寡聚结构变化与DegP蛋白酶的激活

种类繁多的蛋白质所发挥的各种功能对于生命现象是至关重要的,然而蛋白质的结构却总是受到体内外各种因素的干扰甚至破坏。因此,生物体为了维持蛋白质的活性构象,蛋白质质量控制（protein quality control）机制是必不可少的,而这种机制一旦失效将导致各种与蛋白质折叠相关的严重疾病,例如帕金森病（Parkinson’s disease）和阿尔茨海默病（Alzheimer’s disefse）等。分子伴侣和蛋白酶是参与蛋白质质量控制的主要两类蛋白质分子,它们能够结合错误折叠的底物蛋白并辅助其重新折叠或将其降解。DegP蛋白（又称为HtrA）是存在于大肠杆菌的膜间质中的一种热休克蛋白,对于大肠杆菌在高温下的存活是必需的。它的独特之处在于它同时具有分子伴侣和蛋白酶两种活性,因此DegP是研究蛋白质质量控制机制的一种典型样品。DegP同源蛋白（统称为HtrA蛋白家族）几乎存在于所有的生物种类中,它们的功能可能是参与细胞的胁迫反应。     

革兰氏阴性菌膜间质蛋白酶DegP研究进展

膜间质蛋白酶(DegP),是一种广泛存在于真核生物和原核生物细胞中的蛋白。DegP同时具有酶活性和分子伴侣活性,并通过多聚体构成胶囊状结构执行其分子伴侣功能。DegP的酶活性依赖酶切位点与PDZ1结构域双重识别方式识别底物,这种识别模式被称为"分子量尺"。在革兰氏阴性菌中,DegP主要位于膜间质,通过分子伴侣活性与酶活性帮助保护错误折叠蛋白或降解变性蛋白。DegP也参与外膜蛋白的转运,是DegP胞内活性的研究重点。DegP也可以被分泌到胞外,帮助宿主对抗恶劣环境,并参与调节生物被膜的形成。本文将从DegP的结构与活性、胞内功能与胞外功能三大方面对DegP的研究进展进行总结,为革兰氏阴性菌周质中蛋白质质量控制与DegP体外功能的进一步研究提供参考。     

HCV丝氨酸蛋白酶小分子底物构建及GST融合表达

丝氨酸蛋白酶是丙型肝炎病毒重要的功能蛋白和药物作用靶点,其通过分子内（cis）和分子间（trans）方式催化水解前体蛋白,释放病毒功能蛋白。目的：为深入研究病毒蛋白酶活性和抑制剂鉴定需要,实验研究参照丙型肝炎病毒1a亚型菌株蛋白酶天然底物的氨基酸序列特点,设计了一段包含两个天然底物酶切位点的小分子多肽2S,并进行了原核表达。方法：利用PCR方法,合成2S小分子多肽基因,目的基因两端引入BamH I和EcoR I两个限制性酶切位点,双酶切后将基因与表达载体pGEX-4T-2重组,转化大肠杆菌DH5α,经化学诱导进行GST融合蛋白表达,通过亲和层析柱纯化目的蛋白。纯化的GST 2S融合蛋白在体外反应系统进行酶切鉴定,SDS-PAGE和ELISA鉴定酶切结果。结果：PCR合成的小分子底物多肽2S基因,经与表达载体重组后测序,证实基因序列正确。采用0.5mmol/L浓度的IPTG诱导工程菌过夜,获得表达的目的蛋白,经分离纯化得到融合蛋白GST-2S。GST-2S在体外磷酸盐缓冲系统中与丝氨酸蛋白酶反应,15%SDS-PAGE鉴定酶切产物,证实融合蛋白底物条带明显消失,ELISA结果同样说明融合蛋白的底物活性。结论：含有两个天然底物酶切位点的小分子多肽可以替代病毒天然底物,实验结果为丙型肝炎病毒丝氨酸蛋白酶活性研究和酶抑制剂研究奠定了方法学基础。     

棉花病菌Xanthomonas campestris pv.malvacearum的一种具特异肽键专一性的胞外蛋白酶的纯化与鉴定

棉花病原体Xanthomonas campestris pv.malvacearum在酪蛋白(脱脂奶)存在下生长时产生胞外蛋白酶活性,其中至少包含3种蛋白酶,表观分子量分别为29(蛋白酶-1)、38和43kD。 蛋白酶-1被纯化,其最适pH在5.5～7.5之间。抑制研究表明蛋白酶-1可被Phosphoramidone、EDTA及1,10-邻二氮杂菲抑制,然后用锌离子温育重新激活,说明这是一个金属蛋白酶。发现蛋白酶-1特异地裂解肽链的天冬氨酸残基或半胱氯酸残基的氨基端侧,这种高度的肽键专一性预示这个酶在蛋白质链顺序分析中及由较大蛋白质制备特定多肽方面可能十分有用。     

脱脂沙棘籽制备活性多肽的研究

本文比较了用巯基蛋白酶酶解沙棘籽蛋白所得酶解多肽对乙醇脱氢酶(ADH)的激活率效果。结果表明,木瓜蛋白酶的酶解产物对ADH的激活效果最好。该活性多肽的最佳制备条件为,酶解p H为6.5,温度为50℃,酶解时间3.5 h,加酶量为4 000 U/g。体外实验表明,木瓜蛋白酶水解产物中ADH激活率较高的为500~2000 Da的多肽,达57.52%,与未分级的酶解液相比,活性提高显著(P0.01)。分子量在2000~3000 Da的沙棘多肽也具有一定的ADH激活率,为35.09%,说明具有ADH激活率的活性多肽分子量为500~3000 Da。木瓜蛋白酶酶解液经膜分离处理后,收集得到的500~3000 Da的多肽组成占总肽及氨基酸质量的66.50%。     

牙鲆弹性蛋白酶cDNA全长和蛋白质结构分析

为研究牙鲆丝氨酸蛋白酶家族的功能和及其家族的分子进化规律,从本实验室已构建的牙鲆肝胰脏cDNA文库进行了部分测序,从而筛选出一个弹性蛋白酶新成员：弹性蛋白酶5。在此基础上,结合Genbank数据库中已经提交的胰凝乳蛋白酶和胰蛋白酶,对三者蛋白质进行了序列分析和三维结构的比较。牙鲆弹性蛋白酶cDNA包含一个完整的读码框（提交Genbank的登录号为EU873084）。其编码区平均GC含量为54％,推测编码的蛋白质包含296个氨基酸,分子量为29．04KD,等电点为6．14。蛋白序列比较表明它和牙鲆弹性蛋白酶3相似性最高。通过同源建模得到弹性蛋白酶5的三维结构和牛胰凝乳蛋白酶结构相似,包含了2个α螺旋、β个8折叠和13个转角结构。牙鲆弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶中底物结合区的3个关键氨基酸有明显的区别,这些氨基酸的变化改变了底物结合位点开口的大小,胰凝乳蛋白酶2的三个关键氨基酸和牛胰凝乳蛋白酶相同,该区域能接受结构较大的芳香族氨基酸;胰蛋白酶3能更好的结合阳性氨基酸Lys或Arg;而弹性蛋白酶开口很小,只能结合小的残基。上述结果证明了牙鲆丝氨酸蛋白酶家族中的弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶底物结合位点的结构差异决定了其对底物选择的特异性。     

脱脂沙棘籽制备活性多肽的研究北大核心CSCD

本文比较了用巯基蛋白酶酶解沙棘籽蛋白所得酶解多肽对乙醇脱氢酶（ADH）的激活率效果。结果表明,木瓜蛋白酶的酶解产物对ADH的激活效果最好。该活性多肽的最佳制备条件为,酶解pH为6．5,温度为50℃,酶解时间3．5h,加酶量为4000U／g。体外实验表明,木瓜蛋白酶水解产物中ADH激活率较高的为500—2000Da的多肽,达57．52％,与未分级的酶解液相比,活性提高显著（P〈0．01）。分子量在2000～3000Da的沙棘多肽也具有一定的ADH激活率,为35．09％,说明具有ADH激活率的活性多肽分子量为500～3000Da。木瓜蛋白酶酶解液经膜分离处理后,收集得到的500—3000Da的多肽组成占总肽及氨基酸质量的66．50％。     

原卟啉Ⅸ-声动力学疗法诱导S180肿瘤细胞的凋亡

采用频率为2.2MHz,声强为3W/cm2的低强度聚焦超声结合原卟啉Ⅸ对S180肿瘤细胞的损伤以及诱导细胞凋亡的发生进行研究,并探讨其作用的分子机制。超声激活原卟啉Ⅸ作用于S180肿瘤细胞处理后,不同时间段取材,通过Annexin V-PI荧光双染观察凋亡细胞的形态学变化;采用TUNEL末端标记法检测细胞凋亡的发生率;利用间接免疫荧光技术和免疫细胞化学技术检测细胞内凋亡相关蛋白Caspase-8、Caspase-3以及死亡底物聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]的表达活性变化。实验结果显示:超声激活原卟啉Ⅸ可以诱导S180肿瘤细胞凋亡的发生,并且凋亡细胞的比例随着取材时间的延迟明显增加;免疫细胞化学染色表明声动力学处理显著增强了细胞内Caspase-8和Caspase-3的蛋白表达活性,并且其活化程度分别于处理后1h和3h达到最高,而死亡底物PARP也发生时间相关性剪切。研究表明,超声结合原卟啉Ⅸ可以通过诱导细胞凋亡的方式发挥其抗肿瘤活性,其作用的分子机制可能涉及到膜受体介导的Caspase-8、Caspase-3以及PARP依赖性的凋亡信号调节通路     

羧肽酶Y标记蛋白质羧基端方法的优化及应用

蛋白质水解是一种重要的翻译后修饰,它在许多生化过程 (如细胞凋亡和肿瘤细胞转移等) 中起着极其重要的作用。鉴定蛋白质水解位点可以进一步加深我们对这些生化过程的认识。尽管蛋白质氨基端标记方法和蛋白质组学在复杂生物体系中鉴定获得了许多蛋白质的水解位点,但这种方法存在固有的缺陷。羧基端标记方法是另一种可行的鉴定蛋白质水解位点的方法。本文优化了蛋白质羧基端生物酶标记方法,提高了亲和标记效率,从而可以更好地利用正向分离方法对蛋白质羧基端多肽进行分离并用质谱鉴定。我们用优化后的羧基端标记方法来标记大肠杆菌Escherichia coli复杂蛋白样品后鉴定到了120多个蛋白质羧基端多肽和内切多肽。在其所鉴定的蛋白质水解位点中,我们发现了许多已知和未知的位点,这些新的水解位点有可能在正常生化过程的调控发挥着重要的作用。该研究提供了一个可以与蛋白质氨基端组学互为补充、可在复杂体系中鉴定蛋白质水解的方法。     

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。     

Escherichia coli DegP protease cleaves between paired hydrophobic residues in a natural substrate: the PapA pilin

The DegP protein, a multifunctional chaperone and protease, is essential for clearance of denatured or aggregated proteins from the inner-membrane and periplasmic space in Escherichia coli. To date, four natural targets for DegP have been described: colicin A lysis protein, pilin subunits and MalS from E. coli, and high-molecular-weight adherence proteins from Haemophilus influenzae. In vitro, DegP has shown weak protease activity with casein and several other nonnative substrates. We report here the identification of the major pilin subunit of the Pap pilus, PapA, as a natural DegP substrate and demonstrate binding and proteolysis of this substrate in vitro. Using overlapping peptide arrays, we identified three regions in PapA that are preferentially cleaved by DegP. A 7-mer peptide was found to be a suitable substrate for cleavage by DegP in vitro. In vitro proteolysis of model peptide substrates revealed that cleavage is dependent upon the presence of paired hydrophobic amino acids; moreover, cleavage was found to occur between the hydrophobic residues. Finally, we demonstrate that the conserved carboxyl-terminal sequence in pilin subunits, although not a cleavage substrate for DegP, activates the protease and we propose that the activating peptide is recognized by DegP's PDZ domains.     

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.     

Roles of DegP in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in Escherichia coli

Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegP(S210A), a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.     

PDZ domains determine the native oligomeric structure of the DegP (HtrA) protease

DegP (HtrA) is a periplasmic heat shock serine protease of Escherichia coli that degrades misfolded proteins at high temperatures. Biochemical and biophysical experiments have indicated that the purified DegP exists as a hexamer. To examine whether the PDZ domains of DegP were required for oligomerization, we constructed a DegP variant lacking both PDZ domains. This truncated variant, DegPDelta, exhibited no proteolytic activity but exerted a dominant-negative effect on growth at high temperatures by interfering with the functional assembly of oligomeric DegP. Thus, the PDZ domains contain information necessary for proper assembly of the functional hexameric structure of DegP.     

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.     

Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis

The Arabidopsis genome contains 14 genes encoding the serine protease DegP. Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane. We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically. Size-exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers. Proteolytic activity was characterized using beta-casein as a model substrate. DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures. DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen. The results of this work are discussed in the context of the recent elucidation of the structure of the E. coli homolog and the possible physiological role of the protease in the chloroplast lumen.     

The inner cavity of Escherichia coli DegP protein is not essential for molecular chaperone and proteolytic activity

The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28 degrees C, but efficiently degrading unfolded proteins above 28 degrees C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.     

Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus

Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.     

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods-namely, charge optimization and protein design-were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT-RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a 10-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease.