红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

【目的】克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性。【方法】利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构;通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性。【结果】从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh(GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸。序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axono-podis pv. citri str. 306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91%和83%,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str. GPEPC73的肽酶亲缘性最高。基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5°C以上的突变体。【结论】对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程。对高度柔性位点进行饱和突变的策略可以用于提高热稳定性。     

Phosphatidylinositol-3-kinase C2β and TRIM27 function to positively and negatively regulate IgE receptor activation of mast cells

Cross-linking of the IgE receptor (FcεRI) on mast cells plays a critical role in IgE-dependent allergy, including allergic rhinitis, asthma, anaphylaxis, and immediate-type hypersensitivity reactions. Previous studies have demonstrated that the K(+) channel, KCa3.1, plays a critical role in IgE-stimulated Ca(2+) entry and degranulation in both human and mouse mast cells. We now have shown that the class II phosphatidylinositol-3-kinase C2β (PI3KC2β) is necessary for FcεRI-stimulated activation of KCa3.1, Ca(2+) influx, cytokine production, and degranulation of bone marrow-derived mast cells (BMMC). In addition, we found that the E3 ubiquitin ligase, tripartite motif containing protein 27 (TRIM27), negatively regulates FcεRI activation of KCa3.1 and downstream signaling by ubiquitinating and inhibiting PI3KC2β. TRIM27(-/-) mice are also more susceptible in vivo to acute anaphylaxis. These findings identify TRIM27 as an important negative regulator of mast cells in vivo and suggest that PI3KC2β is a potential new pharmacologic target to treat IgE-mediated disease.     

Functional damage of endothelial progenitor cells is attenuated by 14-3-3-n through inhibition of mitochondrial injury and oxidative stress

Endothelial progenitor cells (EPCs) are precursor cells of vascular endothelial cells, which are widely involved in the pathological process of cardiovascular diseases. EPCs apoptosis could accelerate the process of cardiovascular diseases. 14-3-3-η protein has been proved to be a potent antiapoptosis molecule. However, inhibition of EPCs apoptosis by 14-3-3-η and further specific mechanism have not been investigated. EPCs were isolated from human cord blood, and identified using VEGFR2 and CD34. 14-3-3-η overexpression model in vitro was established. Cell invasion, apoptosis, and proliferation were measured by transwell, flow cytometry, and Cell Counting Kit-8, respectively. Expression of 14-3-3-η, Bcl-2, and voltage-dependent anion channel 1 (VDAC1) were measured using quantitative real-time polymerase chain reaction and western blot analysis. Reactive oxygen species (ROS) intensity was measured using 2ʹ-7ʹ dichlorofluorescin diacetate probe. Mitochondrial membrane potential was detected using JC-1 dye. Overexpression of 14-3-3-η significantly promoted invasion and proliferation, but suppressed apoptosis of EPCs. Overexpression of 14-3-3-η remarkably inhibited ROS and promoted antioxidant enzyme levels in EPCs. 14-3-3-η might inhibit apoptosis of EPCs through attenuating mitochondrial injury. This study might provide a new target, 14-3-3-η, for the prevention and treatment of cardiovascular diseases through targeting EPCs.     

GPR39 promotes cardiac hypertrophy by regulating the AMPK–mTOR pathway and protein synthesis

Hypertrophic growth of the cardiomyocytes is one of the core mechanisms underlying cardiac hypertrophy. However, the mechanism underlying cardiac hypertrophy remains not fully understood. Here we provided evidence that G protein-coupled receptor 39 (GPR39) promotes cardiac hypertrophy via inhibiting AMP-activated protein kinase (AMPK) signaling. GRP39 expression is overexpressed in hypertrophic hearts of humans and transverse aortic constriction (TAC)-induced cardiac hypertrophy in mice. In neonatal cardiomyocytes, adenovirus-mediated overexpression of GPR39 promoted angiotensin II-induced cardiac hypertrophy, while GPR39 knockdown repressed hypertrophic response. Adeno-associated virus 9-mediated knockdown of GPR39 suppressed TAC-induced decline in fraction shortening and ejection fraction, increase in heart weight and cardiomyocyte size, as well as overexpression of hypertrophic fetal genes. A mechanism study demonstrated that GPR39 repressed the activation of AMPK to activate the mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase β-1 (S6K1), subsequently promoted de novo protein synthesis. Inhibition of mTOR with rapamycin blocked the effects of GPR39 overexpression on protein synthesis and repressed cardiac hypertrophy. Collectively, our findings demonstrated that GPR39 promoted cardiac hypertrophy via regulating the AMPK–mTOR–S6K1 signaling pathway, and GRP39 can be targeted for the treatment of cardiac hypertrophy.     

WTAP facilitates progression of endometrial cancer via CAV-1/NF-κB axis

The N⁶-methyladenosine (m⁶A) modification is one of the most prevalent methylations in eukaryotic messenger RNA (mRNA), and it is essential for the development of many important biological processes such as multiple types of tumors. One of the most important enzymes catalyzing generation of m⁶A on mRNA is Wilms' tumor 1-associating protein (WTAP); however, the potential role of WTAP in endometrial cancer (EC) still remains unknown. Here, we investigated WTAP expression level in cancer tissue and paracancerous tissue from an EC patient. Subsequently, WTAP was knocked down by small interfering RNA in EC cell line of Ishikawa and HEC-1A, respectively. Cell proliferation, migration, and invasion were studied. The expression of caveolin-1 (CAV-1) was detected by quantitative polymerase chain reaction (qPCR). The enrichments of m⁶A and METTL3 on CAV-1 were detected using RNA immunoprecipitation-qPCR. The activity of nuclear factor-κB (NF-κB) was studied using Western blot. We observed that WTAP was dramatically upregulated in the cancer tissue, and there was an enhancement in cell proliferation, migration, and invasion and a decrease in EC apoptosis in vivo and in vitro, which indicated higher tumor malignancy and worse survival outcome. After WTAP was knocked down in EC cells, CAV-1 was significantly upregulated and the enrichments of m⁶A and METTL3 at 3′-untranslated region (UTR) region of CAV-1 were decreased. Moreover, the activity of NF-κB signaling pathway was inhibited by its regulator CAV-1. Taken together, we concluded that WTAP could methylate 3′-UTR of CAV-1 and downregulate CAV-1 expression to activate NF-κB signaling pathway in EC, which promoted EC progression.     

Pseudomonas aeruginosa survives in epithelia by ExoS‐mediated inhibition of autophagy and mTOR

One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS‐deficient P. aeruginosa but does not affect the survival of ExoS‐containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP‐ribosyltransferase domain. Inhibition of mTOR is caused by ExoS‐mediated ADP ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.     

A type VII secretion system of Streptococcus gallolyticus subsp. gallolyticus contributes to gut colonization and the development of colon tumors

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved in Sgg pathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria and Staphylococcus aureus. Through genome analysis, we identified a locus in Sgg strain TX20005 that encodes a putative type VII secretion system (designated as SggT7SS^T05). We showed that core genes within the SggT7SS^T05 locus are expressed in vitro and in the colon of mice. Western blot analysis showed that SggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that this SggT7SS^T05 is functional. Deletion of SggT7SS^T05 (TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability of Sgg to stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest that SggT7SS^T05-secreted factors are responsible for the pro-proliferative activity of Sgg, whereas Sgg adherence to host cells requires both SggT7SS^T05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesx showed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesx did not. Examination of the Sgg load in the colon in the CRC model suggests that SggT7SS^T05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results reveal SggT7SS^T05 as a previously unrecognized pathogenicity determinant for Sgg colonization of the colon and promotion of colon tumors.