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1.
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
2.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
3.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361059

RESUMO

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.


Assuntos
Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética
4.
Nat Cell Biol ; 23(8): 859-869, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34253896

RESUMO

Dynamin has an important role in clathrin-mediated endocytosis by cutting the neck of nascent vesicles from the cell membrane. Here, using gold nanorods as cargos to image dynamin action during live clathrin-mediated endocytosis, we show that, near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle increases substantially, accompanied by simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by the coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.


Assuntos
Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Dinaminas/fisiologia , Endocitose/fisiologia , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Humanos , Microscopia/métodos , Nanotubos , Torque
5.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
6.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
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.
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
9.
Methods Mol Biol ; 2262: 137-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977475

RESUMO

Various biochemical methods have been introduced to detect and characterize small GTPases and Ras. Luminescence-based techniques cover most of the currently used methods, utilizing single- or multi-luminophore-conjugated molecules and external probes. Here we describe methods enabling Ras activity and activation state monitoring in vitro. This chapter focuses mainly on luminescence-based techniques.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Guanosina Trifosfato/metabolismo , Medições Luminescentes/métodos , Proteínas ras/metabolismo , Humanos
10.
Methods Mol Biol ; 2262: 117-135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977474

RESUMO

Ras and its related small GTPases are important signalling nodes that regulate a wide variety of cellular functions. The active form of these proteins exists in a transient GTP bound state that mediates downstream signalling events. The dysregulation of these GTPases has been associated with the progression of multiple diseases, most prominently cancer and developmental syndromes known as Rasopathies. Determining the activation state of Ras and its relatives has hence been of paramount importance for the investigation of the biochemical functions of small GTPases in the cellular signal transduction network. This chapter describes the most broadly employed approach for the rapid, label-free qualitative and semi-quantitative determination of the Ras GTPase activation state, which can readily be adapted to the analysis of other related GTPases. The method relies on the affinity-based isolation of the active GTP-bound fraction of Ras in cellular extracts, followed by its visualization via western blotting. Specifically, we describe the production of the recombinant affinity probes or baits that bind to the respective active GTPases and the pulldown method for isolating the active GTPase fraction from adherent or non-adherent cells. This method allows for the reproducible measurement of active Ras or Ras family GTPases in a wide variety of cellular contexts.


Assuntos
Guanosina Trifosfato/metabolismo , Imunoprecipitação/métodos , Domínios e Motivos de Interação entre Proteínas , Proteínas ras/metabolismo , Sítios de Ligação , Western Blotting , Humanos , Ligação Proteica
11.
Methods Mol Biol ; 2262: 169-182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977476

RESUMO

RAS oncoproteins exhibit a switch-like behavior to drive diverse signaling cascades. In the active GTP-bound state, a conformational change occurs in these enzymes that enables interaction with downstream effectors. Nucleotide-dependent conformational exchange is easily detected with real-time NMR (RT-NMR) spectroscopy. RT-NMR has been firmly established as an effective assay to measure RAS oncoprotein nucleotide exchange and GTP hydrolysis kinetics and can further determine the regulatory activity of guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). It is now possible to multiplex these assays, allowing for the precise monitoring of activation states for mixtures of RAS oncoproteins or other RAS superfamily GTPases. Here, we describe the protocols necessary to express and purify isotopically labeled RAS and detail how to carry out an RT-NMR assay on a singular RAS protein or on a mixture of small GTPases.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Proteínas ras/metabolismo , Humanos , Hidrólise , Cinética , Ligação Proteica , Transdução de Sinais
12.
Methods Mol Biol ; 2262: 199-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977478

RESUMO

Reaction-diffusion simulations allow us to recapitulate experimentally observed behavior, e.g., from time series of fluorescent micrographs. This is essential to inform our intuitive understanding of the chemical and biophysical interaction of proteins in a cellular context as well as their role in reaction networks. This chapter aims to give a brief introduction to setting up reaction-diffusion simulations and applies these in silico techniques to take apart the spatial cycles that maintain Ras localization in the cell.


Assuntos
Simulação por Computador , Guanosina Trifosfato/metabolismo , Modelos Biológicos , Análise Espacial , Proteínas ras/metabolismo , Difusão , Humanos
13.
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
14.
RNA ; 27(6): 683-693, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33790044

RESUMO

The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3'-5' addition of a single nontemplated guanosine (G-1) to the 5' end of tRNAHis In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3'-5' polymerization of Watson-Crick (WC) base pairs, utilizing nucleotides in the 3'-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3'-5' polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3'-5' polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA "template," addition of a non-WC G-1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5'-3' polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3'-5' polymerases.


Assuntos
Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Pareamento de Bases , Cristalografia por Raios X , Guanosina Trifosfato/metabolismo , Cinética , Nucleotídeos/metabolismo , Nucleotidiltransferases/química , RNA de Transferência de Histidina/metabolismo , Alinhamento de Sequência
15.
Nat Commun ; 12(1): 2448, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907196

RESUMO

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular , Parede Celular/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Expressão Gênica , Hidrólise , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Transporte Proteico
16.
Phys Chem Chem Phys ; 23(15): 9524-9531, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33885101

RESUMO

Cyclic GMP-AMP Synthase (cGAS) is activated upon DNA binding and catalyzes the synthesis of 2',3'-cGAMP from GTP and ATP. This cyclic dinucleotide is a messenger that triggers the autoimmune system of eukaryotic cells. In this study, we propose a Molecular Dynamics (MD) investigation of cGAS activation. We notably provide insights into the motion of the activation loop, both from a mechanical point of view and considering its role in the catalysis of cGAMP production. We finally shed light on the reaction resulting in cGAMP synthesis. Two possible catalytic routes (referred to as GTP-ATP and ATP-GTP) are proposed based on the active site occupancy, paving the way toward further exploration of the reaction mechanism.


Assuntos
DNA/metabolismo , Nucleotidiltransferases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Biocatálise , Domínio Catalítico , DNA/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Ligação Proteica , Conformação Proteica
17.
J Cell Sci ; 134(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912945

RESUMO

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT.


Assuntos
Carioferinas , Proteína ran de Ligação ao GTP , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo
18.
Cells ; 10(2)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672296

RESUMO

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. Common genetic variation in LRRK2 modifies susceptibility to immunological disorders including Crohn's disease and leprosy. Previous studies have reported that LRRK2 is expressed in B lymphocytes and macrophages, suggesting a role for LRRK2 in immunological functions. In this study, we characterized the LRRK2 protein expression and phosphorylation using human lymphoblasts. Lipopolysaccharide (LPS), a proinflammatory agent, induced the increase of LRRK2 expression and kinase activities in human lymphoblasts in a time-dependent manner. Moreover, LPS activated the Toll-like receptor (TLR) signaling pathway, increased TRAF6/LRRK2 interaction, and elevated the phosphorylation levels of MAPK (JNK1/2, p38, and ERK1/2) and IkBα. Treatment with LRRK2 inhibitor 68 reduced LPS-induced TRAF6/LRRK2 interaction and MAPK and IkBα phosphorylation, thereby reducing TNF-α secretion. These results indicate that LRRK2 is actively involved in proinflammatory responses in human lymphoblasts, and inhibition of GTP binding by 68 results in an anti-inflammation effect against proinflammatory stimuli. These findings not only provide novel insights into the mechanisms of LRRK2-linked immune and inflammatory responses in B-cell-like lymphoblasts, but also suggest that 68 may also have potential therapeutic value for LRRK2-linked immunological disorders.


Assuntos
Guanosina Trifosfato/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lipopolissacarídeos/farmacologia , Linfócitos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Linfócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/metabolismo
19.
Cell Biochem Biophys ; 79(2): 221-229, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33733369

RESUMO

Defects in inosine monophosphate dehydrogenase-1 (IMPDH1) lead to insufficient biosyntheses of purine nucleotides. In eyes, these defects are believed to cause retinitis pigmentosa (RP). Major retinal isoforms of IMPDH1 are structurally distinct from those in other tissues, by bearing terminal extensions. Using recombinant mouse IMPDH1 (mH1), we evaluated the kinetics and oligomerization states of the retinal isoforms. Moreover, we adopted molecular simulation tools to study the possible effect of terminal tails on the function of major enzyme isoforms with the aim to find structural evidence in favor of contradictory observations on retinal IMPDH1 function. Our findings indicated higher catalytic activity for the major mouse retinal isoform (mH1603) along with lower fibrillation capacity under the influence of ATP. However, higher mass oligomerization products were formed by the mH1 (603) isoform in the presence of the enzyme inhibitors such as GTP and/or MPA. Collectively, our findings demonstrate that the structural differences between the retinal isoforms have led to functional variations possibly to justify the retinal cells' requirements.


Assuntos
IMP Desidrogenase/metabolismo , Retina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Humanos , Ligação de Hidrogênio , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/química , IMP Desidrogenase/genética , Cinética , Camundongos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química
20.
Biosci Biotechnol Biochem ; 85(6): 1441-1447, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33749776

RESUMO

Glycogen synthesis in bacteria is mainly organized by the products of glgB, glgC, and glgA genes comprising the widely known glg operon. On the genome of extremely halophilic archaeon Haloarcula japonica, there was a gene cluster analogous to the bacterial glg operon. In this study, we focused on a GlgC homolog of Ha. japonica, and its recombinant enzyme was prepared and characterized. The enzyme showed highest activity toward GTP and glucose-1-phosphate as substrates in the presence of 2.6 m KCl and predicted to be work as "GDP-glucose pyrophosphorylase" in Ha. japonica.


Assuntos
Proteínas Arqueais/genética , Haloarcula/genética , Homologia de Sequência do Ácido Nucleico , Proteínas Arqueais/metabolismo , Glicogênio/biossíntese , Guanosina Trifosfato/metabolismo , Haloarcula/metabolismo , Óperon/genética
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