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1.
Ann Transplant ; 29: e943688, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38952007

RESUMO

BACKGROUND The relationship between clonal hematopoiesis (CH)-associated gene mutations and allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been extensively studied since next-generation sequencing (NGS) technology became widely available. However, research has mainly focused on the relationship between donor CH mutations and transplant prognosis, and research into the relationship between CH mutations in the recipient and acute graft-versus-host disease (aGVHD) is lacking. MATERIAL AND METHODS We analyzed NGS results and their correlation with aGVHD and prognosis in 196 AML patients undergoing allo-HSCT. RESULTS A total of 93 (47.4%) patients had CH mutations. The most frequently mutated genes were DNMT3A (28 of 196; 14.3%), TET2 (22 of 196; 11.2%), IDH1 (15 of 196; 7.7%), IDH2 (14 of 196; 7.1%), and ASXL1 (13 of 196; 6.6%). The incidence of aGVHD was higher in patients older than 45 years old with DTA mutations (DNMT3A, TET2 or ASXL1). DNMT3A mutation but not with TET2 or ASXL1 mutation was an independent risk factor for aGVHD in patients receiving allo-HSCT older than 45 years old. With a median follow-up of 42.7 months, CH mutations were not associated with overall survival and leukemia-free survival. CONCLUSIONS DNMT3A mutation, but not TET2 or ASXL1 mutation, was associated with higher incidence of aGVHD.


Assuntos
Hematopoiese Clonal , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Mutação , Humanos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Masculino , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/etiologia , Pessoa de Meia-Idade , Adulto , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Hematopoiese Clonal/genética , Adulto Jovem , Adolescente , DNA Metiltransferase 3A , Dioxigenases , DNA (Citosina-5-)-Metiltransferases/genética , Idoso , Prognóstico , Transplante Homólogo , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Ligação a DNA , Proteínas Repressoras
2.
Hematol Oncol ; 42(4): e3295, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38979860

RESUMO

The biological role of Ten-11 translocation 2 (TET2) and the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the development of extra-nodal natural killer/T-cell lymphoma (ENKTL) remains unclear. The level of 5mC and 5hmC was detected in 112 cases of ENKTL tissue specimens by immunohistochemical (IHC) staining. Subsequently, TET2 knockdown and the overexpression cell models were constructed in ENKTL cell lines. Biochemical analyses were used to assess proliferation, apoptosis, cell cycle and monoclonal formation in cells treated or untreated with L-Ascorbic acid sodium salt (LAASS). Dot-Blots were used to detect levels of genome 5mC and 5hmC. Additionally, the ILLUMINA 850k methylation chip was used to analyze the changes of TET2 regulatory genes. RNA-Seq was used to profile differentially expressed genes regulated by TET2. The global level of 5hmC was significantly decreased, while 5mC was highly expressed in ENKTL tissue. TET2 protein expression was negatively correlated with the ratio of 5mC/5hmC (p < 0.0001). The 5mC/5hmC status were related to the site of disease, clinical stage, PINK score and Ki-67 index, as well as the 5-year OS. TET2 knockdown prolonged the DNA synthesis period, increased the cloning ability of tumor cells, increased the level of 5mC and decreased the level of 5hmC in ENKTL cells. While overexpression of TET2 presented the opposite effect. Furthermore, treatment of ENKTL cells with LAASS significantly induced ENKTL cell apoptosis. These results suggest that TET2 plays an important role in ENKTL development via regulation of 5mC and 5hmC and may serve as a novel therapeutic target for ENKTL.


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA , Dioxigenases , Linfoma Extranodal de Células T-NK , Proteínas Proto-Oncogênicas , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Masculino , Linfoma Extranodal de Células T-NK/metabolismo , Linfoma Extranodal de Células T-NK/patologia , Linfoma Extranodal de Células T-NK/genética , Pessoa de Meia-Idade , Adulto , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Idoso , Linhagem Celular Tumoral , Proliferação de Células
3.
BMJ Case Rep ; 17(6)2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890114

RESUMO

Sarcomas constitute approximately 1% of adult cancers and 8%-10% of paediatric cancers. Undifferentiated pleomorphic sarcoma (UPS) is a type of soft-tissue sarcoma (STS) characterised by dedifferentiated cancer cells. The most common sites of metastasis for UPS include the lungs, liver, bones and regional lymph nodes. Brain metastasis is rare, affecting only 1%-8% of STS patients. This report presents a unique case of a woman in her 80s with a TET2-mutant UPS metastatic to the lung and brain.


Assuntos
Neoplasias Encefálicas , Proteínas de Ligação a DNA , Dioxigenases , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas , Sarcoma , Humanos , Feminino , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/diagnóstico por imagem , Sarcoma/genética , Sarcoma/secundário , Sarcoma/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a DNA/genética , Idoso de 80 Anos ou mais , Mutação , Evolução Fatal
4.
Cell Mol Life Sci ; 81(1): 270, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886218

RESUMO

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.


Assuntos
Diferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Trofoblastos , Feminino , Humanos , Gravidez , Blastocisto/metabolismo , Blastocisto/citologia , Linhagem da Célula/genética , Dioxigenases/metabolismo , Dioxigenases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Trofoblastos/metabolismo , Trofoblastos/citologia
5.
Discov Med ; 36(185): 1289-1297, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38926115

RESUMO

BACKGROUND: Genetic mutations play a crucial role in the development and progression of myelodysplastic syndromes (MDS), impacting the immune microenvironment and influencing the choice of treatment regimen, as well as the efficacy and prognosis of patients. The objective of this study was to examine variations in hematological and immunological characteristics associated with common gene mutations in MDS patients and establish a foundation for the precise treatment of MDS. METHODS: The hematological, immunological, and other clinical features of 71 recently diagnosed MDS patients from January 1, 2019, to July 31, 2023, were retrospectively analyzed. These patients were categorized based on their gene mutations, and the variances in hematological and immunological characteristics among distinct groups were compared. RESULTS: Hematological variances were observed among different gene mutation groups. Specifically, platelet counts in the splicing factor 3B subunit 1 (SF3B1) mutation group were notably higher compared to the wild-type group (p = 0.009). Conversely, in the additional sex combs like 1 (ASXL1) mutation groups, monocyte ratios were significantly elevated in comparison to the wild-type group (p = 0.046), and in the ten-eleven translocation 2 (TET2) mutation group, lymphocyte ratios were significantly lower (p = 0.022). Additionally, the leukocyte (p = 0.005), neutrophil ratio (p = 0.002), and lymphocyte ratio (p = 0.001) were significantly higher in the Runt-related transcription factor 1 (RUNX1) mutation group. Regarding immunological distinctions, the Natural Killer (NK) cell ratio demonstrated a significant increase in the SF3B1 mutation group (p = 0.005). Moreover, the TET2 mutation group exhibited a significantly higher Interleukin-8 (IL-8) level (p = 0.017). In contrast, the U2 small nuclear RNA auxiliary factor 1 (U2AF1) group displayed significantly lower levels of IL-1ß (p = 0.033), IL-10 (p = 0.033), and Tumour Necrosis Factor-α (TNF-α) (p = 0.009). CONCLUSION: Distinct variations exist in the immune microenvironment of MDS associated with different genetic mutations. Further studies are imperative to delve into the underlying mechanisms that drive these differences.


Assuntos
Dioxigenases , Mutação , Síndromes Mielodisplásicas , Fatores de Processamento de RNA , Humanos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/sangue , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Fatores de Processamento de RNA/genética , Estudos Retrospectivos , Adulto , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/genética , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Células Matadoras Naturais/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Contagem de Plaquetas , Proteínas Repressoras
6.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 32(3): 819-824, 2024 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-38926973

RESUMO

OBJECTIVE: To analyze the DTA (DNMT3A, TET2, ASXL1) mutations in patients with myeloproliferative neoplasms (MPN), and preliminarily explore their correlation with thromboembolism. METHODS: Clinical characteristics of 62 patients diagnosed de novo MPN at Central Hospital Affiliated to Shandong First Medical University from September 2016 to September 2022 were retrospectively analyzed. Next-generation sequencing was used to detect 35 MPN-related genes, and the DTA mutations in MPN patients and their relationship with thromboembolic events were analyzed. RESULTS: 75.8% (47/62) of the patients presented pathogenic non-driver mutations, and the mean number of pathogenic non-driver mutations per patient was 1.08. Among them, the most frequently mutated non-driver genes were TET2 (38.7%, 24/62), DNMT3A (9.7%, 6/62) and ASXL1 (6.5%, 4/62). The presence of DTA gene mutations was 50% (31/62) in the total MPN patients, and mainly accompanied by driver mutations. The mutation rate of DTA in patients aged ≥60 years was significantly higher than that in patients <60 years old (P =0.039). The incidence of thromboembolism in patients with DTA mutation was 58.1% (18/31), which was significantly higher than that in patients without DTA mutation (19.4%, 6/31) (P =0.002). The TET2 gene mutation rate in MPN patients with thromboembolism was 66.7% (16/24), which was significantly higher than that in patients without thromboembolism (21.1%, 8/38) (P =0.00). CONCLUSION: Patients with MPN have a higher incidence of DTA mutations, which are mainly accompanied by driver gene mutations. The incidence of thromboembolism in MPN patients with DTA mutations is higher than that in patients without DTA mutations. Especially, the elderly (≥60 years) essential thrombocythemia(ET) and polycythemia vera(PV) patients with TET2 mutation should be vigilant for thromboembolic events.


Assuntos
DNA Metiltransferase 3A , Proteínas de Ligação a DNA , Dioxigenases , Mutação , Transtornos Mieloproliferativos , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Tromboembolia , Humanos , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/complicações , Tromboembolia/genética , Estudos Retrospectivos , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , DNA (Citosina-5-)-Metiltransferases/genética , Masculino , Feminino , Sequenciamento de Nucleotídeos em Larga Escala
7.
Ecotoxicol Environ Saf ; 280: 116579, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38865940

RESUMO

Environmental exposure to the cadmium (Cd) has been shown to be a risk factor for colorectal cancer (CRC) progression, but the exact mechanism has not been fully elucidated. In this study, we found that chronic Cd (3 µM) exposure promoted the proliferation, adhesion, migration, and invasion of CRC cells in vitro, as well as lung metastasis in vivo. RNA-seq and TCGA-COAD datasets revealed that decreased hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) expression may be a crucial factor in Cd-induced CRC progression. Further analysis using qRT-PCR and tissue microarrays from CRC patients showed that HADHB expression was significantly reduced in CRC tissues compared to adjacent normal tissues, and low HADHB expression was associated with adverse clinical features and poor overall survival, either directly or through TNM stage. Furthermore, HADHB was found to play an important role in the Cd-induced malignant metastatic phenotype of CRC cells and lung metastasis in mice. Mechanistically, we discovered that chronic Cd exposure resulted in hypermethylation of the HADHB promoter region via inhibition of DNA demethylase tet methylcytosine dioxygenase 2 (TET2), which then led to decreased HADHB expression and activation of the FAK signaling pathway, and ultimately contributed to CRC progression. In conclusion, this study provided a new potential insight and evaluable biomarker for Cd exposure-induced CRC progression and treatment.


Assuntos
Cádmio , Neoplasias Colorretais , Proteínas de Ligação a DNA , Dioxigenases , Progressão da Doença , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/induzido quimicamente , Humanos , Dioxigenases/genética , Animais , Camundongos , Cádmio/toxicidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular Tumoral , Masculino , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos Nus , Metilação de DNA/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos
8.
J Hazard Mater ; 475: 134889, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38878436

RESUMO

Both polycyclic aromatic hydrocarbons (PAHs) and heavy metals persist in the environment and are toxic to organisms. Their co-occurrence makes any of them difficult to remove during bioremediation and poses challenges to environmental management and public health. Microorganisms capable of effectively degrading PAHs and detoxifying heavy metals concurrently are required to improve the bioremediation process. In this study, we isolated a new strain, Sphingobium sp. SJ10-10, from an abandoned coking plant and demonstrated its capability to simultaneously degrade 92.6 % of 75 mg/L phenanthrene and reduce 90 % of 3.5 mg/L hexavalent chromium [Cr(VI)] within 1.5 days. Strain SJ10-10 encodes Rieske non-heme iron ring-hydroxylating oxygenases (RHOs) to initiate PAH degradation. Additionally, a not-yet-reported protein referred to as Sphingobium chromate reductase (SchR), with low sequence identity to known chromate reductases, was identified to reduce Cr(VI). SchR is distributed across different genera and can be classified into two classes: one from Sphingobium members and the other from non-Sphingobium species. The widespread presence of SchR in those RHO-containing Sphingobium members suggests that they are excellent candidates for bioremediation. In summary, our study demonstrates the simultaneous removal of PAHs and Cr(VI) by strain SJ10-10 and provides valuable insights into microbial strategies for managing complex pollutant mixtures.


Assuntos
Biodegradação Ambiental , Cromatos , Dioxigenases , Oxirredutases , Hidrocarbonetos Policíclicos Aromáticos , Sphingomonadaceae , Sphingomonadaceae/enzimologia , Sphingomonadaceae/metabolismo , Dioxigenases/metabolismo , Dioxigenases/genética , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/química , Cromatos/metabolismo , Oxirredutases/metabolismo , Cromo/metabolismo , Fenantrenos/metabolismo
9.
J Biotechnol ; 391: 92-98, 2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-38880386

RESUMO

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.


Assuntos
Benzaldeídos , Enzimas Imobilizadas , Mutagênese Sítio-Dirigida , Mutação , Benzaldeídos/metabolismo , Benzaldeídos/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/química , Dioxigenases/genética , Dioxigenases/metabolismo , Dioxigenases/química , Eugenol/metabolismo , Eugenol/química , Eugenol/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Engenharia de Proteínas/métodos
10.
Int J Mol Sci ; 25(12)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38928288

RESUMO

Abscisic acid (ABA) plays a crucial role in plant defense mechanisms under adverse environmental conditions, but its metabolism and perception in response to heavy metals are largely unknown. In Pisum sativum exposed to CdCl2, an accumulation of free ABA was detected in leaves at different developmental stages (A, youngest, unexpanded; B1, youngest, fully expanded; B2, mature; C, old), with the highest content found in A and B1 leaves. In turn, the content of ABA conjugates, which was highest in B2 and C leaves under control conditions, increased only in A leaves and decreased in leaves of later developmental stages after Cd treatment. Based on the expression of PsNCED2, PsNCED3 (9-cis-epoxycarotenoid dioxygenase), PsAO3 (aldehyde oxidase) and PsABAUGT1 (ABA-UDP-glucosyltransferase), and the activity of PsAOγ, B2 and C leaves were found to be the main sites of Cd-induced de novo synthesis of ABA from carotenoids and ABA conjugation with glucose. In turn, ß-glucosidase activity and the expression of genes encoding ABA receptors (PsPYL2, PsPYL4, PsPYL8, PsPYL9) suggest that in A and B1 leaves, Cd-induced release of ABA from inactive ABA-glucosyl esters and enhanced ABA perception comes to the forefront when dealing with Cd toxicity. The distinct role of leaves at different developmental stages in defense against the harmful effects of Cd is discussed.


Assuntos
Ácido Abscísico , Cádmio , Regulação da Expressão Gênica de Plantas , Pisum sativum , Folhas de Planta , Proteínas de Plantas , Ácido Abscísico/metabolismo , Pisum sativum/metabolismo , Pisum sativum/efeitos dos fármacos , Pisum sativum/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Cádmio/metabolismo , Cádmio/toxicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Dioxigenases/metabolismo , Dioxigenases/genética , beta-Glucosidase/metabolismo , beta-Glucosidase/genética
11.
J Clin Invest ; 134(11)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38828722

RESUMO

The occurrence of clonal hematopoiesis of indeterminate potential (CHIP), in which advantageous somatic mutations result in the clonal expansion of blood cells, increases with age, as do an increased risk of mortality and detrimental outcomes associated with CHIP. However, the role of CHIP in susceptibility to pulmonary infections, which also increase with age, is unclear. In this issue of the JCI, Quin and colleagues explored the role of CHIP in bacterial pneumonia. Using characterization of immune cells from human donors and mice lacking tet methylcytosine dioxygenase 2 (Tet2), the authors mechanistically link myeloid immune cell dysfunction to CHIP-mediated risk of bacterial pneumonia. The findings suggest that CHIP drives inflammaging and immune senescence, and provide Tet2 status in older adults as a potential prognostic tool for informing treatment options related to immune modulation.


Assuntos
Hematopoiese Clonal , Proteínas de Ligação a DNA , Dioxigenases , Pneumonia Bacteriana , Humanos , Animais , Hematopoiese Clonal/imunologia , Hematopoiese Clonal/genética , Camundongos , Dioxigenases/genética , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo
12.
J Am Chem Soc ; 146(27): 18292-18297, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38941563

RESUMO

We investigated the metal-substituted catalytic activity of human cysteamine dioxygenase (ADO), an enzyme pivotal in regulating thiol metabolism and contributing to oxygen homeostasis. Our findings demonstrate the catalytic competence of cobalt(II)- and nickel(II)-substituted ADO in cysteamine oxygenation. Notably, Co(II)-ADO exhibited superiority over Ni(II)-ADO despite remaining significantly less active than the natural enzyme. Structural analyses through X-ray crystallography and cobalt K-edge excitation confirmed successful metal substitution with minimal structural perturbations. This provided a robust structural basis, supporting a conserved catalytic mechanism tailored to distinct metal centers. This finding challenges the proposed high-valent ferryl-based mechanism for thiol dioxygenases, suggesting a non-high-valent catalytic pathway in the native enzyme. Further investigation of the cysteamine-bound or a peptide mimic of N-terminus RGS5 bound Co(II)-ADO binary complex revealed the metal center's high-spin (S = 3/2) state. Upon reaction with O2, a kinetically and spectroscopically detectable intermediate emerged with a ground spin state of S = 1/2. This intermediate exhibits a characteristic 59Co hyperfine splitting (A = 67 MHz) structure in the EPR spectrum alongside UV-vis features, consistent with known low-spin Co(III)-superoxo complexes. This observation, unique for protein-bound thiolate-ligated cobalt centers in a protein, unveils the capacities for O2 activation in such metal environments. These findings provide valuable insights into the non-heme iron-dependent thiol dioxygenase mechanistic landscape, furthering our understanding of thiol metabolism regulation. The exploration of metal-substituted ADO sheds light on the intricate interplay between metal and catalytic activity in this essential enzyme.


Assuntos
Cobalto , Dioxigenases , Cobalto/química , Cobalto/metabolismo , Dioxigenases/metabolismo , Dioxigenases/química , Humanos , Oxigênio/química , Oxigênio/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo
13.
Exp Biol Med (Maywood) ; 249: 10051, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38881848

RESUMO

Podocyte injury or dysfunction can lead to proteinuria and glomerulosclerosis. Zonula occludens 1 (ZO-1) is a tight junction protein which connects slit diaphragm (SD) proteins to the actin cytoskeleton. Previous studies have shown that the expression of ZO-1 is decreased in chronic kidney disease (CKD). Thus, elucidation of the regulation mechanism of ZO-1 has considerable clinical importance. Triptolide (TP) has been reported to exert a strong antiproteinuric effect by inhibiting podocyte epithelial mesenchymal transition (EMT) and inflammatory response. However, the underlying mechanisms are still unclear. We found that TP upregulates ZO-1 expression and increases the fluorescence intensity of ZO-1 in a puromycin aminonucleoside (PAN)-induced podocyte injury model. Permeablity assay showed TP decreases podocyte permeability in PAN-treated podocyte. TP also upregulates the DNA demethylase TET2. Our results showed that treatment with the DNA methyltransferase inhibitors 5-azacytidine (5-AzaC) and RG108 significantly increased ZO-1 expression in PAN-treated podocytes. Methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA immunoprecipitation (hMeDIP) results showed that TP regulates the methylation status of the ZO-1 promoter. Knockdown of TET2 decreased ZO-1 expression and increased methylation of its promoter, resulting in the increase of podocyte permeability. Altogether, these results indicate that TP upregulates the expression of ZO-1 and decreases podocyte permeability through TET2-mediated 5 mC demethylation. These findings suggest that TP may alleviate podocyte permeability through TET2-mediated hydroxymethylation of ZO-1.


Assuntos
Dioxigenases , Diterpenos , Compostos de Epóxi , Fenantrenos , Podócitos , Proteína da Zônula de Oclusão-1 , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteína da Zônula de Oclusão-1/metabolismo , Fenantrenos/farmacologia , Diterpenos/farmacologia , Compostos de Epóxi/farmacologia , Dioxigenases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Permeabilidade/efeitos dos fármacos , Humanos , Metilação de DNA/efeitos dos fármacos
14.
Appl Environ Microbiol ; 90(6): e0143623, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38709097

RESUMO

Rieske non-heme dioxygenase family enzymes play an important role in the aerobic biodegradation of nitroaromatic pollutants, but no active dioxygenases are available in nature for initial reactions in the degradation of many refractory pollutants like 2,4-dichloronitrobenzene (24DCNB). Here, we report the engineering of hotspots in 2,3-dichloronitrobenzene dioxygenase from Diaphorobacter sp. strain JS3051, achieved through molecular dynamic simulation analysis and site-directed mutagenesis, with the aim of enhancing its catalytic activity toward 24DCNB. The computationally predicted activity scores were largely consistent with the detected activities in wet experiments. Among them, the two most beneficial mutations (E204M and M248I) were obtained, and the combined mutant reached up to a 62-fold increase in activity toward 24DCNB, generating a single product, 3,5-dichlorocatechol, which is a naturally occurring compound. In silico analysis confirmed that residue 204 affected the substrate preference for meta-substituted nitroarenes, while residue 248 may influence substrate preference by interaction with residue 295. Overall, this study provides a framework for manipulating nitroarene dioxygenases using computational methods to address various nitroarene contamination problems.IMPORTANCEAs a result of human activities, various nitroaromatic pollutants continue to enter the biosphere with poor degradability, and dioxygenation is an important kickoff step to remove toxic nitro-groups and convert them into degradable products. The biodegradation of many nitroarenes has been reported over the decades; however, many others still lack corresponding enzymes to initiate their degradation. Although rieske non-heme dioxygenase family enzymes play extraordinarily important roles in the aerobic biodegradation of various nitroaromatic pollutants, prediction of their substrate specificity is difficult. This work greatly improved the catalytic activity of dioxygenase against 2,4-dichloronitrobenzene by computer-aided semi-rational design, paving a new way for the evolution strategy of nitroarene dioxygenase. This study highlights the potential for using enzyme structure-function information with computational pre-screening methods to rapidly tailor the catalytic functions of enzymes toward poorly biodegradable contaminants.


Assuntos
Dioxigenases , Nitrobenzenos , Dioxigenases/metabolismo , Dioxigenases/genética , Dioxigenases/química , Nitrobenzenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Biodegradação Ambiental , Mutagênese Sítio-Dirigida , Simulação de Dinâmica Molecular
15.
Appl Environ Microbiol ; 90(6): e0023324, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38727223

RESUMO

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.


Assuntos
Benzaldeídos , Ácidos Cumáricos , Dioxigenases , Benzaldeídos/metabolismo , Ácidos Cumáricos/metabolismo , Dioxigenases/metabolismo , Dioxigenases/genética , Engenharia Metabólica , Coenzimas/metabolismo , Engenharia de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
16.
Environ Sci Pollut Res Int ; 31(25): 37532-37551, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38777975

RESUMO

Polycyclic aromatic compounds (PAHs) are persistent organic pollutants of environmental concern due to their potential impacts on food chain, with plants being particularly vulnerable. While plants can uptake, transport, and transform PAHs, the precise mechanisms underlying their localization and degradation are not fully understood. Here, a cultivation experiment conducted with Panicum miliaceum exposed different concentrations of phenanthrene (PHE). Intermediate PHE degradation compounds were identified via GC-MS analysis, leading to the proposal of a phytodegradation pathway featuring three significant benzene ring cleavage steps. Our results showed that P. miliaceum exhibited the ability to effectively degrade high levels of PHE, resulting in the production of various intermediate products through several chemical changes. Examination of the localization and anatomical characteristics revealed structural alterations linked to PHE stress, with an observed enhancement in PHE accumulation density in both roots and shoots as treatment levels increased. Following a 2-week aging period, a decrease in the amount of PHE accumulation was observed, along with a change in its localization. Bioinformatics analysis of the P. miliaceum 2-oxoglutarate-dependent dioxygenase (2-ODD) DAO-like protein revealed a 299 amino acid structure with two highly conserved domains, namely 2OG-FeII_Oxy and DIOX_N. Molecular docking analysis aligned with experimental results, strongly affirming the potential link and direct action of 2-ODD DAO-like protein with PHE. Our study highlights P. miliaceum capacity for PAHs degradation and elucidates the mechanisms behind enhanced degradation efficiency. By integrating experimental evidence with bioinformatics analysis, we offer valuable insights into the potential applications of plant-based remediation strategies for PAHs-contaminated environments.


Assuntos
Biodegradação Ambiental , Dioxigenases , Fenantrenos , Fenantrenos/metabolismo , Dioxigenases/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo
17.
Int J Mol Sci ; 25(10)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38791222

RESUMO

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mieloide Aguda , Mutação , Proteínas Proto-Oncogênicas B-raf , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dioxigenases , Proteínas de Ligação a DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Janus Quinase 2/genética , Leucemia Mieloide Aguda/genética , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Repressoras/genética , Estudos Retrospectivos
18.
J Exp Med ; 221(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38805014

RESUMO

Phenotypic plasticity is a rising cancer hallmark, and lung adeno-to-squamous transition (AST) triggered by LKB1 inactivation is significantly associated with drug resistance. Mechanistic insights into AST are urgently needed to identify therapeutic vulnerability in LKB1-deficient lung cancer. Here, we find that ten-eleven translocation (TET)-mediated DNA demethylation is elevated during AST in KrasLSL-G12D/+; Lkb1L/L (KL) mice, and knockout of individual Tet genes reveals that Tet2 is required for squamous transition. TET2 promotes neutrophil infiltration through STAT3-mediated CXCL5 expression. Targeting the STAT3-CXCL5 nexus effectively inhibits squamous transition through reducing neutrophil infiltration. Interestingly, tumor-infiltrating neutrophils are laden with triglycerides and can transfer the lipid to tumor cells to promote cell proliferation and squamous transition. Pharmacological inhibition of macropinocytosis dramatically inhibits neutrophil-to-cancer cell lipid transfer and blocks squamous transition. These data uncover an epigenetic mechanism orchestrating phenotypic plasticity through regulating immune microenvironment and metabolic communication, and identify therapeutic strategies to inhibit AST.


Assuntos
Quimiocina CXCL5 , Proteínas de Ligação a DNA , Dioxigenases , Neoplasias Pulmonares , Neutrófilos , Proteínas Proto-Oncogênicas , Fator de Transcrição STAT3 , Animais , Neutrófilos/metabolismo , Fator de Transcrição STAT3/metabolismo , Camundongos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Quimiocina CXCL5/metabolismo , Quimiocina CXCL5/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Dioxigenases/metabolismo , Pinocitose , Linhagem Celular Tumoral , Infiltração de Neutrófilos , Camundongos Knockout , Camundongos Endogâmicos C57BL , Metabolismo dos Lipídeos
19.
Protein Sci ; 33(6): e4997, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38723110

RESUMO

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3ß3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by ß-ß interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the ß subunit interfaces, with subsequent targeted improvements of the subunits.


Assuntos
Estabilidade Enzimática , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Modelos Moleculares , Dioxigenases/química , Dioxigenases/metabolismo , Dioxigenases/genética , Temperatura , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Concentração de Íons de Hidrogênio , Complexo III da Cadeia de Transporte de Elétrons
20.
Biomolecules ; 14(5)2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38786006

RESUMO

Age is the primary risk factor for neurodegenerative diseases such as Alzheimer's and Huntington's disease. Alzheimer's disease is the most common form of dementia and a leading cause of death in the elderly population of the United States. No effective treatments for these diseases currently exist. Identifying effective treatments for Alzheimer's, Huntington's, and other neurodegenerative diseases is a major current focus of national scientific resources, and there is a critical need for novel therapeutic strategies. Here, we investigate the potential for targeting the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3HAA) using Caenorhabditis elegans expressing amyloid-beta or a polyglutamine peptide in body wall muscle, modeling the proteotoxicity in Alzheimer's and Huntington's disease, respectively. We show that knocking down the enzyme that degrades 3HAA, 3HAA dioxygenase (HAAO), delays the age-associated paralysis in both models. This effect on paralysis was independent of the protein aggregation in the polyglutamine model. We also show that the mechanism of protection against proteotoxicity from HAAO knockdown is mimicked by 3HAA supplementation, supporting elevated 3HAA as the mediating event linking HAAO knockdown to delayed paralysis. This work demonstrates the potential for 3HAA as a targeted therapeutic in neurodegenerative disease, though the mechanism is yet to be explored.


Assuntos
Ácido 3-Hidroxiantranílico , Peptídeos beta-Amiloides , Caenorhabditis elegans , Paralisia , Peptídeos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Animais , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos/farmacologia , Ácido 3-Hidroxiantranílico/metabolismo , Paralisia/induzido quimicamente , Paralisia/metabolismo , Paralisia/genética , Modelos Animais de Doenças , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/tratamento farmacológico , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Doença de Huntington/metabolismo , Doença de Huntington/genética , Dioxigenases/metabolismo , Dioxigenases/genética
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