução de Sinais
13.
Biochim Biophys Acta Mol Cell Res ; 1868(5): 118986, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33581219

RESUMO

Biomolecular condensation through phase separation may be a novel mechanism to regulate bacterial processes, including cell division. Previous work revealed that FtsZ, a protein essential for cytokinesis in most bacteria, forms biomolecular condensates with SlmA, a protein that protects the chromosome from damage inflicted by the division machinery in Escherichia coli. The absence of condensates composed solely of FtsZ under the conditions used in that study suggested this mechanism was restricted to nucleoid occlusion by SlmA or to bacteria containing this protein. Here we report that FtsZ alone, under physiologically relevant conditions, can demix into condensates in bulk and when encapsulated in synthetic cell-like systems generated by microfluidics. Condensate assembly depends on FtsZ being in the GDP-bound state and on conditions mimicking the crowded environment of the cytoplasm that promote its oligomerization. Condensates are dynamic and reversibly convert into filaments upon GTP addition. Notably, FtsZ lacking its C-terminal disordered region, a structural element likely to favor biomolecular condensation, also forms condensates, albeit less efficiently. The inherent tendency of FtsZ to form condensates susceptible to modulation by physiological factors, including binding partners, suggests that such mechanisms may play a more general role in bacterial division than initially envisioned.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Guanosina Difosfato/metabolismo , Proteínas de Bactérias/genética , Citoplasma , Proteínas do Citoesqueleto/genética , Escherichia coli/genética , Técnicas Analíticas Microfluídicas , Microscopia de Fluorescência , Nefelometria e Turbidimetria , Domínios Proteicos , Multimerização Proteica , Deleção de Sequência
14.
J Cell Biol ; 220(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33544140

RESUMO

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the ß subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.


Assuntos
Proteínas de Drosophila/química , Guanosina Trifosfato/química , Microtúbulos/química , Multimerização Proteica , Tubulina (Proteína)/química , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
15.
Int J Biol Macromol ; 171: 59-73, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33412199

RESUMO

Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 µM/min/mg and 39.95±7.7 µM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s-1 and 0.00047±0.0001 µM/s-1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Mycobacterium tuberculosis/genética , Receptores Citoplasmáticos e Nucleares/química , Partícula de Reconhecimento de Sinal/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Cátions Bivalentes , Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Cinética , Magnésio/química , Magnésio/metabolismo , Manganês/química , Manganês/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium tuberculosis/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Transdução de Sinais , Especificidade por Substrato , Termodinâmica
16.
J Cell Biol ; 220(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416861

RESUMO

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase-based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein-dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.


Assuntos
Complexo Dinactina/metabolismo , Dineínas/metabolismo , Septinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Ratos , Septinas/química , Proteínas rab de Ligação ao GTP/metabolismo
17.
FEBS J ; 288(1): 36-55, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32542850

RESUMO

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP-loaded) state and an inactive (GDP-loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post-translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector-recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up-to-date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.


Assuntos
Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Organelas/metabolismo , Processamento de Proteína Pós-Traducional , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/genética , Animais , Transporte Biológico , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/classificação , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Organelas/química , Fosforilação , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terminologia como Assunto , Vesículas Transportadoras/química , Proteínas rab de Ligação ao GTP/classificação , Proteínas rab de Ligação ao GTP/metabolismo
18.
FEBS J ; 288(5): 1565-1585, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32772462

RESUMO

Two small Ras-like GTPases, MglA and SofG, work in synchrony to drive cell polarity and motility in the soil bacterium, Myxococcus xanthus. While MglA regulates two types of motility in Myxococcus and drives cell polarity reversals, SofG regulates social motility enabled by the type IV pili (T4P) machinery. In order to understand the molecular basis of how multiple GTPases act concertedly, we initiated biochemical studies on SofG. A construct of SofG (SofG∆60 ) was purified as a homogenous monomer and could bind to GDP and GTP. Intrinsic GTP hydrolysis by SofG∆60 was negligible. Earlier work from the laboratory revealed that MglB functions both as a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. Biochemical assays of SofG∆60 established that MglB interacts with GTP-bound SofG∆60 and acts as a GAP for SofG∆60 . Interaction of MglB with SofG∆60 in the GDP-bound conformation was not observed, thereby suggesting that MglB might not act as a GEF for SofG∆60 . The existence of a common GAP for both SofG and MglA could potentially contribute to concerted regulation of their GTPase activities, and mediate crosstalk between the two GTPases involved in motility of M. xanthus. Sequence analysis revealed the features for a SofG-like subclass of prokaryotic small Ras-like GTPases that enable MglB to act as a dual-specificity GAP.


Assuntos
Proteínas de Bactérias/química , Fímbrias Bacterianas/genética , GTP Fosfo-Hidrolases/química , Proteínas Ativadoras de GTPase/química , Fatores de Troca do Nucleotídeo Guanina/química , Myxococcus xanthus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Polaridade Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Myxococcus xanthus/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
19.
Mol Cell ; 81(2): 398-407.e4, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33340489

RESUMO

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Treonina-tRNA Ligase/genética , Treonina/metabolismo , Regulação da Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Treonina-tRNA Ligase/antagonistas & inibidores , Treonina-tRNA Ligase/metabolismo
20.
FEBS J ; 288(2): 582-599, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32352209

RESUMO

Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and γ-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG:LG interface. After releasing the cleaved γ-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the ß-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N, 1DG3, 2B92.


Assuntos
Proteínas de Ligação ao GTP/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...