ATP依赖的人Lon蛋白酶的表达、纯化及其活性鉴定

ATP依赖的人Lon蛋白酶是一种同质寡聚、环状的蛋白酶,主要位于细胞线粒体基质中。许多研究表明,Lon蛋白酶对于维护细胞的内环境稳定起着重要作用,并参与线粒体蛋白质量控制和代谢调控。将pPROEX1 His6-Lon重组质粒在Escherichia coli Rosetta 2菌株中诱导表达用Ni2＋柱亲和层析法纯化,获得纯度较高的目的蛋白。经纯化后,Lon蛋白酶的比酶活达到0.17 U/mg。通过多肽底物Rhodamine 110、bis-（CBZ-L-alanyl-L-alanine amide）[（Z-AA）2 Rh110]的降解检测显示,Lon蛋白酶具有肽酶活性,并被ATP所刺激。Casein和线粒体转录因子A降解实验表明,纯化的Lon蛋白酶具有蛋白水解活性,而且蛋白水解活性依赖于ATP。     

细菌Lon蛋白酶研究进展

Lon蛋白酶是首个被鉴定的ATP依赖蛋白酶家族成员,在原核生物中发挥着降解错误折叠蛋白、维持胞内蛋白质平衡的作用。最近研究表明Lon蛋白还可以作为压力应激蛋白,参与降解多种转录调控因子和二元调控系统,改变细菌胞内的生理代谢过程以适应环境的改变。本文从Lon蛋白酶的结构、功能与上下游调控网络作一综述,旨在更全面清楚地了解ATP依赖蛋白酶的生理功能,以期为其胞内调控机制研究提供参考。     

线粒体蛋白酶与人类疾病

线粒体是真核细胞的重要细胞器,在能量转换、细胞应激、脂质合成以及细胞凋亡中具有调节作用.许多线粒体蛋白酶参与蛋白质运输、加工激活和降解过程.其中, ATP依赖性的线粒体蛋白酶通过其AAA+结构域(ATP associated multiple activity domain, AAA domain)利用ATP水解来执行线粒体蛋白质质量控制和调节蛋白降解.线粒体蛋白酶活性的改变会导致线粒体功能障碍,从而导致多种人类疾病,包括心血管疾病、神经退行性疾病、衰老和肿瘤等.本文重点综述线粒体蛋白酶1(Lon protease 1, LONP1)、酪蛋白水解蛋白酶P(caseinolytic protease, ClpP)、m-AAA(IMM-embedded AAA face to matrix)和i-AAA(IMM-embedded AAA face to intermembrane space)蛋白酶四种ATP依赖性线粒体蛋白酶及其功能,并阐述其与人类疾病的相关性和临床意义.     

嗜热菌Lon蛋白酶的逆境应答作用及其功能分析

Lon蛋白水解酶广泛存在于微生物以及动植物体内.它除了水解蛋白质的功能以外,还参与到细胞生理过程的调节当中.以嗜热栖热菌HB8(Thermus thermophilus HB8)的Lon蛋白酶(TTLon)及其两个片段TTlonL和TTlonS为研究对象,采用RT-PCR技术证明了TTLon在嗜热栖热菌HB8的逆境应答过程中发挥着重要作用,它的蛋白酶活性是受到Mg2+影响的,失去C端的TTlonL和TTlonS没有了蛋白水解酶活性和分子伴侣活性,但是它们仍然具有一定的ATP结合能力.     

蛋白酶B.P与几种试剂蛋白酶的比较

蛋白酶B.P与国内外几种试剂蛋白酶比较,证明其酶种单一,只含有蛋白酶,不含DNA酶和RNA酶,与蛋白酶K和蛋白酶E相似。蛋白酶B.P除含有碱性蛋白酶外还含有较高的中性蛋白酶活性,它可以广泛地水解多种天然蛋白,应用于微生物细胞蛋白的水解,提取DNA和RNA,还可水解叶肉细胞蛋白,提取叶绿体DNA,是我国第一个碱性生化试剂蛋白酶。     

植物中的金属蛋白酶FtsH

FtsH是一种对ATP和Zn^2＋依赖型金属蛋白酶,广泛存在于原核生物和真核生物中。具有高度保守的AAA结构域和Zn^2＋结合模块,在真核生物中是多基因家族。FtsH具有ATP酶活性,蛋白水解活性和分子伴侣活性,参与蛋白质质量平衡控制,还与热激、高渗、光胁迫、低温、病害等胁迫响应有联系。文章介绍FtsH基因的发现和分布,结构、FtsH的底物识别机制以及FtsH功能的研究概况。     

一种新的丙型肝炎病毒单链丝氨酸蛋白酶基因的构建、表达与鉴定

根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .     

酶解法制备草鱼抗氧化多肽工艺的建立

目的:筛选优化合适的可用于水解草鱼蛋白制备活性多肽的蛋白酶制剂及其工艺.方法:选取不同特性来源的商品化蛋白酶制剂与实验室分离的枯草蛋白酶BPN,对比分析它们在水解草鱼蛋白过程中的水解度变化,及其酶解产物的抗氧化活性.结果:大多数蛋白酶在水解反应最初的60min内,水解度迅速增加,之后2h内曲线变化不大.其中细菌来源的水解蛋白酶水解能力最强,其2h水解产物水解度可达15.17％;木瓜蛋白酶水解产物的抗氧化活性较强,经测定其DPPH清除能力为96.24％.结论:不同特性的蛋白酶水解草鱼蛋白的水解速度和产物的活性成分具有明显差异,总的来说,木瓜蛋白酶在草鱼蛋白加工中制备活性多肽是一个比较理想的选择.     

钙蛋白酶的结构及活性调节

钙蛋白酶广泛存在于各组织,广泛表达的钙蛋白酶有两种,钙蛋白酶Ⅰ和钙蛋白酶Ⅱ,它们激活所需的Ca^2＋浓度不同.这两种酶都有大、小两个亚基,分子质量分别为80 ku和30 ku.大亚基有4个结构域,小亚基由2个结构域构成.新近还发现了几种组织特异表达的钙蛋白酶.钙蛋白酶抑制蛋白是钙蛋白酶的内源抑制蛋白,它由5个结构域组成,其中4个为重复序列,均具有独立抑制钙蛋白酶活性的功能.体内钙蛋白酶活性受到严格调控,贴膜反应可以降低钙蛋白酶对Ca^2＋的依赖性,膜磷脂头部所带的磷酸基团与激活作用有关,自溶也可以降低对Ca^2＋的依赖,而钙蛋白酶抑制蛋白则起专一的抑制作用.     

响应面法优化蚕豆蛋白酶解工艺条件的研究

考察了碱性蛋白酶、胰蛋白酶和中性蛋白酶对蚕豆蛋白的酶解效果,探讨了水解度（DH）与酶解产物抗氧化活性间的关系。通过单因素试验和响应面分析法,得到碱性蛋白酶酶解工艺的最佳条件。结果表明,温度50℃、pH8.0、酶底比8%、底物浓度3%条件下酶解3h,水解度0~22%内,碱性蛋白酶较胰蛋白酶和中性蛋白酶水解蚕豆蛋白效果好;DH与还原能力（R2=0.68~0.81）及ABTS清除能力（R2=0.98~0.99）具有较好的相关性,碱性蛋白酶酶解液较其他2个酶解液有较好的还原能力和ABTS清除能力;优化后的最佳酶解工艺参数为：酶底比8%,温度50℃、pH 7.6,对蚕豆蛋白还原能力的影响顺序为酶底比＞pH＞温度;在此条件下,蚕豆蛋白酶解液的还原能力理论值为0.174,验证试验测得还原能力为0.173,与理论值接近。     

Towards the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.     

Coupling of Proteolysis to ATP Hydrolysis upon Escherichia coliLon Protease Functioning: II. Hydrolysis of ATP and Activity of the Enzyme Peptide Hydrolase Sites

The absence of direct correlation between the efficiency of functioning of ATPase and peptide hydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptide hydrolase sites is provided by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme ATPase sites. It was revealed that the ADP–Mg complex, an inhibitor of the native enzyme, is an activator of the Lon-K362Q (the Lon protease mutant in the ATPase site). Variants of functional contacts between different sites of the enzyme are considered. It was established that two ways of signal transduction from the ATPase sites to peptide hydrolase ones exist in the Lon protease oligomer--intra- and intersubunit ways. The enzyme ATPase sites are suggested to be located in the areas of the complementary surfaces of subunits. It is hypothesized that upon degradation of protein substrates by the E. coliLon protease in vivoATP hydrolysis acts as a factor of limitation of the enzyme degrading activity.     

The N-terminal sequence after residue 247 plays an important role in structure and function of Lon protease from Brevibacillus thermoruber WR-249

Previous studies on the N-terminal domain of Lon proteases have not clearly identified its function. Here we constructed randomly chosen N-terminal-truncated mutants of the Lon protease from Brevibacillus thermoruber WR-249 to elucidate the structure-function relationship of this domain. Mutants lacking amino acids from 1 to 247 of N terminus retained significant peptidase and ATPase activities, but lost ∼90% of protease activity. Further truncation of the protein resulted in the loss of all three activities. Mutants lacking amino acids 246-259 or 248-256 also lost all activities and quaternary structure. Our results indicated that amino acids 248-256 (SEVDELRAQ) are important for the full function of the Lon protease.     

Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates

Lon, also known as the protease La, is a homo-oligomeric ATP-dependent protease, which is highly conserved in archaea, eubacteria and eukaryotic mitochondria and peroxisomes. Since its discovery, studies have shown that Lon activity is essential for cellular homeostasis, mediating protein quality control and metabolic regulation. This article highlights the discoveries made over the past decade demonstrating that Lon selectively degrades abnormal as well as certain regulatory proteins and thus plays significant roles in maintaining bacterial and mitochondrial function and integrity. In addition, Lon is required in certain pathogenic bacteria, for rendering pathogenicity and host infectivity. Recent research endeavors have been directed toward elucidating the reaction mechanism of the Lon protease by different biochemical and structural biological techniques. In this mini-review, the authors survey the diverse biological roles of Lon, and also place special emphasis on recent findings that clarify the mechanistic aspects of the Lon reaction cycle.     

Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli

Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.     

Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains

ATP-dependent Lon proteases are multi-domain enzymes found in all living organisms. All Lon proteases contain an ATPase domain belonging to the AAA(+) superfamily of molecular machines and a proteolytic domain with a serine-lysine catalytic dyad. Lon proteases can be divided into two subfamilies, LonA and LonB, exemplified by the Escherichia coli and Archaeoglobus fulgidus paralogs, respectively. The LonA subfamily is defined by the presence of a large N-terminal domain, whereas the LonB subfamily has no such domain, but has a membrane-spanning domain that anchors the protein to the cytoplasmic side of the membrane. The two subfamilies also differ in their consensus sequences. Recent crystal structures for several individual domains and sub-fragments of Lon proteases have begun to illuminate similarities and differences in structure-function relationships between the two subfamilies. Differences in orientation of the active site residues in several isolated Lon protease domains point to possible roles for the AAA(+) domains and/or substrates in positioning the catalytic residues within the active site. Structures of the proteolytic domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. The structure of a large segment of the N-terminal domain has revealed a folding motif present in other protein families of unknown function and should lead to new insights regarding ways in which Lon interacts with substrates or other cellular factors. These first glimpses of the structure of Lon are heralding an exciting new era of research on this ancient family of proteases.     

Recent developments in the mechanistic enzymology of the ATP-dependent Lon protease from Escherichia coli: highlights from kinetic studies

Lon protease, also known as protease La, is one of the simplest ATP-dependent proteases that plays vital roles in maintaining cellular functions by selectively eliminating misfolded, damaged and certain short-lived regulatory proteins. Although Lon is a homo-oligomer, each subunit of Lon contains both an ATPase and a protease active site. This relatively simple architecture compared to other hetero-oligomeric ATP-dependent proteases such as the proteasome makes Lon a useful paradigm for studying the mechanism of ATP-dependent proteolysis. In this article, we survey some recent developments in the mechanistic characterization of Lon with an emphasis on the utilization of pre-steady-state enzyme kinetic techniques to determine the timing of the ATPase and peptidase activities of the enzyme.