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1.
Biochem J ; 480(1): 1-23, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36607281

RESUMO

RAS proteins regulate most aspects of cellular physiology. They are mutated in 30% of human cancers and 4% of developmental disorders termed Rasopathies. They cycle between active GTP-bound and inactive GDP-bound states. When active, they can interact with a wide range of effectors that control fundamental biochemical and biological processes. Emerging evidence suggests that RAS proteins are not simple on/off switches but sophisticated information processing devices that compute cell fate decisions by integrating external and internal cues. A critical component of this compute function is the dynamic regulation of RAS activation and downstream signaling that allows RAS to produce a rich and nuanced spectrum of biological outputs. We discuss recent findings how the dynamics of RAS and its downstream signaling is regulated. Starting from the structural and biochemical properties of wild-type and mutant RAS proteins and their activation cycle, we examine higher molecular assemblies, effector interactions and downstream signaling outputs, all under the aspect of dynamic regulation. We also consider how computational and mathematical modeling approaches contribute to analyze and understand the pleiotropic functions of RAS in health and disease.


Assuntos
Neoplasias , Transdução de Sinais , Humanos , Proteínas ras/química , Guanosina Trifosfato/metabolismo
2.
Elife ; 122023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36598133

RESUMO

The small GTPase Arl3 is important for the enrichment of lipidated proteins to primary cilia, including the outer segment of photoreceptors. Human mutations in the small GTPase Arl3 cause both autosomal recessive and dominant inherited retinal dystrophies. We discovered that dominant mutations result in increased active G-protein-Arl3-D67V has constitutive activity and Arl3-Y90C is fast cycling-and their expression in mouse rods resulted in a displaced nuclear phenotype due to an aberrant Arl3-GTP gradient. Using multiple strategies, we go on to show that removing or restoring the Arl3-GTP gradient within the cilium is sufficient to rescue the nuclear migration defect. Together, our results reveal that an Arl3 ciliary gradient is involved in proper positioning of photoreceptor nuclei during retinal development.


Assuntos
Fatores de Ribosilação do ADP , Proteínas de Membrana , Células Fotorreceptoras Retinianas Bastonetes , Animais , Humanos , Camundongos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Células Fotorreceptoras Retinianas Bastonetes/metabolismo
3.
Eur J Med Chem ; 248: 115104, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36641861

RESUMO

To search more therapeutic strategies for Ras-mutant tumors, regulators of the Ras superfamily involved in the GTP/GDP (guanosine triphosphate/guanosine diphosphate) cycle have been well concerned for their anti-tumor potentials. GTPase activating proteins (GAPs) provide the catalytic group necessary for the hydrolysis of GTPs, which accelerate the switch by cycling between GTP-bound active and GDP-bound inactive forms. Inactivated GAPs lose their function in activating GTPase, leading to the continuous activation of downstream signaling pathways, uncontrolled cell proliferation, and eventually carcinogenesis. A growing number of evidence has shown the close link between GAPs and human tumors, and as a result, GAPs are believed as potential anti-tumor targets. The present review mainly summarizes the critically important role of GAPs in human tumors by introducing the classification, function and regulatory mechanism. Moreover, we comprehensively describe the relationship between dysregulated GAPs and the certain type of tumor. Finally, the current status, research progress, and clinical value of GAPs as therapeutic targets are also discussed, as well as the challenges and future direction in the cancer therapy.


Assuntos
Neoplasias , Proteínas Ativadoras de ras GTPase , Humanos , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de GTPase , GTP Fosfo-Hidrolases , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismo , Neoplasias/tratamento farmacológico
4.
Biochem Biophys Res Commun ; 641: 27-33, 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36516586

RESUMO

KRAS mutations occur in a quarter of all human cancers. When activated in its GTP-bound form, RAS stimulates diverse cellular systems, such as cell division, differentiation, growth, and apoptosis through the activations of various signaling pathways, which include mitogen-activated protein kinase (MAPK), phosphoinositide 3 kinases (PI3K), and RAL-GEFs pathways. We found that GJ101 (65LYDVA69) binds directly to the KRAS mutant (G12V) and showed tumor-suppressive activity. In addition, the GJ101 peptide inhibited KRAS mutant as determined by a [α-32P] guanosine triphosphate (GTP) binding assay and suppressed pancreatic cell line in a cell proliferation assay. Herein, the complex structure of KRAS and GJ101 was clarified by X-ray crystallography. Isothermal titration calorimetry showed that GJ101 binds highly with KRAS mutant and the complex structure of KRAS G12V.GJ101 complex presented that the residue of Q61 directly interacted with L65 of GJ101. Overall, the results suggest GJ101 be considered a developmental starting point for KRAS G12V inhibitor.


Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linhagem Celular , Mutação , Guanosina Trifosfato/metabolismo , Linhagem Celular Tumoral
5.
Curr Biol ; 33(1): 122-133.e4, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36565699

RESUMO

Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 µm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.


Assuntos
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Citoplasma/metabolismo , Polímeros/metabolismo , Citoesqueleto de Actina/metabolismo , Guanosina Trifosfato/metabolismo
6.
Methods Mol Biol ; 2557: 507-518, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512233

RESUMO

Rab GTPases are key regulators of membrane trafficking. When GTP-bound, or "active," Rabs are anchored to membranes and recruit effector proteins that mediate vesicle formation, transport, and fusion. Rabs are inactivated by GTPase-activating proteins (Rab-GAPs), which catalyze GTP hydrolysis, rendering Rabs cytosolic. In vivo, C-terminal prenylation modifications link activated Rabs to organelle and vesicle membranes, yet historically, in vitro Rab-GAP activity assays have been performed in the absence of membranes. We have developed a method for assaying Rab-GAP activity in a physiological context, with dissociation of the Rab from the membrane serving as a readout for Rab-GAP activity. Given that membrane-binding status is a key consequence of Rab activation state, this assay will be useful for the study of a wide range of Rab/Rab-GAP pairs.


Assuntos
Proteínas Ativadoras de GTPase , Proteínas rab de Ligação ao GTP , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Membranas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Guanosina Trifosfato/metabolismo
7.
Anal Biochem ; 663: 115019, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36526022

RESUMO

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas ras , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/química , Ligação Proteica , Guanosina Trifosfato/metabolismo , Fosfatidilinositóis
8.
Int J Mol Sci ; 23(24)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36555100

RESUMO

RhoA, a member of Rho GTPases, regulates myriad cellular processes. Abnormal expression of RhoA has been implicated in various diseases, including cancers, developmental disorders and bacterial infections. RhoA mutations G14V and Q63L have been reported to constitutively activate RhoA. To figure out the mechanisms, in total, 1.8 µs molecular dynamics (MD) simulations were performed here on RhoAWT and mutants G14V and Q63L in GTP-bound forms, followed by dynamic analysis. Both mutations were found to affect the conformational dynamics of RhoA switch regions, especially switch I, shifting the whole ensemble from the wild type's open inactive state to different active-like states, where T37 and Mg2+ played important roles. In RhoAG14V, both switches underwent thorough state transition, whereas in RhoAQ63L, only switch I was sustained in a much more closed conformation with additional hydrophobic interactions introduced by L63. Moreover, significantly decreased solvent exposure of the GTP-binding site was observed in both mutants with the surrounding hydrophobic regions expanded, which furnished access to water molecules required for hydrolysis more difficult and thereby impaired GTP hydrolysis. These structural and dynamic differences first suggested the potential activation mechanism of RhoAG14V and RhoAQ63L. Together, our findings complemented the understanding of RhoA activation at the atomic level and can be utilized in the development of novel therapies for RhoA-related diseases.


Assuntos
Proteínas rho de Ligação ao GTP , Proteína rhoA de Ligação ao GTP , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Transdução de Sinais , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Mutação
9.
Proc Natl Acad Sci U S A ; 119(46): e2208294119, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36343235

RESUMO

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used in vitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.


Assuntos
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Guanosina Trifosfato/metabolismo , Método de Monte Carlo
10.
Biochemistry ; 61(22): 2482-2489, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36315857

RESUMO

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.


Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto , Proteínas de Escherichia coli , Ânions/metabolismo , Condensados Biomoleculares , Proteínas de Transporte/metabolismo , Divisão Celular , Proteínas do Citoesqueleto/metabolismo , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Guanosina Trifosfato/metabolismo , Bicamadas Lipídicas/metabolismo
11.
Anal Chem ; 94(41): 14410-14418, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36206384

RESUMO

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.


Assuntos
Nucleotídeos de Guanina , Proteínas Heterotriméricas de Ligação ao GTP , Polarização de Fluorescência , Nucleotídeos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Ligação Proteica , Subunidades Proteicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
12.
Elife ; 112022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36264623

RESUMO

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.


Assuntos
Produtos Biológicos , Aminoacil-RNA de Transferência , Animais , Humanos , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo , Códon/metabolismo , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/genética , Produtos Biológicos/metabolismo , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo
13.
J Phys Chem B ; 126(42): 8486-8494, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36251789

RESUMO

Hydrotropes are small amphiphilic compounds that increase the aqueous solubility of hydrophobic molecules. Recent evidence suggests that adenosine triphosphate (ATP), which is the primary energy carrier in cells, also assumes hydrotropic properties to prevent the aggregation of hydrophobic proteins, but the mechanism of hydrotropy is unknown. Here, we compare the hydrotropic behavior of all four biological nucleoside triphosphates (NTPs) using molecular dynamics (MD) simulations. We launch all atom MD simulations of aqueous solutions of NTPs [ATP, guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP)] with pyrene, which acts both as a model hydrophobic compound and as a spectroscopic reporter for aggregation. GTP prevents pyrene aggregation effectively. Dissolution is not achieved in the presence of CTP and UTP. The higher stability of the base stacking in guanine is responsible for the higher hydrotropic efficiency of GTP. Consistent with the simulations, spectroscopic measurements also suggest that the hydrotropic activity of GTP is higher than ATP. Stacking of aromatic pyrene with the aromatic base of NTPs is a characteristic feature of this hydrotropic property. Both ATP and GTP also dissolve clusters of di- and tripeptides containing tryptophan but with equal potency. Importantly, the presence of aromatic amino acids is a necessary condition for the hydrotropic potency of ATP and GTP. Our results can have broad implications for hydrotrope design in the pharmaceutical industry, as well as the possibility of cells employing GTP as a hydrotrope to regulate the hydrophobic protein aggregation in membrane-less biological condensates.


Assuntos
Trifosfato de Adenosina , Agregados Proteicos , Guanosina Trifosfato/metabolismo , Uridina Trifosfato , Trifosfato de Adenosina/metabolismo , Citidina Trifosfato , Triptofano , Nucleosídeos , Pirenos , Guanina
14.
Nat Commun ; 13(1): 6004, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224181

RESUMO

Aberrant activation of EGFR due to overexpression or mutation is associated with poor prognosis in many types of tumors. Here we show that blocking the sorting system that directs EGFR to plasma membrane is a potent strategy to treat EGFR-dependent tumors. We find that EGFR palmitoylation by DHHC13 is critical for its plasma membrane localization and identify ARF6 as a key factor in this process. N-myristoylated ARF6 recognizes palmitoylated EGFR via lipid-lipid interaction, recruits the exocyst complex to promote EGFR budding from Golgi, and facilitates EGFR transporting to plasma membrane in a GTP-bound form. To evaluate the therapeutic potential of this sorting system, we design a cell-permeable peptide, N-myristoylated GKVL-TAT, and find it effectively disrupts plasma membrane localization of EGFR and significantly inhibits progression of EGFR-dependent tumors. Our findings shed lights on the underlying mechanism of how palmitoylation directs protein sorting and provide an potential strategy to manage EGFR-dependent tumors.


Assuntos
Fatores de Ribosilação do ADP , Neoplasias , Fatores de Ribosilação do ADP/metabolismo , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lipídeos , Neoplasias/metabolismo , Transporte Proteico
15.
Proc Natl Acad Sci U S A ; 119(42): e2211178119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36215496

RESUMO

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.


Assuntos
Bacillus subtilis , Proteínas do Citoesqueleto , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Divisão Celular , Proteínas do Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Peptídeos/metabolismo
16.
Sci Rep ; 12(1): 16991, 2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216916

RESUMO

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.


Assuntos
Acetilglucosamina , Fosfotransferases (Aceptor do Grupo Álcool) , Saccharomyces cerevisiae , Acetilglucosamina/metabolismo , Trifosfato de Adenosina/metabolismo , Citidina Trifosfato/metabolismo , Glucose/metabolismo , Glicoproteínas/metabolismo , Guanosina Trifosfato/metabolismo , Nucleosídeos/metabolismo , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Uridina Trifosfato/metabolismo
17.
Cells ; 11(19)2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36231021

RESUMO

Rab GTPases, the largest family of small GTPases, are ubiquitously expressed proteins that control various aspects of cellular function, from cell survival to exocytosis. Rabs cycle between the GDP-bound inactive form and the GTP-bound active form. When activated, specific Rab GTPase-positive vesicles mediate cellular networks involved in intracellular trafficking, recycling, and/or exocytosis of cargo proteins. Dysfunctional Rab signaling pathways have been implicated in various disease processes. The precise cellular functions of several members of the Rab GTPase family are still unknown. A lack of pharmacological tools and the lethality of gene knockouts have made more detailed characterizations of their protein interaction networks difficult. Nevertheless, available evidence suggests that these proteins are vital for normal cell function. Endothelial and smooth muscle cells control vascular lumen diameter and modulate blood flow. Endothelial cells also secrete several pro- and antithrombotic factors and vasoactive substances to coordinate local inflammatory responses and angiogenesis. Rab GTPase function in endothelial cells has been relatively well-explored, while only a handful of reports are available on these proteins in vascular smooth muscle. This review summarizes the present knowledge on Rab GTPases in the vasculature.


Assuntos
Células Endoteliais , Proteínas rab de Ligação ao GTP , Células Endoteliais/metabolismo , Exocitose , Fibrinolíticos , Guanosina Trifosfato/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
18.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 363-370, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189720

RESUMO

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Šresolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Šresolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.


Assuntos
ATP Citrato (pro-S)-Liase , Succinato-CoA Ligases , ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetilcoenzima A , Acil Coenzima A , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , Difosfatos , Guanosina Trifosfato/metabolismo , Humanos , Magnésio , Complexos Multienzimáticos , Nucleosídeos , Oxo-Ácido-Liases , Succinato-CoA Ligases/química , Succinatos , Ácido Succínico/metabolismo
19.
Nucleic Acids Res ; 50(17): 10015-10025, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36107775

RESUMO

tRNAHis guanylyltransferase (Thg1) catalyzes the 3'-5' incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74-76), how HsThg1 prevents successive 3'-5' incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.


Assuntos
Nucleotidiltransferases/metabolismo , RNA de Transferência de Histidina , Trifosfato de Adenosina , GTP Fosfo-Hidrolases/genética , Guanosina , Guanosina Trifosfato/metabolismo , Histidina-tRNA Ligase , Humanos , RNA de Transferência , RNA de Transferência de Histidina/genética , RNA de Transferência de Histidina/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
J Immunol ; 209(8): 1574-1585, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36165184

RESUMO

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting ß2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced ß2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and ß2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.


Assuntos
Antígenos CD18 , Neutrófilos , Animais , Antígenos CD18/metabolismo , Cálcio/metabolismo , Adesão Celular , Guanosina , Guanosina Trifosfato/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Polifosfatos , Proteínas rac1 de Ligação ao GTP/metabolismo
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