Transcription factor EB attenuates neuronal damage following cerebral ischemia via regulating autophagy-lysosomal pathway / 国际脑血管病杂志

The disorder of autophagy lysosomal pathway (ALP) is an important pathogenesis of neuronal damage after cerebral ischemia, and the restoration of ALP may alleviate neuronal damage after cerebral ischemia. As the main transcription factor regulating ALP, transcription factor EB (TFEB) can directly regulate autophagosome generation, autophagosome-lysosome fusion, and autophagic flux by regulating the expression of autophagic genes and lysosomal genes. Therefore, regulating TFEB can alleviate ALP dysfunction and thereby reduce cerebral ischemic damage. This article reviews the structure, biological function of TFEB and its role in regulating ALP to alleviate neuronal damage after cerebral ischemia.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Soluble expression, purification and structural analysis of the bHLH transcription factor Bmsage of Bombyx mori / 生物工程学报

Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra. The results showed that NusA and MBP could significantly enhance the soluble expression of Bmsage in E. coli, but it was difficult to separate Bmsage from these tags. SUMO could not only increase the soluble expression of Bmsage in E. coli to a certain degree, but also be effectively separated from Bmsage. Other tags did not effectively promote the soluble expression of Bmsage in E. coli. Circular dichroism spectra showed that the purified Bmsage had well-defined α-helix structure in solution, indicating that SUMO may promote the correct folding of Bmsage into native-like structure. These work not only establish a foundation for further study of the property, structure and function of Bmsage, but also provide a reference for the expression and purification of other similar proteins.

Effects of hypoxia-inducible factor -2α on the expression of tight junction proteins and permeability in rat glomerular endothelial cells under hypoxia condition / 中华肾脏病杂志

Objective To investigate the role of hypoxia?inducible factor?2α(HIF?2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF?2α and tight junction proteins such as occludin and ZO?1 of rGENCs were examined after exposed to 5%oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF?2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF?2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO?1 and HIF?2α were assessed by Western blotting. The permeability of rGENCs was measured using trans?epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF?2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P0.05). Conclusion Hypoxia may promote HIF?2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO?1.

Análise molecular do gene HES1 em pacientes portadores de hipopituitarismo congênito / Molecular analysis of the HES1 gene in patients with congenital hypopituitarism

Estudos em modelos animais transgênicos possibilitaram o conhecimento de parte dos genes envolvidos na embriogênese hipofisária e da etiologia genética do hipopituitarismo em humanos. Entretanto, a etiologia da maior parte dos casos de hipopituitarismo congênito, principalmente os associados à neuro-hipófise ectópica (NE), ainda é pouco definida. Mutações no gene PROP1 são a causa genética mais comum de hipopituitarismo descritas até o momento, mas estão sempre associadas à neuro-hipófise tópica. Estudos destinados a esclarecer o mecanismo molecular da mutação do gene Prop1 em camundongos demonstraram a participação da via de sinalização Notch e de seus componentes, dentre eles, o gene Hes1. O HES1 é um gene que codifica um fator de transcrição que participa de estágios precoces do desenvolvimento hipofisário e está envolvido com a morfogênese da neuro-hipófise. A avaliação do camundongo com nocaute em homozigose deste gene acarreta uma hipoplasia da adeno-hipófise e ausência da neuro-hipófise; e sua expressão constitutiva está associada ao hipopituitarismo. Como a NE é um achado comum no hipopituitarismo congênito e o gene HES1 pode estar relacionado a sua fisiopatologia, a região codificadora do gene HES1 foi avaliada em 192 pacientes com hipopituitarismo congênito. A variante alélica c.578G > A (p.G193D) em heterozigose foi encontrada em um paciente com hipopituitarismo congênito associado à NE. A avaliação da predição in silico do efeito funcional da variante pela ferramenta MutationTaster sugere que a troca do aminoácido glicina, altamente conservado entre os mamíferos, por ácido aspártico, seja deletéria. No estudo da segregação familiar, quatro irmãos aparentemente normais apresentam a mesma variante, sendo que dois deles possuem alterações discretas na imagem da hipófise. Em conclusão, esta é uma nova variante alélica descrita no gene HES1, ausente em grandes bancos de dados e controles saudáveis da população brasileira, mas presente em irmãos não...

Studies of transgenic animal models have allowed for the discovery of genes involved in human pituitary embryogenesis and the genetic etiology of hypopituitarism. However, the genetic causes of most cases of congenital hypopituitarism, especially those associated with an ectopic posterior pituitary, remain poorly defined. Mutations in the gene PROP1 are the most common genetic causes of hypopituitarism described to date, and are always associated with an ectopic posterior pituitary. Studies to elucidate the molecular mechanisms of Prop1 mutations in mice have demonstrated the involvement of the Notch signaling pathway, including its downstream target Hes1. The HES1 gene encodes a transcription factor that participates in early stages of pituitary development and is involved in posterior pituitary morphogenesis. Hes1 knockout mice exhibit a hypoplastic anterior pituitary and absence of a posterior pituitary. Conversely, constitutive expression of Hes1 is associated with hypopituitarism. Since an ectopic posterior pituitary is commonly found in congenital hypopituitarism and the HES1 gene may be related to its pathophysiology, the coding region of gene HES1 was screened in 192 patients with congenital hypopituitarism. A heterozygous allelic variant c.578G >A (p.G193D) was identified in a patient with congenital hypopituitarism associated with an ectopic posterior pituitary. Assessment by MutationTaster, a bioinformatic tool for in silico prediction of functional effect of missense variants, suggests that substitution of glycine (a highly conserved amino acid in this position among mammals) for aspartic acid is deleterious. In the genetic study of family members, we identified four apparently normal siblings with the same variant, two of which have discrete changes in their pituitary MRI. In conclusion, we described a new allelic variant in the gene HES1, absent in large databases and healthy Brazilian controls, but present in the unaffected...