RESUMO
A nanohybrid-modified glassy carbon electrode based on conducting polypyrrole doped with carbon quantum dots (QDs) was developed and used for the electrochemical detection of anti-tissue transglutaminase (anti-tTG) antibodies. To improve the polypyrrole conductivity, carrier mobility, and carrier concentration, four types of carbon nanoparticles were tested. Furthermore, a polypyrrole-modified electrode doped with QDs was functionalized with a PAMAM dendrimer and transglutaminase 2 protein by cross-linking with N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The steps of electrode surface modification were surveyed via electrochemical measurements (differential pulse voltammetry (DPV), impedance spectroscopy, and X-ray photoelectron spectroscopy (XPS)). The surface characteristics were observed by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and contact angle measurements. The obtained modified electrode exhibited good stability and repeatability. DPV between - 0.1 and 0.6 V (vs. Ag/AgCl 3 M KCl reference electrode) was used to evaluate the electrochemical alterations that occur after the antibody interacts with the antigen (transglutaminase 2 protein), for which the limit of detection was 0.79 U/mL. Without the use of a secondary label, (anti-tTG) antibodies may be detected at low concentrations because of these modified electrode features.
Assuntos
Dendrímeros , Proteína 2 Glutamina gama-Glutamiltransferase , Pirróis , Pontos Quânticos , Transglutaminases , Humanos , Anticorpos/imunologia , Anticorpos/química , Técnicas Biossensoriais/métodos , Carbono/química , Dendrímeros/química , Técnicas Eletroquímicas/métodos , Eletrodos , Proteínas de Ligação ao GTP/imunologia , Polímeros/química , Pirróis/química , Pontos Quânticos/química , Transglutaminases/imunologia , Transglutaminases/químicaRESUMO
Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.
Assuntos
Sobrevivência Celular , Proteínas de Ligação ao GTP , Conformação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Humanos , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Transglutaminases/metabolismo , Transglutaminases/química , Transglutaminases/genética , Sobrevivência Celular/efeitos dos fármacos , Microscopia Crioeletrônica , Linhagem Celular Tumoral , Morte Celular/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Difração de Raios X , Cálcio/metabolismoRESUMO
Kisspeptin receptor (KISS1R), belonging to the class A peptide-GPCR family, plays a key role in the regulation of reproductive physiology after stimulation by kisspeptin and is regarded as an attractive drug target for reproductive diseases. Here, we demonstrated that KISS1R can couple to the Gi/o pathway besides the well-known Gq/11 pathway. We further resolved the cryo-electron microscopy (cryo-EM) structure of KISS1R-Gq and KISS1R-Gi complexes bound to the synthetic agonist TAK448 and structure of KISS1R-Gq complex bound to the endogenous agonist KP54. The high-resolution structures provided clear insights into mechanism of KISS1R recognition by its ligand and can facilitate the design of targeted drugs with high affinity to improve treatment effects. Moreover, the structural and functional analyses indicated that conformational differences in the extracellular loops (ECLs), intracellular loops (ICLs) of the receptor, and the "wavy hook" of the Gα subunit may account for the specificity of G protein coupling for KISS1R signaling.
Assuntos
Microscopia Crioeletrônica , Receptores de Kisspeptina-1 , Humanos , Ligantes , Receptores de Kisspeptina-1/metabolismo , Receptores de Kisspeptina-1/química , Ligação Proteica , Kisspeptinas/metabolismo , Kisspeptinas/química , Modelos Moleculares , Células HEK293 , Conformação Proteica , Transdução de Sinais , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/química , Relação Estrutura-AtividadeRESUMO
Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.
Assuntos
Epigênese Genética , Histonas , Neoplasias , Proteína 2 Glutamina gama-Glutamiltransferase , Processamento de Proteína Pós-Traducional , Humanos , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/enzimologia , Neoplasias/patologia , Histonas/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Transglutaminases/metabolismo , Transglutaminases/genética , Regulação Neoplásica da Expressão Gênica , Transdução de SinaisRESUMO
Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.
Assuntos
Microscopia Crioeletrônica , Guanosina Difosfato , Guanosina Trifosfato , Mutação , Humanos , Células HEK293 , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Ligação Proteica , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genéticaRESUMO
ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.
Assuntos
Proteínas de Ciclo Celular , Centrossomo , Microtúbulos , Proteínas do Tecido Nervoso , Células-Tronco Neurais , Neurogênese , Animais , Microtúbulos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neurogênese/genética , Células-Tronco Neurais/metabolismo , Centrossomo/metabolismo , Proliferação de Células , Movimento Celular , Córtex Cerebral/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Tubulina (Proteína)/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genéticaRESUMO
Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.
Assuntos
Ácidos Cumáricos , Proteínas de Ligação ao GTP , Glioblastoma , Nanopartículas , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Ácidos Cumáricos/farmacologia , Humanos , Transglutaminases/metabolismo , Transglutaminases/genética , Glioblastoma/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Nanopartículas/química , Portadores de Fármacos/química , Apoptose/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
RHOBTB2 was first described as epileptogenic when it presents a missense variant in 2016 and studied more specifically in 2018. It is a gene that causes rare, but potentially severe childhood epileptic encephalopathy. In 2021, research confirmed that heterozygous mutations of RHOBTB2 included other clinical signs besides these encephalopathies. Thus, these infantile epilepsies are mainly associated with highly variable phenotypes, with developmental delay, post-traumatic encephalitis, paroxysmal movement disorders and iconographic brain damage. In this work, after presenting a clinical case, we will recall the role of RhoGTPases on neuronal development. We will then discuss a study which highlighted the neurodevelopmental impact of mutations on the RHOBTB2 gene by carrying out work on Drosophila melanogaster flies. Finally, we will compare the presented clinical case with a literature review.
Le gène RHOBTB2 est décrit pour la première fois comme épileptogène alors qu'il présente un variant faux-sens en 2016, puis est étudié plus précisément en 2018. Il s'agit d'un gène qui est à l'origine d'encéphalopathies épileptiques infantiles rares, mais pouvant être sévères. En 2021, des recherches ont confirmé que les mutations hétérozygotes de RHOBTB2 englobaient d'autres signes cliniques que ces encéphalopathies. Ainsi, ces épilepsies infantiles sont associées, principalement, avec des phénotypes fortement variables, à un retard développemental, à des encéphalites post-traumatiques, à des troubles paroxystiques des mouvements et à des atteintes iconographiques de l'encéphale. Dans ce travail, après avoir présenté un cas clinique, nous rappellerons le rôle des RhoGTPases sur le développement neuronal. Nous discuterons ensuite d'une étude qui a mis en évidence l'impact neurodéveloppemental de mutations sur le gène RHOBTB2 en réalisant des travaux sur des mouches Drosophila melanogaster. Pour terminer, nous mettrons le cas clinique présenté en parallèle avec une revue de la littérature réalisée par rapport à ce gène.
Assuntos
Mutação , Humanos , Animais , Proteínas Supressoras de Tumor/genética , Proteínas de Ligação ao GTP/genética , Masculino , Drosophila melanogaster/genética , Feminino , LactenteRESUMO
BACKGROUND: Lung adenocarcinoma (LUAD) is a leading cause of tumor-associated mortality, and it is needed to find new target to combat this disease. Guanine nucleotide-binding -protein-like 3 (GNL3) mediates cell proliferation and apoptosis in several cancers, but its role in LUAD remains unclear. OBJECTIVE: To explore the expression and function of Guanine nucleotide-binding protein-like 3 (GNL3) in lung adenocarcinoma (LUAD) and its potential mechanism in inhibiting the growth of LUAD cells. METHODS: We evaluated the expression of GNL3 in LUAD tissues and its association with patient prognosis using databases and immunohistochemistry. Cell proliferation was assessed by CCK-8 assay as well as colony formation, while apoptosis was evaluated by FCM. The effect of GNL3 knockdown on the Wnt/ß-catenin axis was investigated by Immunoblot analysis. RESULTS: GNL3 is overexpressed in LUAD tissues and is correlated with poor prognosis. Knockdown of GNL3 significantly inhibited the growth as well as induced apoptosis in A549 as well as H1299 cells. Furthermore, we found that the inhibitory effect of GNL3 knockdown on LUAD cell growth is associated with the downregulation of the Wnt/ß-catenin axis. CONCLUSION: GNL3 is key in the progression of LUAD by metiating Wnt/ß-catenin axis. Targeting GNL3 may represent a novel therapeutic method for LUAD treatment.
Assuntos
Adenocarcinoma de Pulmão , Apoptose , Proliferação de Células , Técnicas de Silenciamento de Genes , Neoplasias Pulmonares , Via de Sinalização Wnt , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Prognóstico , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , beta Catenina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células A549 , Proteínas NuclearesRESUMO
Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.
Assuntos
Proteínas de Ligação ao GTP , Mitocôndrias , Ribossomos Mitocondriais , Fosforilação Oxidativa , Humanos , Ribossomos Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Células HEK293 , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Células HeLaRESUMO
Mathematical models of biochemical reaction networks are an important and emerging tool for the study of cell signaling networks involved in disease processes. One promising potential application of such mathematical models is the study of how disease-causing mutations promote the signaling phenotype that contributes to the disease. It is commonly assumed that one must have a thorough characterization of the network readily available for mathematical modeling to be useful, but we hypothesized that mathematical modeling could be useful when there is incomplete knowledge and that it could be a tool for discovery that opens new areas for further exploration. In the present study, we first develop a mechanistic mathematical model of a G-protein coupled receptor signaling network that is mutated in almost all cases of uveal melanoma and use model-driven explorations to uncover and explore multiple new areas for investigating this disease. Modeling the two major, mutually-exclusive, oncogenic mutations (Gαq/11 and CysLT2R) revealed the potential for previously unknown qualitative differences between seemingly interchangeable disease-promoting mutations, and our experiments confirmed oncogenic CysLT2R was impaired at activating the FAK/YAP/TAZ pathway relative to Gαq/11. This led us to hypothesize that CYSLTR2 mutations in UM must co-occur with other mutations to activate FAK/YAP/TAZ signaling, and our bioinformatic analysis uncovers a role for co-occurring mutations involving the plexin/semaphorin pathway, which has been shown capable of activating this pathway. Overall, this work highlights the power of mechanism-based computational systems biology as a discovery tool that can leverage available information to open new research areas.
Assuntos
Mutação , Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Mutação/genética , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Biologia de Sistemas/métodos , Modelos Biológicos , Melanoma/genética , Melanoma/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismoAssuntos
Proteínas de Ligação ao GTP , Guanosina Trifosfato , Transdução de Sinais , Transdução de Sinais/genética , Humanos , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/genética , Ligação Proteica , AnimaisAssuntos
Clatrina , Endocitose , Proteínas de Ligação ao GTP , Vírus Hantaan , Internalização do Vírus , Humanos , Endocitose/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Clatrina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Vírus Hantaan/fisiologia , Vírus Hantaan/efeitos dos fármacos , Interações Hospedeiro-Patógeno , AnimaisRESUMO
Restoring and maintaining the function of endothelial cells is critical for acute respiratory distress syndrome (ARDS). Guanylate binding protein 1(GBP1) is proved to elevated in ARDS patients, but its role and mechanism remains unclear. The objective of this study is to investigate the internal mechanism of GBP1 in lung injury. Our study showed that when the LPS and IFN-γ induced human Pulmonary Microvascular Endothelial Cells (HPMECs) injury model was established, cell viability was significantly reduced, and the levels of GBP1 levels and inflammatory factors were significantly increased. When transfection with si-GBP1, low expression of GBP1 promoted cell proliferation and migration, and decreased the expression of downstream inflammatory factors. Furthermore, the inhibition of GBP1 significantly reduced the occurrence of cell pyroptosis and the expression of NLRP3 and STAT1. Our study indicated that GBP1 alleviates endothelial pyroptosis and inflammation through STAT1 / NLRP3/GSDMD signaling pathway, and GBP1 may be a new target in the treatment of lung injury in the future.
Assuntos
Células Endoteliais , Proteínas de Ligação ao GTP , Pulmão , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Fator de Transcrição STAT1 , Transdução de Sinais , Humanos , Piroptose/fisiologia , Fator de Transcrição STAT1/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Endoteliais/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Pulmão/metabolismo , Pulmão/irrigação sanguínea , Pulmão/patologia , Proliferação de Células , Lipopolissacarídeos/farmacologia , Microvasos , Inflamação/metabolismoRESUMO
The metal-resistant beta-proteobacterium Cupriavidus metallidurans is also able to survive conditions of metal starvation. We show that zinc-starved cells can substitute some of the required zinc with cobalt but not with nickel ions. The zinc importer ZupT was necessary for this process but was not essential for either zinc or cobalt import. The cellular cobalt content was also influenced by the two COG0523-family proteins, CobW2 and CobW3. Pulse-chase experiments with radioactive and isotope-enriched zinc demonstrated that both proteins interacted with ZupT to control the cellular flow-equilibrium of zinc, a central process of zinc homeostasis. Moreover, an antagonistic interplay of CobW2 and CobW3 in the presence of added cobalt caused a growth defect in mutant cells devoid of the cobalt efflux system DmeF. Full cobalt resistance also required a synergistic interaction of ZupT and DmeF. Thus, the two transporters along with CobW2 and CobW3 interact to control cobalt homeostasis in a process that depends on zinc availability. Because ZupT, CobW2, and CobW3 also direct zinc homeostasis, this process links the control of cobalt and zinc homeostasis, which subsequently protects C. metallidurans against cadmium stress and general metal starvation.IMPORTANCEIn bacterial cells, zinc ions need to be allocated to zinc-dependent proteins without disturbance of this process by other transition metal cations. Under zinc-starvation conditions, C. metallidurans floods the cell with cobalt ions, which protect the cell against cadmium toxicity, help withstand metal starvation, and provide cobalt to metal-promiscuous paralogs of essential zinc-dependent proteins. The number of cobalt ions needs to be carefully controlled to avoid a toxic cobalt overload. This is accomplished by an interplay of the zinc importer ZupT with the COG0523-family proteins, CobW3, and CobW2. At high external cobalt concentrations, this trio of proteins additionally interacts with the cobalt efflux system, DmeF, so that these four proteins form an inextricable link between zinc and cobalt homeostasis.
Assuntos
Proteínas de Bactérias , Cobalto , Cupriavidus , Homeostase , Zinco , Cobalto/metabolismo , Zinco/metabolismo , Cupriavidus/metabolismo , Cupriavidus/genética , Cupriavidus/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genéticaRESUMO
Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.
Assuntos
Cálcio , Proteínas de Ligação ao GTP , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Transglutaminases/metabolismo , Transglutaminases/química , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Humanos , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/química , Cristalografia por Raios X , Glutens/metabolismo , Glutens/química , Modelos Moleculares , Conformação Proteica , Doença Celíaca/metabolismo , Ligação ProteicaRESUMO
Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.
Assuntos
Bleomicina , Pulmão , Proteína 2 Glutamina gama-Glutamiltransferase , Fibrose Pulmonar , Transglutaminases , Animais , Masculino , Camundongos , Glicólise , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Pulmão/patologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transglutaminases/metabolismo , Transglutaminases/genéticaRESUMO
The function of glioma stem cells (GSCs) is closely related to the progression of glioblastoma multiforme (GBM). Centromere protein A (CENPA) has been confirmed to be related to the poor prognosis of GBM patients. However, whether CENPA regulates GSCs function to mediate GBM progression is still unclear. GSCs were isolated from GBM cells. The expression of CENPA and guanylate-binding protein 2 (GBP2) was examined by quantitative real-time PCR and western blot. GSCs proliferation and stemness were assessed using EdU assay and sphere formation assay. Cell ferroptosis was evaluated by detecting related factors. The interaction between CENPA and GBP2 was analyzed by ChIP assay and dual-luciferase reporter assay. Animal experiments were conducted to measure the effect of CENPA knockdown on the tumorigenicity of GSCs in vivo. CENPA was upregulated in GBM tissues and GSCs. CENPA knockdown inhibited GSCs proliferation, stemnness, and promoted ferroptosis. GBP2 was overexpressed in GBM tissues and GSCs, and CENPA enhanced GBP2 transcription by binding to its promoter region. CENPA overexpression accelerated GSCs proliferation and stemnness and suppressed ferroptosis, while GBP2 knockdown reversed these effects. Downregulation of CENPA reduced the tumorigenicity of GSCs by decreasing GBP2 expression in vivo. In conclusion, CENPA enhanced GBP2 transcription to increase its expression, thus accelerating GSCs proliferation and stemnness and repressing ferroptosis. Our findings promote a new idea for GBM treatment.
Assuntos
Neoplasias Encefálicas , Ferroptose , Glioblastoma , Células-Tronco Neoplásicas , Ferroptose/genética , Ferroptose/fisiologia , Humanos , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Progressão da Doença , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Proliferação de Células/genética , Linhagem Celular Tumoral , Camundongos NusRESUMO
Inherited retinal diseases encompass a genetically diverse group of conditions caused by variants in genes critical to retinal function, including handful of ribosome-associated genes. This study focuses on the HBS1L gene, which encodes for the HBS1-like translational GTPase that is crucial for ribosomal rescue. We have reported a female child carrying biallelic HBS1L variants, manifesting with poor growth and neurodevelopmental delay. Here, we describe the ophthalmologic findings in the patient and in Hbs1ltm1a/tm1a hypomorph mice and describe the associated microscopic and molecular perturbations. The patient has impaired visual function, showing dampened amplitudes of a- and b-waves in both rod- and cone-mediated responses. Hbs1ltm1a/tm1a mice exhibited profound thinning of the entire retina, specifically of the outer photoreceptor layer, due to extensive photoreceptor cell apoptosis. Loss of Hbs1l resulted in comprehensive proteomic alterations by mass spectrometry analysis, with an increase in the levels of 169 proteins and a decrease in the levels of 480 proteins, including rhodopsin (Rho) and peripherin 2 (Prph2). Gene Ontology biological process and gene set enrichment analyses reveal that the downregulated proteins are primarily involved in phototransduction, cilium assembly and photoreceptor cell development. These findings underscore the importance of ribosomal rescue proteins in maintaining retinal health, particularly in photoreceptor cells.
Assuntos
Modelos Animais de Doenças , Distrofias Retinianas , Animais , Distrofias Retinianas/patologia , Distrofias Retinianas/genética , Feminino , Humanos , Camundongos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Apoptose , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , GTP Fosfo-Hidrolases/deficiência , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , CriançaRESUMO
Kappa opioid receptor (KOR) agonists represent promising therapeutics for pain relief due to their analgesic properties along with lower abuse potential than opioids that act at the mu opioid receptor. However, typical KOR agonists produce sedation and dysphoria. Previous studies have shown that G protein signaling-biased KOR agonists may present a means to untangle the desired analgesic properties from undesired side effects. In this paper, we report a new series of G protein signaling-biased KOR agonists entailing -S- â -CH2- replacement in a previously reported KOR agonist, triazole 1.1. With an optimized carbon linker in hand, further development of the scaffold was undertaken to investigate the appendages of the triazole core. The structure-activity relationship study of this series is described, including several analogues that display enhanced potency while maintaining G protein-signaling bias compared to triazole 1.1.