动脉内膜损伤后血管平滑肌细胞表型转化及MKP-1表达的变化

目的:观察动脉内膜损伤后血管平滑肌细胞（vascu lar smooth musc le cells,VSMC）表型转化和丝裂原激活蛋白激酶磷酸酶-1（m itogen-activated prote in k inase phosphatase-1,MKP-1）表达的动态变化。方法:分别用HE染色、免疫组化和逆转录-聚合酶链（RT-PCR）方法检测假损伤组（S组）和损伤后不同时间点血管形态学改变及血管壁中增殖细胞核抗原（PCNA）、平滑肌α肌动蛋白（SMα-actin）和MKP-1 mRNA及蛋白表达的变化。结果:①损伤后1 d中膜腔侧、3 d管腔内表面可见增殖的VSMC,57 d新生内膜（neointim a,NI）形成并逐渐增厚,1435 d NI进行性增厚;各组中膜均有增殖的VSMC向腔面集聚。②S组中膜VSMC及内皮细胞PCNA为阴性;中膜于损伤后114d,NI于514 d PCNA阳性细胞率逐渐增多,14 d达高峰,28 d后开始逐渐减少,但NI阳性率多于中膜。③S组中膜SMα-actin表达为阳性,内皮为阴性;中膜阳性面积于损伤后1 d开始减少,3 d最为明显,5 d后开始逐渐增加,NI阳性表达弱于中膜。④S组中膜MKP-1呈弱阳性或阳性表达,损伤后1d即开始下降,57 d达最低,14 d稍有回升,至35 d仍未回到假损伤组水平;NI阳性表达弱于中膜。MKP-1表达变化与PCNA表达变化呈负相关。结论:VSMC增殖能力与其表型转化密切相关,MKP-1参与了损伤后VSMC表型转化的调节。     

粉防己碱对内膜损伤后平滑肌细胞表型转化和MKP-1表达的影响

目的研究粉防己碱（tetrandrine,Tet）在防治血管内膜损伤后再狭窄（RS）与血管平滑肌细胞（VSMC）表型转化及丝裂原激活蛋白激酶磷酸酶-1（MKP-1）表达之间的关系。方法采用HE染色检测假损伤组（S组）、损伤组（I组）和损伤+Tet治疗组（Tet组）28 d血管形态学改变;分别使用免疫组化和RT-PCR技术检测I组和Tet组7、14、28 d增殖细胞核抗原（PCNA）、平滑肌α-肌动蛋白（SMα-actin）和MKP-1表达的变化。结果①28 d血管形态观察,S组血管壁各层结构完整;I组新生内膜（neointim a,NI）面积显著增加,管腔面积（LA）显著缩小;Tet组NI增殖较I组明显减轻,LA增加。②损伤后7 d,Tet组与I组之间血管壁SMα-actin、PCNA和MKP-1表达变化无显著差异,NI增殖程度亦基本相同。Tet组14 d和28 d血管壁中PCNA表达均低于I组,而MKP-1表达均高于I组;14d SMα-actin表达略高于I组,28 d两组间无差异。结论Tet可不同程度地拮抗内膜损伤后VSMC表型转化及其调节,继而减缓NI增殖。     

大鼠颈动脉球囊损伤后血管平滑肌细胞E1A激活基因阻遏子的表达变化

目的探讨血管再狭窄发生的病理生理机制及E1A激活基因细胞阻遏子(CREG)在新生内膜增殖中的调控作用, 为研究CREG防治增生性血管疾病的作用奠定基础.方法采用大鼠颈动脉球囊损伤后血管再狭窄的动物模型, 以免疫组织化学染色、逆转录-聚合酶链反应(RT-PCR)方法, 检测新生内膜中增殖细胞核抗原(PCNA)和平滑肌α肌动蛋白(SM α-actin)的表达变化及血管壁中CREG mRNA水平、蛋白表达的动态变化.结果大鼠颈动脉球囊损伤后1 d血管壁 CREG mRNA水平开始下降, 至损伤后5 d达最低, 损伤后7 d CREG mRNA表达回升, 至28 d时仍未回到正常对照组的水平.血管损伤后3 d血管内表面可见增殖的血管平滑肌细胞(VSMC), PCNA染色阳性, 其胞浆内SM α-actin和CREG染色均为阴性; 损伤后5 d新生内膜形成并增厚, PCNA阳性细胞数达到高峰, 部分VSMC胞浆内SM α-actin和CREG染色均呈阳性; 损伤后28 d管腔严重狭窄, 新生内膜中PCNA表达已较低, SM α-actin和CREG表达均明显增加, 新生内膜SM α-actin表达程度仍弱于中膜, CREG表达程度接近中膜.损伤后不同时间点VSMC增殖程度与血管壁中CREG mRNA水平的变化呈负相关(r=-0.80, P<0.05), CREG mRNA的表达为先降低, 后回升, 而细胞增殖指数为先升高, 后回降.结论 VSMC的表型转化、增殖、迁移和分泌细胞外基质导致新生内膜过度增生和管腔狭窄, CREG参与VSMC增殖的调控.     

p38和丝裂原活化蛋白激酶磷酸酶1在内膜损伤后血管平滑肌细胞表型转化过程中的表达变化

目的观察动脉内膜损伤后血管平滑肌细胞表型转化和p38及丝裂原活化蛋白激酶磷酸酶1表达的动态变化。方法分别用免疫组织化学、免疫印迹和逆转录聚合酶链反应方法检测假损伤组和损伤后不同时间点血管壁中增殖细胞核抗原、平滑肌α肌动蛋白、p38蛋白和丝裂原活化蛋白激酶磷酸酶1蛋白及其mRNA的表达。结果假损伤组血管中膜平滑肌细胞及内皮细胞增殖细胞核抗原为阴性表达;损伤后5~14天,新生内膜阳性细胞率逐渐增加,28天后开始逐渐减少,且新生内膜阳性率均略高于中膜。假损伤组血管中膜平滑肌α肌动蛋白表达为阳性,内皮为阴性;中膜阳性面积于损伤后1天开始减少,3天最为明显,5天后开始逐渐增加,且新生内膜阳性表达略低于中膜。假损伤组血管中膜p38呈阴性或弱阳性着色;损伤后1~35天呈持续高表达,新生内膜阳性表达高于中膜。p38表达变化与增殖细胞核抗原表达变化呈正相关。假损伤组血管中膜丝裂原活化蛋白激酶磷酸酶1呈弱阳性或阳性表达;损伤后1天即开始下降,14~28天稍有回升,至35天仍未回到假损伤组水平,且新生内膜阳性面积稍低于中膜。其表达变化与增殖细胞核抗原表达变化呈负相关。结论内膜损伤后血管平滑肌细胞增殖能力与其表型转化密切相关,p38和丝裂原活化蛋白激酶磷酸酶1参与了损伤后血管平滑肌细胞表型转化的信号转导及调节。     

MiR-92a-3p_R+1和miR-92a-1-5p在ox-LDL诱导大鼠VSMC表型转化和增殖中的作用

目的探讨miR-92a-3p_R+1和miR-92a-1-5p在氧化型低密度脂蛋白(ox-LDL)诱导大鼠血管平滑肌细胞(VSMC)表型转化和增殖中的作用及其与ERK1/2信号通路的相关性。方法分离、培养大鼠VSMC,以ox-LDL(50 mg/L)诱导VSMC,采用ERK1/2特异性抑制剂U0126(10μmol/L)阻断ox-LDL(50 mg/L)诱导VSMC的ERK1/2信号激活,MicroRNA微阵列分析VSMC的miR-92a-3p_R+1和miR-92a-1-5p表达,CCK-8法和Brdu流式细胞术检测细胞增殖;免疫荧光法检测VSMC收缩表型标志蛋白SM22α的变化;Western blot检测VSMC的ERK1/2通路信号激活情况(ERK、p-ERK)、表型标志蛋白SM22α、细胞周期相关蛋白(PCNA、cyclin D1、p21、p27)的表达情况。结果 ox-LDL诱导下,VSMC的miR-92a-3p_R+1和miR-92a-1-5p表达明显上调,VSMC的ERK1/2磷酸化水平明显增加,SM22α的表达降低,同时细胞周期相关蛋白PCNA、cyclin D1高表达,p21、p27低表达;ERK1/2通路特异性抑制剂U0126干预后,ERK1/2磷酸化水平受抑制,相应的VSMC miR-92a-3p_R+1和miR-92a-1-5p表达明显下调(P0.05),VSMC增殖显著下降,SM22α的表达上调(P0.05),提示VSMC由合成表型转化为收缩表型,并下调PCNA、cyclin D1的表达,上调p21、p27蛋白的表达(P0.05),说明其表型转化和增殖明显受抑制。结论 miR-92a-3p_R+1和miR-92a-1-5p在ox-LDL诱导VSMC表型转化和增殖中起重要作用,并与ERK1/2信号通路密切相关。     

miR-145抑制VSMC增殖的作用及其相关信号通路研究

目的探讨miR-145对PDGF-BB诱导的大鼠原代血管平滑肌细胞(VSMC)的作用及丝裂原激活的蛋白激酶(MAPK)信号转导途径的作用。方法体外培养大鼠原代VSMC,再将细胞分为空白对照组、miR-NC组、PDGF-BB+miR-145组及PDGF-BB组。CCK-8法检测各组细胞的增殖情况;实时RT-PCR方法检测PCNA、c-Jun及SM22a的表达水平;Western印迹方法检测ERK1/2和p-ERK1/2的表达、JNK和p-JNK的表达以及p38MAPK和p-p38MAPK的表达。结果 miR-145过表达后能够抑制PDGF诱导的大鼠原代VSMC增殖,并下调VSMC增殖相关基因PCNA、c-Jun的表达、上调分化相关基因SM22a的表达;PDGF-BB诱导VSMC后,ERK、JNK、p38MAPK的磷酸化水平均明显上调,而转染miR-145慢病毒后再加PDGF刺激,ERK、JNK、p38MAPK的磷酸化水平均明显下调。结论 miR-145能够抑制去分化型VSMC中的MAPK信号通路,进而抑制VSMC的增殖。     

大鼠microRNA-145慢病毒表达载体的构建及其对血管平滑肌细胞表型转化的影响

目的构建针对Rno-miR-145的慢病毒表达载体并探讨其在血小板源生长因子(PDGF)诱导的血管平滑肌细胞(VSMC)表型转化中的作用。方法人工合成含有酶切位点粘端miR-145 shDNA双链模板序列,克隆于LV3 pGLV/H1/GFP+Puro-miRNA慢病毒穿梭载体中,转染293T细胞,收获并浓缩慢病毒颗粒,感染大鼠原代VSMC,倒置荧光显微镜下观察VSMC感染后的荧光表达情况,实时荧光定量PCR检测miR-145的表达情况;实验分为空白对照组、PDGF组、PDGF+miR-145组和细胞转染阴性慢病毒载体组(miR-NC组);采用实时荧光定量PCR测定miR-145对VSMC增殖相关基因PCNA、c-Jun及分化相关基因SM22αmRNA表达水平的影响。结果成功构建了microRNA-145慢病毒载体,测定病毒滴度为1×109TU/mL。倒置荧光显微镜下观察大鼠microRNA-145慢病毒表达载体感染成功,MOI值为50,感染72 h时感染率最高。实时荧光定量PCR结果显示PDGF可使PCNA、c-Jun表达增加,而使SM22α表达降低;miR-145可使PDGF诱导的去分化型VSMC增殖相关基因PCNA、c-Jun表达降低,分化相关基因SM22α表达增加。结论 miR-145慢病毒载体可高效感染大鼠原代VSMC。感染miR-145慢病毒后可抑制VSMC的表型转化。     

黄芪、当归对血管内皮剥脱后内膜增生的影响及作用机制

目的 研究黄芪和当归防治血管内皮剥脱后再狭窄与血管平滑肌细胞(VSMC)表型转化之问的关系。方法 建立大鼠主动脉内皮剥脱模型，分别采用形态学和Northern印迹分析技术，观察大鼠血管内皮剥脱后内膜增生情况及VSMC表型标志基因SM α—肌动蛋白、平滑肌胚胎型肌聋蛋白重链(SMemb)表达活性。结果 术后7d模型组内膜明显增生，14～21d内膜呈进行性弥漫性增厚，与模型组相比，两种中药治疗组的内膜增生程度均明显藏轻1分化标志SM α—肌动蛋白基因表达增高，去分化标志SMemb表达降低。结论 黄芪和当归通过抑制VSMC表型转化而减缓血管内膜增生。     

p38MAPK信号途径与血管损伤后平滑肌细胞增殖关系的研究

目的检测大鼠颈动脉球囊损伤后平滑肌细胞增殖及p38丝裂素活化蛋白激酶(MAPK)的表达,探讨血管损伤后平滑肌细胞增殖的信号途径.方法雄性Sprague-dawley大鼠30只,体重250～300 g.随机分为假手术组、手术后7 d组、手术后14d组,每组10只大鼠,进行球囊血管损伤术,术后7、14 d分别取15 mm颈动脉作为实验标本.血管组织行苏木精-伊红染色、免疫组化和免疫印迹,检测血管壁增生、增殖细胞核抗原(PCNA)和p38MAPK表达情况.结果术后7、14d组有动脉壁增厚、新生内膜形成和中膜平滑肌胶原化,PCNA细胞阳性率分别是(39.5±7.9)%和(44.8±9.9)%,与假手术组[(1.3±0.4)%]相比明显增加(P＜0.01);p38MAPK表达明显高于假手术组(P＜0.05).p38MAPK表达与血管平滑肌细胞增殖率呈正相关(r=0.714,P＜0.05).结论球囊损伤血管能促进血管平滑肌细胞增殖和血管内膜增生;血管损伤后p38MAPK表达明显增强,与平滑肌细胞增殖呈正相关;p38MAPK信号途径参与了血管平滑肌细胞的增殖.     

AT2R基因转染对血管平滑肌细胞增殖核抗原表达的影响

目的:探讨血管紧张素Ⅱ2型受体(AT2R)基因体外及体内转染对血管平滑肌细胞(VSMC)增殖核抗原(PCNA)表达的影响。方法:细胞爬片及大鼠颈动脉球囊损伤后,局部转染带有绿色荧光蛋白报告基因的AT2R重组腺病毒载体(pAd-AT2R)或病毒载体(pAd-GFP),于细胞转染后48h及术后14d取材,应用免疫组织化学染色、免疫荧光双标染色和激光共聚焦技术检测了VSMC及血管中AT2R与PCNA的表达关系。结果:pAd-AT2R转染VSMC后,在激光共聚焦显微镜下胞质及胞核有绿色荧光蛋白表达;同时在胞质有红色荧光AT2R表达,胞核无蓝色荧光,PCNA表达阴性,无红色荧光AT2R表达的VSMC胞核有蓝色荧光,PCNA表达阳性。pAd-AT2R在体转染大鼠颈动脉后,新生内膜及中膜中PCNA阳性表达率显著低于未转染组和pAd-GFP组[(27.29±5.81)%∶(72.25±4.47)%,(68.43±9.12)%,P<0.01];在激光共聚焦显微镜下,绿色荧光及红色荧光AT2R主要分布于新生内膜、中膜、外膜,内膜散在蓝色荧光PCNA表达,在红、绿色荧光处无蓝色荧光,PCNA表达阴性,无红、绿色荧光处,有蓝色荧光,PCNA表达阳性。结论:AT2R基因体外及体内转染可抑制VSMCPCNA表达,ATR基因转染能抑制新生内膜增生。     

A thermodynamic definition of protein domains

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.     

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1’s involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ_lag) and maximum growth rate (ν_max) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ_lag/∂[urea] and ∂ log ν_max/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.     

Experimental support for the evolution of symmetric protein architecture from a simple peptide motif

The majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down symmetric deconstruction" strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed "conserved architecture" model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.     

Retro-translocation of mitochondrial intermembrane space proteins

The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.Mitochondrial biogenesis is essential for eukaryotic cells. Because most mitochondrial proteins originate in the cytosol, mitochondria had to develop a protein import system. Given the complex architecture of these organelles, with two membranes and two aqueous compartments, protein import and sorting require the cooperation of several pathways. The main entry gate for precursor proteins is the translocase of the outer mitochondrial membrane (TOM) complex. Upon entering mitochondria, proteins are routed to different sorting machineries (1–5).Reaching the final location is one step in the maturation of mitochondrial proteins that must be accompanied by their proper folding. The mitochondrial intermembrane space assembly (MIA) pathway for intermembrane space (IMS) proteins illustrates the importance of coupling these processes because this pathway links protein import with oxidative folding (6–10). Upon protein synthesis in the cytosol, the cysteine residues of IMS proteins remain in a reduced state, owing to the reducing properties of the cytosolic environment (11, 12). After entering the TOM channel, precursor proteins are specifically recognized by Mia40 protein, and their cysteine residues are oxidized through the cooperative action of Mia40 and Erv1 proteins (7, 13–17). Mia40 is a receptor, folding catalyst, and disulfide carrier, and the Erv1 protein serves as a sulfhydryl oxidase. The oxidative folding is believed to provide a trapping mechanism that prevents the escape of proteins from the IMS back to the cytosol (10, 13, 18). Our initial result raised a possibility that the reverse process can also occur, as we observed the relocation of in vitro imported Tim8 from mitochondria to the incubation buffer (13). Thus, we sought to establish whether and how this process can proceed in the presence of the intact outer membrane (OM). Our study provides, to our knowledge, the first characterization of the mitochondrial protein retro-translocation. The protein retro-translocation serves as a regulatory and quality control mechanism for the mitochondrial IMS proteome.     

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.     

Folding pathways of proteins with increasing degree of sequence identities but different structure and function

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G_A (a 3-α helix fold) and G_B (an α/β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G_A domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G_Bs, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.     

Functional modulation of a protein folding landscape via side-chain distortion

Ultrahigh-resolution (< 1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6° distortion along the side-chain, estimated to cost 4 kcal/mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.     

Multimolecule test-tube simulations of protein unfolding and aggregation

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.     

Golden triangle for folding rates of globular proteins

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured rates of spontaneous folding of single-domain globular proteins range from microseconds to hours: the difference (11 orders of magnitude) is akin to the difference between the life span of a mosquito and the age of the universe. Here, we show that physical theory with biological constraints outlines a “golden triangle” limiting the possible range of folding rates for single-domain globular proteins of various size and stability, and that the experimentally measured folding rates fall within this narrow triangle built without any adjustable parameters, filling it almost completely. In addition, the golden triangle predicts the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control. It also predicts the maximal allowed size of the “foldable” protein domains, and the size of domains found in known protein structures is in a good agreement with this limit.     

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.The phylogenetically oldest living organisms identified that possess most major components of a vertebrate adaptive immune system are cartilaginous fish (Chondrichthyes) such as sharks, skates, and rays (1, 2). They shared the last common ancestor with other jawed vertebrates roughly 500 million years ago (2, 3). Accordingly, shark antibodies can provide unique insights into the molecular evolution of the immune system. Furthermore, shark antibodies have evolved under challenging conditions; for example, the high osmolarity of shark blood is partially sustained by the protein denaturant urea (4, 5). Even though it is partially counteracted by other osmolytes (6), shark antibodies are believed to be particularly stable (7). Insights into the structural features that provide this increased stability may provide attractive applications for biotechnology (8). Sharks and other Elasmobranchs have two conventional antibodies, IgM and IgW, but the structurally simplest antibody molecule in sharks is the so-called Ig new antigen receptor (IgNAR) (9). In its secreted form, it consists of two identical heavy chains (HCs) composed of one variable domain (V) and five constant domains (C1–C5) each (4, 9) (Fig. 1A). Similar to camelid antibodies, IgNARs are devoid of light chains (LCs) (9, 10), an example of convergent evolution (11). The variable domain of IgNAR, whose structure had been solved (12, 13), shows similarity to the variable domains of evolutionarily more recent immunoglobulins (12, 13). In contrast, its constant domains (C1–C5; Fig. 1A) are most homologous to the primordial IgW of sharks (14). Of the five human antibody classes, IgA, IgD, IgE, IgG, and IgM, IgW is most closely related to IgD, which, along with IgM, are the oldest Ig isotypes (14–17). Except for low-resolution electron microscopic images (18), no structural data are available for any of the constant IgNAR domains.Open in a separate windowFig. 1.Sequence and structure of IgNAR domains C1–C4. (A) Schematic of the secreted dimeric IgNAR molecule, comprising one variable (V) and five constant (C1–C5) domains. Predicted glycosylation sites are shown as gray hexagons. Cysteines that are not part of the intradomain disulfide bridges are indicated (–SH). The secretory tail is C terminally of the C5 domain. (B) Sequence alignment of IgNAR C1–C5 with the human IgG1 HC domains C_H1–C_H3. Conserved cysteines are highlighted in red, and conserved hydrophobic residues of a YxCxY (Y, hydrophobic residue) motif around the disulfide bridge are highlighted in orange. Conserved tryptophans in strand c and the second helix are highlighted in blue, and the cis-proline residue in the loop between strand b and c is depicted in cyan. Secondary structure elements are indicated above the alignment. Black arrows indicate strictly conserved residues, and gray arrows homologous residues. (C) Ribbon diagram of the isolated constant IgNAR domains C1–C4 (C1, cyan; C2, blue; C3, red; C4, green; colors like in A). Residues marked in the alignment are shown in stick representation, the small helices are indicated. (D) Superposition of the IgNAR C1-4 domains (C1, cyan; C2, blue; C3, red; C4, green) on a human IgG C_H3 domain (gray, Protein Data Bank ID code 1HZH).Here, we determined the structures of the four N-terminal IgNAR constant domains (C1–C4) at atomic resolution and present a model for the complete IgNAR molecule that reveals key adaptations of HC-only antibodies. We identified structural elements that contribute to the high stability of some IgNAR domains and transferred these to human antibodies to improve their stability and secretion.