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1.
World J Gastroenterol ; 27(38): 6348-6356, 2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34720526

RESUMO

Thiopurines are immunomodulators used in the treatment of acute lymphoblastic leukemia and inflammatory bowel diseases. Adverse reactions to these agents are one of the main causes of treatment discontinuation or interruption. Myelosuppression is the most frequent adverse effect; however, approximately 5%-20% of patients develop gastrointestinal toxicity. The identification of biomarkers able to prevent and/or monitor these adverse reactions would be useful for clinicians for the proactive management of long-term thiopurine therapy. In this editorial, we discuss evidence supporting the use of PACSIN2, RAC1, and ITPA genes, in addition to TPMT and NUDT15, as possible biomarkers for thiopurine-related gastrointestinal toxicity.


Assuntos
Mercaptopurina , Pirofosfatases , Azatioprina/efeitos adversos , Biomarcadores , Humanos , Fatores Imunológicos , Mercaptopurina/efeitos adversos , Metiltransferases/genética
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(12): 1258-1261, 2021 Dec 10.
Artigo em Chinês | MEDLINE | ID: mdl-34839520

RESUMO

OBJECTIVE: To investigate the association between single nucleotide polymorphism of NUDT15 gene (SNP rs116855232) and hepatotoxicity in children with acute lymphocytic leukemia (ALL). METHODS: A total of 135 children with ALL in Shandong Province were recruited in this study, and patients were divided into two groups based on the presence of liver injury. Genotypes of each patient were detected using PCR and Sanger sequencing. Clinical data and the average dose of 6-mercaptopurine (6-MP) were collected and analyzed by SPSS 19.0 software. RESULTS: Respectively, 99 patients were found with CC genotype, 32 patients with CT genotype and 4 patients with TT genotype. Compared with ALL patients without hepatotoxicity, there was a difference in genotypes between the two groups in the initial stage of chemotherapy for leukemia (Chi2 = 7.583, P = 0.023). In maintenance therapy stage there was also a difference between the two groups (Chi2 = 10.591, P = 0.005), and T allele was a risk factor for hepatotoxicity. CONCLUSION: The polymorphism of rs116855232 in NUDT15 gene was associated with hepatotoxicity induced by 6-mercaptopurine in children with ALL, and ALL patients with TT genotype should take a lower dose of 6-MP to avoided hepatotoxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Antimetabólitos Antineoplásicos/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/genética , Criança , Genótipo , Humanos , Mercaptopurina/efeitos adversos , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirofosfatases/genética
3.
Metab Eng ; 68: 210-219, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34673235

RESUMO

Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol's seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.


Assuntos
Proteínas de Escherichia coli , Ácido Mevalônico , Biocombustíveis , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Engenharia Metabólica , Pirofosfatases
4.
Biochemistry ; 60(40): 3027-3039, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34569786

RESUMO

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-ß phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-ß-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in Escherichia coli (Foor, F., Brown, G. M. J. Biol. Chem., 1975, 250, 9, 3545-3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA from E. coli with the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point 31P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , GTP Cicloidrolase/química , Biocatálise , Difosfatos/metabolismo , Proteínas de Escherichia coli/metabolismo , GTP Cicloidrolase/metabolismo , Guanosina Trifosfato/metabolismo , Inosina Trifosfato/metabolismo , Cinética , Ligação Proteica , Pirofosfatases/química , Pirofosfatases/metabolismo
5.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575984

RESUMO

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H+ and Na+ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a "direct coupling" mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na+ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg2+ (essential cofactor). The replacement of K+, the second metal cofactor, by Na+ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PPi hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell's direct coupling with conformational coupling to catalyze cation transport across the membrane.


Assuntos
Catálise , Difosfatos/química , Pirofosfatase Inorgânica/química , Canais Iônicos/química , Membrana Celular/enzimologia , Dimerização , Hidrólise , Canais Iônicos/genética , Transporte de Íons/genética , Cinética , Potássio/química , Prótons , Pirofosfatases
6.
Aliment Pharmacol Ther ; 54(9): 1124-1133, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34563096

RESUMO

INTRODUCTION: Thiopurine S-methyltransferase (TPTM) is a well known biomarker for thiopurine-induced leucopenia, which has limited value in Asia. Instead, NUDT15 C415T is a promising predictor in Asia. AIMS: To explore whether an optimised strategy based on NUDT15 C415T genotypes affects thiopurine-induced leucopenia, as well as efficacy in Chinese patients with Crohn's disease. METHODS: Patients with Crohn's disease and indications for thiopurines were included from two hospitals in China. They were randomly assigned to either the intervention or the control group. In the intervention group, those with genotype CC received a standard dose (control group), those with CT genotype received 50% of the standard dose, those with TT genotype received alternative drugs. The primary endpoint was thiopurine-induced leucopenia (<3.5 × 109 /L). Secondary outcomes were the incidence of other adverse events and the efficacy for maintaining steroid-free remission at week 36. RESULTS: The rate of thiopurine-induced leucopenia was lower in the intervention group (n = 52) than in the control group (n = 66) (23.7% vs 32.4%, P = 0.049, RR = 0.73, 95% CI 0.53-1.00). In CT subgroup, the incidence of leucopenia in the intervention group (n = 10) was significantly lower than in the control group (n = 28) (31.3% vs 65.1%, RR = 0.48, 95% CI 0.28-0.84). Neither other adverse events nor treatment efficacy was significantly different between the two groups during follow-up. CONCLUSIONS: Among Chinese patients with Crohn's disease, dose optimisation by NUDT15 C415T reduced the rate of thiopurine-induced leucopenia, without significant influence on efficacy. Using 50% dose reduction for heterozygotes, and alternative drugs for homozygotes, are practicable strategies. Clinical trial number: NCT02929706.


Assuntos
Anemia , Doença de Crohn , Leucopenia , Azatioprina , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Genótipo , Humanos , Leucopenia/induzido quimicamente , Mercaptopurina/efeitos adversos , Pirofosfatases/genética
7.
ACS Chem Biol ; 16(9): 1680-1691, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34477366

RESUMO

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.


Assuntos
Nucleotídeos de Adenina/metabolismo , Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Nucleotídeos de Adenina/síntese química , Sítio Alostérico , Bacillus subtilis , Desoxirribonucleotídeos , Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Pirofosfatases/metabolismo
8.
BMC Gastroenterol ; 21(1): 327, 2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34425754

RESUMO

BACKGROUND: Polymorphisms in thiopurine methyltransferase (TPMT) and Nudix hydrolase-15 (NUDT15) have been implicated as the predominant cause of thiopurine induced leukopenia in the Western countries and East Asia respectively. Exact role of these polymorphisms in South Asian population with inflammatory bowel disease (IBD) is uncertain. METHODS: We included consecutive patients with IBD who were initiated on thiopurines at a center in North India. The dosage of thiopurines was titrated using regular monitoring of hemogram and liver function tests. Three TPMT polymorphisms (c.238 G > C, c.460 G > A, and c.719A > G) and one NUDT15 polymorphism (c.415 C > T) were assessed. Comparison regarding incidence of leukopenia and maximum tolerated thiopurine dosage was performed between those with wild polymorphism and those with TPMT and NUDT15 polymorphisms, respectively. RESULTS: Of the 119 patients (61 males, mean age 36.8 ± 13.5 years), 105 (88.2%) had ulcerative colitis and 14 (11.8%) had Crohn's disease. Leukopenia was noted in 33 (27.7%), gastrointestinal intolerance in 5 (4.2%) and pancreatitis in 2 (1.6%). TPMT polymorphisms were detected amongst five patients of whom 1 developed leukopenia. NUDT15 polymorphism was noted in 13 patients of whom 7 had leukopenia. The odds of developing leukopenia in TPMT polymorphism were non-significant (0.77, 95% CI:0.0822 to 7.2134, P = 0.819) but were significantly higher in those with NUDT15 polymorphism (3.5933, 1.1041 to 11.6951, P value: = 0.0336). CONCLUSION: NUDT15 polymorphism was more frequent than TPMT polymorphisms and was associated with thiopurine induced leukopenia. However, the tested polymorphisms account for only 24.2% of the risk of thiopurine induced leukopenia.


Assuntos
Doenças Inflamatórias Intestinais , Leucopenia , Metiltransferases/genética , Pirofosfatases/genética , Adulto , Humanos , Índia/epidemiologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Leucopenia/induzido quimicamente , Leucopenia/epidemiologia , Leucopenia/genética , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
9.
Mol Cell ; 81(16): 3310-3322.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416138

RESUMO

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.


Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismo
11.
J Oral Biosci ; 63(3): 259-264, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34391947

RESUMO

OBJECTIVE: This study aimed to demonstrate the immunolocalization and gene expression of tissue nonspecific alkaline phosphatase (TNALP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in osteoblasts, preosteoblasts, and osteocytes of murine bone to provide clues for a better understanding of the supply of phosphate ions (Pi) during bone mineralization. METHODS: Six-week-old male C57BL/6J mice (n = 6) were fixed with a paraformaldehyde solution, and the right femora were extracted for immunodetection of TNALP and ENPP1, while the left tibiae were used for reverse transcription polymerase chain reaction to evaluate Tnalp and Enpp1 gene expression. RESULTS: TNALP was intensely localized on the basolateral cell membranes of mature osteoblasts and preosteoblastic cells. There was little immunoreactivity of TNALP on the secretory surface of the osteoblasts and no TNALP reactivity in the osteocytes. In contrast, ENPP1 was observed throughout the cytoplasm of mature osteoblasts and osteocytes embedded in bone but was not observed in preosteoblasts. Together, despite the fact that the osteoid is a site of matrix vesicle-mediated mineralization, ENPP1, which inhibits mineralization by providing pyrophosphates, was localized in close proximity of the osteoid, whereas TNALP, which facilitates mineralization by providing Pi, was relatively distant from the osteoid. CONCLUSION: It seems likely that the differential localization of TNALP and ENPP1 around the osteoid observed at the microscopic level may provide preferential micro-circumstance for a balanced concentration of Pi and pyrophosphate for bone mineralization.


Assuntos
Fosfatase Alcalina , Pirofosfatases , Fosfatase Alcalina/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteócitos , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética
12.
Nat Commun ; 12(1): 4181, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234136

RESUMO

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.


Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , Infecções por Citomegalovirus/prevenção & controle , Ganciclovir/análogos & derivados , Pirofosfatases/genética , Aciclovir/uso terapêutico , Adolescente , Adulto , Idoso , Animais , Antibioticoprofilaxia , Antivirais/uso terapêutico , Variação Biológica da População/genética , Linhagem Celular , Criança , Pré-Escolar , Cristalografia por Raios X , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/virologia , DNA Viral/sangue , DNA Viral/isolamento & purificação , Modelos Animais de Doenças , Farmacorresistência Viral , Feminino , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Muromegalovirus/isolamento & purificação , Muromegalovirus/patogenicidade , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Pirofosfatases/metabolismo , Pirofosfatases/ultraestrutura , Resultado do Tratamento , Adulto Jovem
14.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299012

RESUMO

Atopic dermatitis (AD) represents a severe global burden on physical, physiological and mental health. Innate immune cell basophils are essential for provoking allergic inflammation in AD. However, the roles of novel immunoregulatory cytokine IL-37 in basophils remain elusive. We employed in vitro co-culture of human basophils and human keratinocyte HaCaT cells and an in vivo MC903-induced AD murine model to investigate the anti-inflammatory mechanism of IL-37. In the in vitro model, IL-37b significantly decreased Der p1-induced thymic stromal lymphopoietin (TSLP) overexpression in HaCaT cells and decreased the expression of TSLP receptor as well as basophil activation marker CD203c on basophils. IL-37 could also reduce Th2 cytokine IL-4 release from TSLP-primed basophils ex vivo. In the in vivo model, alternative depletion of basophils ameliorated AD symptoms and significantly lowered the Th2 cell and eosinophil populations in the ear and spleen of the mice. Blocking TSLP alleviated the AD-like symptoms and reduced the infiltration of basophils in the spleen. In CRISPR/Cas9 human IL-37b knock-in mice or mice with direct treatment by human IL-37b antibody, AD symptoms including ear swelling and itching were significantly alleviated upon MC903 challenge. Notably, IL-37b presence significantly reduced the basophil infiltration in ear lesions. In summary, IL-37b could regulate the TSLP-mediated activation of basophils and reduce the release of IL-4. The results, therefore, suggest that IL-37 may target TSLP-primed basophils to alleviate AD.


Assuntos
Basófilos/imunologia , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Interleucina-1/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Basófilos/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Citocinas/antagonistas & inibidores , Citocinas/farmacologia , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Regulação para Baixo , Orelha/patologia , Eosinófilos/metabolismo , Técnicas de Introdução de Genes , Humanos , Interleucina-1/genética , Interleucina-1/farmacologia , Interleucina-1/uso terapêutico , Interleucina-4/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Baço/imunologia , Baço/metabolismo , Células Th2/imunologia , Regulação para Cima
15.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299254

RESUMO

The transient receptor potential (TRP) melastatin-like subfamily member 2 (TRPM2) is a non-selective calcium-permeable cation channel. It is expressed by many mammalian tissues, including bone marrow, spleen, lungs, heart, liver, neutrophils, and endothelial cells. The best-known mechanism of TRPM2 activation is related to the binding of ADP-ribose to the nudix-box sequence motif (NUDT9-H) in the C-terminal domain of the channel. In cells, the production of ADP-ribose is a result of increased oxidative stress. In the context of endothelial function, TRPM2-dependent calcium influx seems to be particularly interesting as it participates in the regulation of barrier function, cell death, cell migration, and angiogenesis. Any impairments of these functions may result in endothelial dysfunction observed in such conditions as atherosclerosis or hypertension. Thus, TRPM2 seems to be an attractive therapeutic target for the conditions connected with the increased production of reactive oxygen species. However, before the application of TRPM2 inhibitors will be possible, some issues need to be resolved. The main issues are the lack of specificity, poor membrane permeabilization, and low stability in in vivo conditions. The article aims to summarize the latest findings on a role of TRPM2 in endothelial cells. We also show some future perspectives for the application of TRPM2 inhibitors in cardiovascular system diseases.


Assuntos
Células Endoteliais/metabolismo , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Cálcio/metabolismo , Morte Celular , Movimento Celular , Células Endoteliais/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Estresse Oxidativo/fisiologia , Pirofosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/fisiologia
16.
Elife ; 102021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34250901

RESUMO

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single-molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.


Assuntos
Proteínas de Bactérias/metabolismo , Citidina Trifosfato/metabolismo , DNA Bacteriano/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Centrômero/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Hidrólise , Ligação Proteica , Pirofosfatases/metabolismo
17.
Parasitol Int ; 85: 102423, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34298165

RESUMO

Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.


Assuntos
Regulação da Expressão Gênica , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Leishmaniose Tegumentar Difusa/fisiopatologia , Proteínas de Protozoários/genética , Pirofosfatases/genética , Animais , Leishmania mexicana/enzimologia , Camundongos , Proteínas de Protozoários/metabolismo , Pirofosfatases/metabolismo , Virulência
18.
Bone ; 153: 116111, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252603

RESUMO

Awareness for hypophosphatemic rickets has increased in the last years, based on the availability of specific medical treatments. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is a rare form of hypophosphatemic rickets, which is known to develop in survivors of generalized arterial calcification of infancy (GACI). Both disorders are based on a deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and present with a high clinical variability and a lack of a phenotype-genotype association. ARHR2 is characterized by phosphate wasting due to elevated fibroblast growth factor 23 (FGF23) levels and might represent a response of the organism to minimize ectopic calcification in individuals with ENPP1-deficiency. This report reviews the recent clinical and preclinical data on this ultra-rare disease in childhood.


Assuntos
Raquitismo Hipofosfatêmico Familiar , Raquitismo Hipofosfatêmico , Raquitismo Hipofosfatêmico Familiar/genética , Fatores de Crescimento de Fibroblastos , Humanos , Fosfatos , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Raquitismo Hipofosfatêmico/genética
19.
Mol Cell ; 81(17): 3623-3636.e6, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34270916

RESUMO

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.


Assuntos
Bacillus subtilis/citologia , Citidina Trifosfato/metabolismo , Pirofosfatases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Cromossomos Bacterianos/genética , Citidina Trifosfato/fisiologia , Proteínas do Citoesqueleto/genética , Pirofosfatases/fisiologia
20.
Elife ; 102021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34286695

RESUMO

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Citidina Trifosfato/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Centrômero/metabolismo , Cromossomos Bacterianos , Citidina Trifosfato/genética , DNA Primase , DNA Bacteriano , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Plasmídeos , Ligação Proteica , Pirofosfatases
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