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1.
Bioorg Chem ; 90: 103018, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31220674

RESUMO

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate vmax was 55.56 µmol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.


Assuntos
Aldo-Ceto Redutases/química , Caproatos/química , Proteínas Fúngicas/química , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/metabolismo , Sequência de Aminoácidos , Bacillales/enzimologia , Proteínas de Bactérias/química , Biocatálise , Caproatos/metabolismo , Domínio Catalítico , Exiguobacterium , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose 1-Desidrogenase/química , Cinética , Kluyveromyces/enzimologia , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Engenharia de Proteínas
3.
Biochem Biophys Res Commun ; 488(2): 425-431, 2017 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-28511795

RESUMO

Cancer immunotherapy has many great achievements in recent years. One of the most promising cancer immunotherapies is PD-1/PD-L1 pathway blockade. miRNAs (MicroRNAs) belongs to small noncoding RNA and can regulate gene expression by binding to the 3'UTR. Many miRNAs can inhibit cancer growth by regulating the PD-L1 expression in cancer cells. Herein, we firstly found that PD-L1 could be the target of miR-142-5p by using bioinformatics methods, then we conduct luciferase activity assay, RT-PCR and western blot experiments to demonstrate that miR-142-5p can regulate PD-L1 expression by binding to its 3'UTR. And in vivo experiments certified that miR-142-5p overexpression can inhibit pancreatic cancer growth. Flow cytometry and RT-PCR experiment demonstrated that miR-142-5p overexpression on tumor cells inhibits the expression of PD-L1 on tumor cells which result in the increase of CD4+ T lymphocytes and CD8+ T lymphocytes, the decrease of PD-1+ T lymphocytes and increase of IFN-γ and TNF-α. So, miR-142-5p overexpression can enhance anti-tumor immunity by blocking PD-L1/PD-1 pathway. Our results identify a novel mechanism by which PD-L1 is regulated by miR-142-5p and overexpression of miR-142-5p could enhance the anti-tumor immunity.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , MicroRNAs/farmacologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Proliferação de Células/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Imunidade , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Células Tumorais Cultivadas
5.
Nat Commun ; 12(1): 4300, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262035

RESUMO

Common fragile sites (CFSs) are specific breakage-prone genomic regions and are present frequently in cancer cells. The (E2-independent) E3 ubiquitin-conjugating enzyme FATS (fragile site-associated tumor suppressor) has antitumor activity in cancer cells, but the function of FATS in immune cells is unknown. Here, we report a function of FATS in tumor development via regulation of tumor immunity. Fats-/- mice show reduced subcutaneous B16 melanoma and H7 pancreatic tumor growth compared with WT controls. The reduced tumor growth in Fats-/- mice is macrophage dependent and is associated with a phenotypic shift of macrophages within the tumor from tumor-promoting M2-like to antitumor M1-like macrophages. In addition, FATS deficiency promotes M1 polarization by stimulating and prolonging NF-κB activation by disrupting NF-κB/IκBα negative feedback loops and indirectly enhances both CD4+ T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) adaptive immune responses to promote tumor regression. Notably, transfer of Fats-/- macrophages protects mice against B16 melanoma. Together, these data suggest that FATS functions as an immune regulator and is a potential target in cancer immunotherapy.


Assuntos
Proteínas de Ciclo Celular/imunologia , Macrófagos/imunologia , Neoplasias/imunologia , Proteínas Supressoras de Tumor/imunologia , Enzimas de Conjugação de Ubiquitina/imunologia , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Humanos , Imunoterapia , Ativação de Macrófagos , Camundongos , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Proteínas Supressoras de Tumor/genética , Enzimas de Conjugação de Ubiquitina/genética
6.
Appl Biochem Biotechnol ; 190(4): 1257-1270, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31741208

RESUMO

Echinocandin B deacylase (EBDA), from Actinoplanes utahensis ZJB-08196, is capable of cleaving the linoleoyl group from echinocandin B (ECB), forming the echinocandin B nucleus (ECBN), which is a key precursor of semisynthetic antifungal antibiotics. In the present study, molecular evolution of AuEBDA by random mutagenesis combined with site-directed mutagenesis (SDM) and screening was performed. Random mutagenesis on the wild-type (WT) AuEBDA generated two beneficial substitutions of G287Q, R527V. The "best" variant AuEBDA-G287Q/R527V was obtained by combining G287Q with R527V through SDM, which was most active at 35 °C, pH 7.5, with Km and vmax values of 0.68 mM and 395.26 U/mg, respectively. Mutation of G287Q/R527V markedly increased the catalytic efficiency kcat/Km by 290% compared with the WT-AuEBDA.


Assuntos
Actinoplanes/enzimologia , Amidoidrolases/genética , Antifúngicos/farmacologia , Equinocandinas/química , Proteínas Fúngicas/química , Mutagênese Sítio-Dirigida , Catálise , Escherichia coli/enzimologia , Escherichia coli/genética , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Cinética , Mutação , Streptomyces lividans/genética , Temperatura
7.
Biomed Pharmacother ; 102: 464-471, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29579707

RESUMO

As a phenylpropanoid and dibenzylbutyrolactone lignan present in medical plants, such as those used in traditional Chinese herbal medicine, including Arctium lappa (Niubang), arctigenin exhibits antimicrobial, anti-inflammatory, and anticancer activities. In this study, we investigated the protective role of arctigenin in Concanavalin A (ConA)-induced acute hepatitis in mice. Arctigenin remarkably reduced the congestion and necroinflammation of livers, and improved hepatic function (ALT and AST) in ConA-induced acute hepatitis in vivo. The infiltration of CD4 T, NKT and macrophages into the livers was found to be reduced with arctigenin treatment. Arctigenin suppressed ConA-induced T lymphocyte proliferations that might have resulted from enhanced IL-10 production by macrophages and CD4 T cells. These results suggested that arctigenin could be a powerful drug candidate for acute hepatitis through immune suppression.


Assuntos
Furanos/farmacologia , Hepatite/complicações , Hepatite/imunologia , Lignanas/farmacologia , Fígado/imunologia , Fígado/lesões , Substâncias Protetoras/farmacologia , Doença Aguda , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Concanavalina A , Hepatite/tratamento farmacológico , Inflamação/tratamento farmacológico , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-10/biossíntese , Interleucina-10/genética , Fígado/efeitos dos fármacos , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células RAW 264.7
8.
Oncotarget ; 8(59): 99693-99706, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29245934

RESUMO

Pancreatic cancer is an aggressive malignancy that is unresponsive to conventional radiation and chemotherapy. Therefore, development of novel immune therapeutic strategies is urgently needed. L-4F, an Apolipoprotein A-I (ApoA-I) mimetic peptide, is engineered to mimic the anti-inflammatory and anti-oxidative functionalities of ApoA-I. In this work, H7 cells were orthotopically implanted in C57BL/6 mice and treated with L-4F. Then, pancreatic cancer progression and the inflammatory microenvironment were investigated in vivo. The cytotoxicity of L-4F toward H7 cells was assessed in vitro. Furthermore, we investigated the effects of L-4F on macrophage polarization by analyzing the polarization and genes of mouse bone marrow-derived macrophages in vitro. The results show that L-4F substantially reduced the tumorigenicity of H7 cells. L-4F inhibited inflammation by reducing the accumulation of inflammatory cells, such as IL-17A-, IL-4-, GM-CSF-, IL-1ß-, and IL-6-producing cells and Th1 and Th17. Notably, L-4F also decreased the percentage of macrophages in tumor tissues, especially M2 macrophages (CD11b+F4/80+CD206+), which was also confirmed in vitro. Additionally, the expression of the M2 marker genes Arg1, MRC1, and CCL22 and the inflammatory genes IL-6, iNOS, and IL-12 was decreased by L-4F, indicating that L-4F prevents M2 type macrophage polarization. However, L-4F could not directly attenuate H7 cell invasion or proliferation and did not induce apoptosis. In addition, L-4F potently down-regulated STAT3, JNK and ERK signaling pathways but not affects the phosphorylation of p38 in RAW 264.7 cells. These results suggest that L-4F exhibits an effective therapeutic effect on pancreatic cancer progression by inhibiting tumor-associated macrophages and inflammation.

9.
Oncotarget ; 8(43): 74178-74187, 2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-29088777

RESUMO

Esophageal carcinoma (EC) is a malignancy with high metastatic potential. Chromosomal helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21, and it is amplified in many solid tumors. However, the status of CHD1L protein expression in EC and its clinical significance is uncertain. This study was designed to investigate the significance of CHD1L expression in human EC and its biological function in EC cells. The expression of CHD1L was examined by immunohistochemistry in 191 surgically resected ECs. The associations between CHD1L expression and clinical pathological parameters and the prognostic value of CHD1L were analyzed. Western blot analysis showed that CHD1L was overexpressed in EC cell lines. In addition, positive CHD1L expression was strongly related to advanced clinical stage (P<0.01), and lymph node metastasis (P<0.01) of EC. The Kaplan-Meier curve indicated that high expression of CHD1L may result in poor prognosis of EC patients (P<0.01), and multivariate analysis showed that CHD1L overexpression was an independent predictor of overall survival. Furthermore, suppression of CHD1L in EC cells increased apoptosis and decreased cell proliferation invasion ability. Our results suggest that CHD1L is a target oncogene with the potential to serve as a novel prognostic biomarker in EC pathogenesis.

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