肝细胞生长因子通过鞘氨醇激酶途径诱导内皮细胞迁移作用的研究

目的：阐明鞘氨醇激酶（SPK）在肝细胞生长因子（HGF）诱导的内皮细胞迁移中的调节作用。方法：构建携带野生型SPK（SPK^WT）及负显性SPK（SPKDN）基因的重组腺病毒载体并包装获得重组腺病毒;用重组腺病毒感染ECV304细胞,检测感染效率及目的基因的表达;以^32P标记产物S1P测定细胞内SPK酶活性;用扩散盒技术观察高表达SPK^WT及SPK^ND对HGF诱导的内皮细胞迁移的影响。结果：野生型SPK基因表达可明显增强细胞内SPK的活性,并促进HGF诱导的内皮细胞迁移;而SPK负显性基因则显著抑制HGF诱导的内皮细胞迁移。结论：HGF通过SPK调控内皮细胞的迁移。     

Crystal Structures of a Populus tomentosa 4-Coumarate:CoA Ligase Shed Light on Its Enzymatic Mechanisms

4-Coumaric acid:CoA ligase (4CL) is the central enzyme of the plant-specific phenylpropanoid pathway. It catalyzes the synthesis of hydroxycinnamate-CoA thioesters, the precursors of lignin and other important phenylpropanoids, in two-step reactions involving the formation of hydroxycinnamate-AMP anhydride and then the nucleophilic substitution of AMP by CoA. In this study, we determined the crystal structures of Populus tomentosa 4CL1 in the unmodified (apo) form and in forms complexed with AMP and adenosine 5′-(3-(4-hydroxyphenyl)propyl)phosphate (APP), an intermediate analog, at 2.4, 2.5, and 1.9 Å resolution, respectively. 4CL1 consists of two globular domains connected by a flexible linker region. The larger N-domain contains a substrate binding pocket, while the C-domain contains catalytic residues. Upon binding of APP, the C-domain rotates 81° relative to the N-domain. The crystal structure of 4CL1-APP reveals its substrate binding pocket. We identified residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) based on their crystal structures and by means of mutagenesis and enzymatic activity studies. We also demonstrated that the size of the binding pocket is the most important factor in determining the substrate specificities of 4CL1. These findings shed light on the enzymatic mechanisms of 4CLs and provide a solid foundation for the bioengineering of these enzymes.     

Insights into the oligomeric states, conformational changes, and helicase activities of SV40 large tumor antigen

The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N- and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg(2+). The most C-terminal fragment (residues 628-708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N- and C-terminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.     

Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen

The large tumor antigen (LTag) of simian virus 40, an AAA(+) protein, is a hexameric helicase essential for viral DNA replication in eukaryotic cells. LTag functions as an efficient molecular machine powered by ATP binding and hydrolysis for origin DNA melting and replication fork unwinding. To understand how ATP binding and hydrolysis are coupled to conformational changes, we have determined high-resolution structures ( approximately 1.9 A) of LTag hexamers in distinct nucleotide binding states. The structural differences of LTag in various nucleotide states detail the molecular mechanisms of conformational changes triggered by ATP binding/hydrolysis and reveal a potential mechanism of concerted nucleotide binding and hydrolysis. During these conformational changes, the angles and orientations between domains of a monomer alter, creating an "iris"-like motion in the hexamer. Additionally, six unique beta hairpins on the channel surface move longitudinally along the central channel, possibly serving as a motor for pulling DNA into the LTag double hexamer for unwinding.     

雪衣藻Chlamydomonas nivalis适应温度周期性变化的生理响应和分子机制

【目的】雪衣藻(Chlamydomonas nivalis)分布于高山积雪和两极地区,可耐受低温胁迫和温度骤变,其适应温度变化的分子机制目前尚不清楚。【方法】本研究基于温度周期性变化下雪衣藻生理指标的响应规律,选择10个取样点进行转录组测序。运用加权基因共表达网络分析(weighted gene co-expression network analysis, WGCNA)划分得到17个共表达模块,从中找到5个与样品处理显著关联的模块,并对温度骤变的时间点进行基因差异表达分析。最后对筛选得到的基因集进行功能注释分析。【结果】转录组学分析显示,C. nivalis在温度周期变化下基因表达量发生了全局变化,其中果糖和甘露糖代谢通路、淀粉和蔗糖代谢通路、谷胱甘肽代谢通路以及抗坏血酸和醛酸代谢通路中关键酶的编码基因在低温下上调表达。研究还发现在温度周期变化下,C. nivalis中蛋白质质量控制系统、光合作用系统、DNA修复系统相关基因响应温度变化。【结论】本研究为揭示雪衣藻适应温度胁迫的分子机制提供了重要线索,丰富了生物抗逆基因资源库。     

α-Synuclein Immunoisolation of Glial Inclusions from Multiple System Atrophy Brain Tissue Reveals Multiprotein Components

Immunohistochemical studies have shown that oligodendroglial inclusions in multiple system atrophy contain alpha-synuclein, a synaptic protein also found in Lewy bodies in Parkinson's disease. We have now used density gradient enrichment and an anti-alpha-synuclein immunomagnetic technique to isolate pure and morphologically intact oligodendroglial inclusions from brain white matter of patients dying with multiple system atrophy. Filamentous inclusion structures were obtained only from multiple system atrophy tissue, but not from normal brain tissues, or from multiple system atrophy tissue processed without anti-alpha-synuclein antibody. We confirmed the purity and morphology of isolated inclusions by electron microscopy. The inclusions comprised multiple protein bands after separation by polyacrylamide gel electrophoresis. Immunoblotting demonstrated that these proteins included alpha-synuclein, alphaB-crystallin, tubulins, ubiquitin, and prominent, possibly truncated alpha-synuclein species as high-molecular-weight aggregates. Our study provides the first biochemical evidence that oligodendroglial inclusion filaments consist of multiple protein components, suggesting that these inclusions may form as a result of multiprotein interactions with alpha-synuclein.