EMA3C/SEMA3D基因错义突变对蛋白稳定性和受体亲合力的影响

目的：明确先天性巨结肠患者携带的5 个基因错义突变对Semaphorin 3（Sema3）蛋白自身稳定性和受 体亲合力的影响作用。方法：构建Sema3-Neuropilin-Plexin 配体-受体复合物蛋白质模型,对全部5个错义突变进行定位,通过计 算标准能量功能赋值（驻驻G）和复合物界面值（驻I_sc）预测突变对Sema3 的影响作用。将野生型和突变型AP-tagged Sema3 质粒分 别转染HEK293T 细胞,72 h后收集含有融合蛋白的细胞培养液上清并与分别表达Neuropilin 1（Nrp-1）或Neuropilin 2（Nrp-2）的 COS-7 细胞孵育,洗脱未结合的蛋白后加入碱性磷酸酶底物显色拍片,或提取细胞总蛋白,利用融合蛋白N- 末端含有的碱性磷 酸酶在底物PNPP 存在时可以发生颜色变化的特性,对与受体结合的野生型和突变型AP-Sema3 蛋白进行定量。结果：5 个错义突 变中的4 个都会不同程度地影响相应Semaphorin 3蛋白与其受体Neuropilin 的结合（与Nrp-1 的结合：SEMA3C S329G,V337M, SEMA3D H424Q,V457I,P615T 分别与野生型相比：1.12± 0.15,0.37± 0.03,0.56± 0.07,0.51± 0.05,0.66± 0.05;与Nrp-2 的结合： SEMA3C S329G,V337M,SEMA3D H424Q,V457I,P615T 分别与野生型相比：1.18 ± 0.09,0.37 ± 0.03,0.76 ± 0.01,0.65 ± 0.06,0.85± 0.03,n=3,单因素方差分析,差异有统计学意义),说明它们可能通过严重影响分子通路的信号转导而妨碍蛋白功能的 正常行使。结论：先天性巨结肠患者携带的基因错义突变可不同程度影响蛋白与其受体的结合,提示 Semaphorin 3这类经典的神经元轴突导向因子在功能失常的情况下可能参与先天性巨结肠的发生。     

Semaphorin4D的作用机制与功能

Semaphorins家族是一类以结构中具有sema区域为共同特征的蛋白,Semaphorin4D(Sema4D)是其成员之一.Sema4D与受体丛状蛋白B1(PlexinB1)和分化抗原簇72(cluster of differentiation antigen72,CD72)结合,通过多种信号转导途径,在神经系统的轴突导向,免疫系统中T、B细胞的活化和免疫调节中发挥关键作用.最近发现,Sema4D在许多人体肿瘤组织中高表达,且对血管发生及肿瘤侵袭转移起重要作用.本文旨在对Sema4D的结构、作用机制及生物学功能的研究最新进展作一综述.     

小鼠Agouti基因变异及生物信息学分析

目的分析候选基因Agouti的多态性,揭示染色体工程小鼠毛色差异的分子机制。方法首先,用测色仪检测小鼠的毛色差异。其次,利用DNA芯片进行全基因组扫描确定候选基因Agouti。最后,运用生物信息学软件分析Agouti基因cDNA序列和氨基酸序列的多态性,预测突变对蛋白结构和功能产生的影响。结果发现Agouti基因cDNA序列上存在5个SNPs,使Agouti信号蛋白产生了3个错义突变。生物信息学分析发现,其中一个错义突变使该蛋白丢失了一个β折叠,并且其三级结构发生改变,最终导致与受体结合的能力相较于野生型有所下降。结论在毛色基因Agouti的编码区发现了一个新的错义突变,该突变使Agouti信号蛋白与受体结合能力下降,最终导致小鼠的毛色由浅灰色变为深灰色。     

肠毒素C2中Met24对其超抗原活性无重要作用

目的:分析金黄色葡萄球菌肠毒素C2的T细胞受体结合区域α3结构中,第24位甲硫氨酸对其超抗原活性的影响作用.方法:应用定点突变技术对金黄色葡萄球菌肠毒素C2的Met24进行无意义突变,获得突变蛋白SEC2(M24A),对突变蛋白和野生型蛋白的体内、体外超抗原活性进行比较.结果:分别对野生型蛋白及突变蛋白的免疫刺激活性、肿瘤抑制活性、体内致热毒性进行了比较,发现突变蛋白的增值指数(PI)、抑瘤率、热源效应与野生型相比无显著差异(P＞0.05),这表明对第24位甲硫氨酸的替换没有对金黄色葡萄球菌肠毒素C2的超抗原活性造成明显影响.结论:在金黄色葡萄球菌肠毒素C2的α3结构中,Met24并不是决定其超抗原活性的重要氨基酸残基.     

人载脂蛋白A-Ⅰ半胱氨酸突变体的二级结构和脂质结合能力的比较

为探讨人载脂蛋白A-Ⅰ(apoA-Ⅰ,apolipoproteinA-Ⅰ)α螺旋不同位点的半胱氨酸突变后,对蛋白二级结构和脂质结合能力的影响,利用定点诱变技术构建apoA-Ⅰ的天然半胱氨酸突变体apoA-ⅠMilano(R173C),及其它α螺旋片段上的半胱氨酸突变体,分别为apoA-Ⅰ(S52C),apoA-Ⅰ(N74C),apoA-Ⅰ(L107C),apoA-Ⅰ(K129C),和apoA-Ⅰ(L195C).观察比较各种野生型及突变apoA-Ⅰ单体蛋白的α螺旋含量和二级结构稳定性及其脂质结合能力.结果显示,野生型apoA-Ⅰ,apoA-Ⅰ(S52C),apoA-Ⅰ(N74C),apoA-Ⅰ(L107C),apoA-Ⅰ(K129C),apoA-ⅠMilano和apoA-Ⅰ(L195C)的α螺旋含量分别为54±4%,49±4%,50±2%,51±6%,56±4%,52±3%,和54±1%,各种蛋白的α螺旋含量无显著性差异(P>0.05).野生型apoA-Ⅰ的变性标准自由能(ΔG0D)为10.5kJ/mol;apoA-Ⅰ(S52C)和apoA-ⅠMilano的ΔG0D比野生型低2.1kJ/mol;而apoA-Ⅰ(K129C)的ΔG0D比野生型apoA-Ⅰ高1.6kJ/mol.与野生型apoA-Ⅰ相比,apoA-Ⅰ(K129C)和apoA-Ⅰ(L195C)两个突变体与脂质结合能力明显下降(P<0.05),而其它半胱氨酸突变体(包括apoA-ⅠMilano)在脂质结合动力学方面与野生型apoA-Ⅰ无明显差异.以上结果提示,不同位点发生的半胱氨酸突变对apoA-Ⅰ单体蛋白的α螺旋含量无明显影响,但对蛋白的二级结构稳定性和脂质结合能力影响不尽相同.     

EDNRB基因编码区点突变及多态性在浙江地区散发性先天性巨结肠症中的检测与分析

本实验应用聚合酶链反应-单链构象多态性(single strand confbrmation polymorphism analysis of polymerase chain reaction products,PCR-SSCP)和DNA直接测序技术,对75例浙江地区散发性先天性巨结肠病例EDNRB基因编码区的全部7个外显子,进行了点突变与单核苷酸多态性的检测与分析,探讨浙江地区先天性巨结肠患者EDNRB基因的突变特征,阐明EDNRB基因与散发性先天性巨结肠症发病之间可能存在的关系。结果有6例患者在第4外显子上检测到密码子277位点 CTG→CTA的置换,导致亮氨酸的同义突变(L277L),属于单核苷酸多态性,发生率为8％(6/75)。有 2例患者在第2外显子上检测到密码子185位点GTG→ATG的置换,导致缬氨酸到蛋氨酸的错义突变(V185M),此突变型未在国内外文献中报道过,认为是新的基因突变型,突变率2．7％(2/75)。研究结果表明,浙江地区先天性巨结肠群体可发生EDNRB基因的杂合性突变,提示EDNRB基因与先天性巨结肠症的发病存在一定程度的关联。     

一个Ⅰ型遗传性淋巴水肿中国家系的VEGFR3基因的新突变方式

通过对国人Ⅰ型遗传性淋巴水肿一家系分子遗传学检测,报告VEGFR-3基因新突变.首先在Ⅰ型遗传性淋巴水肿对该家系进行致病基因的连锁分析,然后用DNA直接测序方法进行基因突变分析.连锁分析和单倍体分析确定该家系致病基因位于5q35.3,与Ⅰ型遗传性淋巴水肿连锁.VEGFR-3基因突变分析发现了一个新的错义突变D1055V.该错义突变在家系中共分离,且在100个正常对照组中未发现该序列改变.本研究首次报告了国内Ⅰ型遗传性淋巴水肿VEGFR-3基因新的错义突变D1055V,丰富了VEGFR-3基因基因突变谱,为今后开展遗传性淋巴水肿的基因诊断和遗传咨询奠定基础.     

一个Ⅰ型遗传性淋巴水肿中国家系的VEGFR3基因的新突变方式

通过对国人Ⅰ型遗传性淋巴水肿一家系分子遗传学检测,报告VEGFR-3基因新突变。首先在Ⅰ型遗传性淋巴水肿对该家系进行致病基因的连锁分析,然后用DNA直接测序方法进行基因突变分析。连锁分析和单倍体分析确定该家系致病基因位于5q35.3,与Ⅰ型遗传性淋巴水肿连锁。VEGFR-3基因突变分析发现了一个新的错义突变D1055V,该错义突变在家系中共分离,且在100个正常对照组中未发现该序列改变。本研究首次报告了国内Ⅰ型遗传性淋巴水肿VEGFR-3基因新的错义突变D1055V,丰富了VEGFR-3基因基因突变谱,为今后开展遗传性淋巴水肿的基因诊断和遗传咨询奠定基础。     

中国人先天性巨结肠RET基因突变特征研究

为了研究中国人先天性巨结肠(HD)RET基因突变特征,从分子水平探讨先天性巨结肠症(HD)的发病机理,揭示中国人群HD与RET基因突变的关系。我们应用聚合酶链反应-单链构象多态性(PCR-SSCP)-DNA测序方法,对50例HD患儿和30例无便秘正常对照者,进行RET基因第13外显子突变筛查,并对第13外显子突变阳性患儿家系作研究。结果发现在50例HD患儿中,有7例第13外显子扩增片段在SSCP分析时出现泳动变位。经DNA测序证实,有3种点突变类型(错义、同义和移码),第13外显子突变率为14%(7/50)。在7例第13外显子突变的患儿中,发现2例患儿的父亲存在与其子相同的突变。实验表明中国先天性巨结肠人群存在着RET基因突变,且以杂和性点突变为主,HD具有一定的遗传性。     

中国人先天性巨结肠RET基因突变特征研究

为了研究中国人先天性巨结肠(HD)RET基因突变特征,从分子水平探讨先天性巨结肠症(HD)的发病机理,揭示中国人群HD与RET基因突变的关系。我们应用聚合酶链反应-单链构象多态性(PCR—SSCP)-DNA测序方法,对50例HD患儿和30例无便秘正常对照者,进行RET基因第13外显子突变筛查,并对第13外显子突变阳性患儿家系作研究。结果发现在50例HD患儿中,有7例第13外显子扩增片段在SSCP分析时出现泳动变位。经DNA测序证实,有3种点突变类型(错义、同义和移码),第13外显子突变率为14％(7／50)。在7例第13外显子突变的患儿中,发现2例患儿的父亲存在与其子相同的突变。实验表明中国先天性巨结肠人群存在着RET基因突变,且以杂和性点突变为主,HD具有一定的遗传性。     

SEMA3A Signaling Controls Layer-Specific Interneuron Branching in the Cerebellum

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SEMA3A regulates developing sensory projections in the chicken spinal cord

The present study explores the role of SEMA3A (collapsin-1) in the temporal and spatial regulation of developing sensory projections in the chick spinal cord. During development, SEMA3A mRNA (SEMA3A) is first expressed throughout the spinal gray matter, but disappears from the dorsal region when small caliber (trkA(+)) sensory axon collaterals first grow into the dorsal horn. In explant cultures of spinal cord segments with attached sensory ganglia, the spatial extent of SEMA3A expression varied in different explants, but in each case the growth of trkA(+) sensory collaterals was largely excluded from areas of SEMA3A expression. To test if SEMA3A had a direct effect on sensory axon growth, we injected recombinant protein into the explants before placing them in culture. Increased levels of SEMA3A substantially reduced the ingrowth of trkA(+) axons, whereas trkC(+) axon collaterals were not affected. Consistent with the insensitivity of trkC(+) collaterals to SEMA3A, these collaterals did not express neuropilin-1, a receptor for SEMA3A. The inhibitory effects of SEMA3A on trkA(+) axons within the spinal cord suggests that the fall in SEMA3A expression in the dorsal horn may contribute to the initiation of growth of these axons into gray matter. In addition, the observation that trkA(+) axons frequently grew close to but rarely over areas of SEMA3A expression suggests that semaphorin may act principally as a short-range guidance cue within the spinal cord.     

Control of Intracellular Localization and Function of Cx43 by SEMA3F

Connexin genes are considered to form a family of tumor-suppressor genes. However, the mechanism of connexin-mediated growth control is not well understood. We now provide several lines of evidence which suggest that SEMA3F, a member of the class 3 semaphorin family, which is also reported to be a tumor suppressor, controls the intracellular localization and function of connexin 43 (Cx43). We employed a series of rat liver epithelial cell lines, among which we previously found that the level of expression of malignant phenotypes (IAR20 < IAR27E < IAR6-1 < IAR27F) is inversely related to that of gap junctional intercellular communication (GJIC). When we immunostained SEMA3F and Cx43 in these cell lines, the extent of immunostaining in the plasma membrane of both proteins decreased in the order of IAR20 > IAR27E > IAR6-1 > IAR27F, suggesting a close relationship between Cx43 and SEMA3F. Further studies revealed a partial colocalization of SEMA3F and Cx43 in the plasma membrane of IAR20 cells. We also found that both SEMA3F and Cx43 moved from the cytoplasm to the plasma membrane in a mouse papilloma cell line when E-cadherin became functional after transferring the cells from low- to high-calcium conditions. When SEMA3F gene expression was inhibited by siRNA in IAR20 cells, Cx43 localization in the plasma membrane and GJIC ability were reduced. Moreover, we found that SEMA3F binds with the cytoplasmic loop domain of Cx43, employing the yeast two-hybrid complementation and screening assays. Taken together, these results strongly suggest that SEMA3F directly associates with Cx43 and controls its intracellular localization and function.     

SEMA3B基因在鼻咽癌组织中的表达、杂合性丢失和甲基化分析

SEMA3B基因定位于鼻咽癌高频缺失区域3p21.3上,最近被证明具有抑瘤基因的功能.分析了鼻咽癌组织中SEMA3B基因的表达、杂合性丢失(LOH)和甲基化情况.首先应用逆转录-聚合酶链式反应(RT-PCR)方法检测了33例鼻咽癌组织和15例慢性鼻咽炎组织中SEMA3B基因的表达,结果显示75.8%(25/33)鼻咽癌组织中SEMA3B基因表达缺失或下调,显著低于慢性鼻咽炎组织中的表达(P=0.001).进一步选取3个微卫星位点D3S1568、D3S1621和D3S4597分析了20例鼻咽癌组织中SEMA3B基因LOH的情况,结果表明3个位点的丢失率分别为10%、20%和15%,总的丢失率为45%,统计分析发现LOH与基因表达之间存在明显相关(P=0.023).最后,采用甲基化特异性PCR方法分析了SEMA3B基因启动子区甲基化,结果发现在100%的鼻咽癌组织和73.3%的慢性鼻咽炎组织中检测到SEMA3B基因启动子区高甲基化.由此得出结论,SEMA3B基因在鼻咽癌组织中表达缺失或下调,LOH是引起其表达异常的原因之一.     

Suppression of miR-221 inhibits glioma cells proliferation and invasion via targeting SEMA3B

Background

Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant.

Results

In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes.

Conclusion

Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.     

Tumor Suppressor Function of the SEMA3B Gene in Human Lung and Renal Cancers

The SEMA3B gene is located in the 3p21.3 LUCA region, which is frequently affected in different types of cancer. The objective of our study was to expand our knowledge of the SEMA3B gene as a tumor suppressor and the mechanisms of its inactivation. In this study, several experimental approaches were used: tumor growth analyses and apoptosis assays in vitro and in SCID mice, expression and methylation assays and other. With the use of the small cell lung cancer cell line U2020 we confirmed the function of SEMA3B as a tumor suppressor, and showed that the suppression can be realized through the induction of apoptosis and, possibly, associated with the inhibition of angiogenesis. In addition, for the first time, high methylation frequencies have been observed in both intronic (32-39%) and promoter (44-52%) CpG-islands in 38 non-small cell lung carcinomas, including 16 squamous cell carcinomas (SCC) and 22 adenocarcinomas (ADC), and in 83 clear cell renal cell carcinomas (ccRCC). Correlations between the methylation frequencies of the promoter and the intronic CpG-islands of SEMA3B with tumor stage and grade have been revealed for SCC, ADC and ccRCC. The association between the decrease of the SEMA3B mRNA level and hypermethylation of the promoter and the intronic CpG-islands has been estimated in renal primary tumors (P < 0.01). Using qPCR, we observed on the average 10- and 14-fold decrease of the SEMA3B mRNA level in SCC and ADC, respectively, and a 4-fold decrease in ccRCC. The frequency of this effect was high in both lung (92-95%) and renal (84%) tumor samples. Moreover, we showed a clear difference (P < 0.05) of the SEMA3B relative mRNA levels in ADC with and without lymph node metastases. We conclude that aberrant expression and methylation of SEMA3B could be suggested as markers of lung and renal cancer progression.     

The crucial role of SEMA3F in suppressing the progression of oral squamous cell carcinoma

Background

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancy. Semaphorin 3F (SEMA3F) is highly conserved but present at a lower level in various cancers than in healthy tissues. While it has been reported that SEMA3F is involved in cancer cell proliferation, migration and invasion, its function in OSCC remains unknown.

Methods

The expression of SEMA3F in OSCC tissues and OSCC-derived cells was analyzed using qRT-PCR and western blotting. Using SAS and HSC2 cells, we also monitored the effect of SEMA3F on OSCC cell proliferation, migration and invasion using MTT, colony formation and transwell assays. The function of SEMA3F in OSCC tumor formation was also assessed in vivo.

Results

SEMA3F was significantly downregulated in OSCC tissues and OSCC-derived cells. SEMA3F shows growth inhibitory activity in SAS and HSC2 cells and may act as a tumor suppressor. It can inhibit the migration and invasion potential of OSCC cells. Our results also demonstrate that SEMA3F can suppress the growth of OSCC cells in vivo.

Conclusions

This study revealed that SEMA3F plays a role as a tumor suppressor in OSCC cell proliferation, migration and invasion. Our finding provides new insight into the progression of OSCC. Therapeutically, SEMA3F has some potential as a target for OSCC treatment, given sufficient future research.

     

SEMA3B-AS1-inhibited osteogenic differentiation of human mesenchymal stem cells revealed by quantitative proteomics analysis

Human mesenchymal stem cells (hMSCs) are fibroblastoid multipotent adult stem cells with capacities of differentiation into osteoblasts and chondrocytes and show great potential in new bone formation and bone repair-related clinical settings, such as osteoporosis. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in various biological processes. Here, we report an antisense lncRNA SEMA3B-AS1 regulating hMSCs osteogenesis. SEMA3B-AS1 is proximal to a member of the semaphorin family Sema3b. Overexpression of SEMA3B-AS1 using the lentivirus system markedly inhibits the proliferation of hMSCs and meanwhile reduces osteogenic differentiation. Using a comprehensive proteomic technique named isobaric tag for relative and absolute quantitation, we found that SEMA3B-AS1 significantly alters the process of osteogenesis through downregulating the expression of proteins involved in actin cytoskeleton, focal adhesion, and extracellular matrix–receptor interaction, while increasing the expression of proteins in the spliceosome. Collectively, we find that SEMA3B-AS1 is a target for controlling osteogenesis of hMSCs.