HBx基因下调p21对HepG2细胞增殖与凋亡的影响

目的:构建转基因细胞模型HepG2/HBx,观察HBx基因对HepG2细胞增殖、周期和凋亡的影响, 探讨细胞周期蛋白P21在其中的作用和意义.方法:应用脂质体转染和G418筛选构建稳定表达HBx的转基因细胞HepG2/HBx,RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.分别以四唑蓝(MTT)比色法、流式细胞术检测HepG2/HBx细胞及对照组HepG2与HepG2/pcDNA3.1细胞(转染空载体pcDNA3.1的HepG2细胞)的增殖、周期和凋亡.另半定量RT-PCR检测各组细胞中细胞周期蛋白P21与抑癌基因p53mRNA的表达.结果:HepG2/HBx细胞中有HBx mRNA和蛋白的表达.HepG2/HBx细胞生长速度加快.HepG2/HBx中G0/G1期细胞比例较对照组显著减少(43.34%±3.11%vs57.69±4.28%,P<0.01),S期细胞比例明显增加(28.69%±1.17%vs22.41%±1.99%,P<0.05),同时还发现与对照组相比其凋亡率也显著降低(1.19%±0.06%vs 5.43%±0.42%, P<0.001).细胞周期蛋白p21 mRNA在HepG2/HBx细胞中的表达较对照组细胞显著降低(0.16±0.05vs0.78±0.15,P<0.001),而p53表达则无显著变化.结论:HBx基因可下调细胞周期蛋白P21mRNA的表达,可能参与HBx基因加速HepG2细胞周期进程、促进细胞增殖以及抑制细胞凋亡的作用.     

马尾松花粉多糖硫酸酯对肝癌HepG2细胞的诱导分化

目的:观察马尾松花粉多糖硫酸酯(SPPM60)对人肝癌细胞株HepG2的诱导分化作用, 并对其机制进行初步探讨.方法:MTT法检测HepG2细胞增殖活力; 倒置显微镜、HE染色及透射电镜观察细胞形态;流式细胞技术检测细胞周期; 荧光分光光度计检测细胞内钙离子浓度的变化; 免疫细胞化学方法检测甲胎蛋白的表达, 溴甲酚绿法测定白蛋白含量; 重氮反应比色法测定γ-谷氨酰转肽酶活力, 比色法测定碱性磷酸酶活力, 考马斯亮蓝法测定总蛋白含量.结果:经SPPM60处理后, HepG2细胞的增殖受到抑制; 细胞形态趋于正常; 与对照组相比, SPPM60组G0/G1期细胞数增加(76.97%±0.91% vs 64.62%±0.18%, P<0.05), S期细胞数减少(17.21%±0.71% vs 24.26%±0.15%,P<0.05), G2/M细胞数减少(5.82%±0.20% vs11.13%±0.34%, P<0.01); [Ca2+]i降低; 甲胎蛋白表达降低, 白蛋白分泌量上升(6.77±0.33 mg/106细胞vs 4.87±0.30 mg/106细胞,P<0.05), γ-谷氨酰转肽酶与碱性磷酸酶活力降低(16.3±0.7 U/g vs 22.3±1.2 U/g; 223.3±15.7 U/g vs 311.1±13.4 U/g P<0.05或0.01), 胎盘型碱性磷酸酶的比例降低(46.4%±1.5% vs62.5%±2.3%, P<0.05). PPM60组细胞没有明显变化.结论:硫酸酯化赋予了SPPM60抑制HepG2细胞的活性, 其作用机理可能是降低细胞内钙离子浓度, 阻滞细胞周期于G0/G1期, 诱导HepG2细胞分化逆转其恶性.     

转基因细胞模型L02/HBx的构建及HBx对细胞周期的影响

目的:构建L02/HBx转基因细胞模型并研究HBx对肝细胞周期的影响.方法:运用脂质体转染和G418筛选获得L02/ HBx阳性克隆,并分别用RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.进一步用四唑蓝(MTT)比色试验、流式细胞仪检测L02/HBx的增殖、凋亡和细胞周期.结果:RT-PCR和Western blot分别检测到L02/ HBx细胞中HBx mRNA和蛋白的表达.MTT比色试验显示L02/HBx生长速度加快,流式细胞仪检测发现L02/HBx凋亡率低(0.09%±0.13% vs 3.74%±1.29%,P<0.05),G1期细胞比例减少(61.35%±0.82% vs 67.80±6.84%,P<0.05),S期细胞比例相应增加(36.59%±2.54% vs 22.37%±2.17%,P<0.05).经阿霉素(ADM)培养后,L02/HBx的凋亡率显著增加(34.91%±5.85% vs 0.09%±0.13%,P<0.05),G1期细胞比例明显增加但低于对照组(82.81%±6.48% vs 61.35%±0.82%,P<0.05;82.81%±6.48% vs 87.19%±1.92%,P<0.05),S期细胞比例降低但较对照组高(13.84%±6.16% vs 36.59%±2.54%,P<0.05;13.84%±6.16% vs 2.22%±1.26%,P<0.05).结论:L02/HBx构建成功,HBx能促进细胞周期进程,加快细胞的生长并抑制细胞的凋亡;转染HBx基因的肝细胞凋亡更易受凋亡因子所触发,表明HBx可能会增加正常肝细胞对诱导凋亡因素的敏感性.     

胰岛素抵抗肝癌细胞IGF-1R/NF-κB表达及多药耐药机制研究

目的 探讨胰岛素抵抗（IR）肝癌细胞胰岛素样生长因子1受体（IGF-1R）和核因子-κB（NF-κB）表达变化及多药耐药（MDR）发生机制。方法 采用高浓度胰岛素诱导人肝癌细胞（HepG2和HepG2.2.15）建立胰岛素抵抗（IR）细胞模型。采用Western blot 法检测胰岛素受体（InsR）、IGF-1R、NF-κB 和 P-糖蛋白（P-gp）表达变化。使用流式细胞仪（Annexin V-FITC法）检测阿霉素对细胞凋亡的影响。结果 分别用100 nmol/L 和 1 000 nmol/L 胰岛素培养 HepG2 和 HepG2.2.15 细胞 48 h,成功建立 IR 肝癌细胞模型;IR 肝癌细胞 IGF-1R、NF-κB、P-gp 表达上调,而InsR 表达下调;应用 25μg/mL 阿霉素作用细胞 24 h 后,IR-HepG2 细胞组凋亡率（31.1%±1.9%）显著低于HepG2 细胞组【（49.7%±2.2%）,P＜0.01】,IR-HepG2.2.15细胞凋亡率【（20.1±1.7） %】显著低于 HepG2.2.15 细胞【（33.8±1.8）%,P＜0.01】;HepG2.2.15 和 IR-HepG2.2.15 细胞凋亡率分别较 HepG2 和 IR-HepG2 细胞显著降低（P＜0.01）。结论 IGF-1R/NF-κB/P-gp 过表达可能介导 IR 肝癌细胞对阿霉素的多药耐药。     

肝癌细胞对5-氮杂-2'-脱氧胞苷的敏感性与细胞总DNA甲基化水平无关

目的 比较不同肝癌细胞株对5-氮杂-2'-脱氧胞苷(5-aza-dC)的敏感性,探讨肝癌细胞对5-aza-dC的敏感性是否与细胞总DNA甲基化水平有关.方法 用不同剂量(0.5、5.0、10.0μmol/L)的5-aza-dC处理肝癌细胞株(HepG2、QGY7701和HepG2.2.15细胞)及正常肝细胞株L02,比较不同浓度处理前后的细胞增殖抑制率,比较10 μmol/L 5-aza-dC处理前后的Caspase-3活性及细胞DNA片段化水平(5-溴脱氧尿嘧啶核苷掺入率),比较不同细胞总DNA甲基化水平.组间检测结果比较采用t检验.结果 5-aza-dC对HepG2、QGY7701、HepG2.2.15、L02细胞的半数抑制浓度分别为0.5、0.5、4.5、11.4μmol/L,与HepG2细胞和QGY7701细胞相比,HepG2.2.15绌胞和L02细胞对5-aza-dC不敏感.HepG2和QGY7701细胞中Caspase-3的活性升高较L02和HepG2.2.15细胞明显(P值均＜0.05),QGY7701细胞中5-溴脱氧尿嘧啶核苷掺入率升高较L02细胞明显(P＜0.05).L02、HepG2、QGY7701和HepG 2.2.15细胞的DNA总甲基化水平分别为11.7%±0.9%、10.9%±1.3%、11.7%±1.7%和12.2%±1.0%,差异无统计学意义(P值均＞0.05).结论 细胞对5-aza-dC的敏感性与细胞总DNA甲基化水平无关.     

丹皮酚联合5-FU对食管癌EC9706细胞增殖及凋亡的影响

目的:探讨丹皮酚(paeonol,Pae)单独及联合5-Fu对人食管癌EC9706细胞的增殖抑制及凋亡诱导作用.方法:采用6种浓度的Pae(7.81、15.63、31.25、62.50、125.00、250.00 mg/L)、3种浓度的5-FU(12.50、25.00、50.00 mg/L)及Pae(31.25 mg/L)和5-FU(12.50 mg/L)联合分别处理EC9706细胞24、48、72 h.同时设对照组(细胞不做处理),采用MTT法检测各个时间段细胞的增殖情况:采用流式细胞术检测4种浓度的Pae(31-25、62.50、125.00、250.00 mg/L)处理EC9706细胞72 h后细胞周期的变化:倒置显微镜下观察各Pae组细胞各时间段形态学变化,HE染色光镜下观察凋亡细胞:采用免疫细胞化学法检测经Pae(31.25 mg/L)、5-FU(12.50mg/L)单独和联合作用48 h后细胞中凋亡相关蛋白Bcl-2及Bax的表达.结果:Pae、5-FU可明显抑制EC9706细胞增殖,并随着浓度的增加和作用时间的延长而增强(P<0.05),Pae与5-FU联合用药比单用Pae或5.FU抑制效果更明显(P<0.05);Pae作用后EC9706细胞中G0/G1期和G2/M期细胞比例下降、S期细胞比例上升(Pae 125.00 mg/L组:G0/G1期21.18%±2.28% vs 62.17%±5.23%、G2/M期0.76%±0.54% vs 9.92%±3.10%、S期78.06%±2.82% vs 27.91%±2.13%,均P<0.05):HE染色光镜下可见典型的肿瘤细胞凋亡改变:Pae、5-FU可下调EC9706细胞中Bcl-2蛋白表达,同时增强EC9706细胞中Bax蛋白的表达,联合用药组较单药组作用更为明显(2.21±0.14 vs 5.67±0.30,4.22±0.34;8.55±0.33 vs 3.90±0.27,6.28±0.26,均P<0.05).结论:Pae可明显抑制人食管癌EC9706细胞的增殖.促进其凋亡,Pae联合5-FU作用更为明显.     

丹参酮ⅡA介导p38MAPK信号转导诱导人肝癌细胞凋亡

目的: 研究丹参酮ⅡA诱导人肝癌细胞凋亡和凋亡相关基因表达的p38MAPK信号转导通路, 揭示其抗肝癌的部分机制.方法: 4、8、16 mg/L丹参酮ⅡA分别作用人肝癌SMMC-7721细胞48 h后, 免疫荧光染色观察细胞凋亡情况; 琼脂糖凝胶电泳观察凋亡细胞特征性DNA条带; 流式细胞仪法(Flowcytometry, FCM)检测细胞凋亡和细胞周期; 荧光定量PCR检测Fas和Caspase-3基因mRNA的表达水平; 并比较阻断p38MAPK信号通路后丹参酮ⅡA对肝癌细胞凋亡和Fas和Caspase-3基因mRNA的表达.结果: 丹参酮ⅡA作用48 h后, 荧光显微镜下观察到经Hoechst染色的典型凋亡细胞. 琼脂糖凝胶电泳可见凋亡细胞DNA呈规律性的梯状条带. 4、8、16 mg/L浓度丹参酮ⅡA作用人肝癌细胞后的细胞凋亡率分别为12.83%±1.51%, 17.86%±2.70%和29.24%±7.58%, 与对照组6.30%±2.08%比较均有显著性差异( P<0.01); 阻断p38MAPK信号通路后, 凋亡率和G0/G1期细胞比例明显降低( P<0.01). 8 mg/L丹参酮ⅡA作用人肝癌细胞48 h后Fas mRNA和Caspase-3 mRNA的表达明显上升; 阻断p38MAPK信号通路后, 丹参酮ⅡA作用人肝癌细胞的Fas mRNA和Caspase-3 mRNA的表达明显下降.结论: 丹参酮Ⅱ A 能诱导人肝癌细胞株SMMC-7721凋亡, 阻滞肝癌细胞于G0/G1期. 通过p38MAPK信号转导通路上调Fas、Caspase-3 mRNA的表达可能是其诱导肝癌细胞凋亡的重要机制.     

PI3K/Akt/p27kip1通道介导胃癌细胞对阿霉素、足叶乙甙化疗的耐药性及其机制

目的:探讨PI3K/Akt(PKB)/p27kip1通道对胃癌细胞BGC-823化疗的效果和作用机制.方法:将培养的胃癌细胞BGC-823分为对照组,P13K/Akt/p27kip1通道抑制剂Wort组(Wort组),足叶乙甙组(Eto组),阿霉素组(Dox组),Eto Wort组和Dox Wort组:MTT法检测细胞生存率,流式细胞仪检测细胞周期和凋亡,Western印迹法检测p27Kip1蛋白表达水平,RT-PCR检测p27基因mRNA表达水平.结果:与对照组相比,Wort组,Eto Wort组和Dox Wort组24h细胞生存率降低(57.8%、46.5%、44.3%vs 46.5%、44.3%,P＜0.01),G0-G1期的比例增多(85.0±3.54,91.5±3.63,92.4±3.64 vs71.5±3.25,P＜0.01),12h和24h蛋白表达升高;Eto组和Dox组细胞生成率较对照组升高,但G0-G1期的比例无明显变化;各组p27基因mRNA表达水平一致.结论:P13K/Akt/p27/通道激活能介导肿瘤细胞化疗耐药.     

没药甾酮对人肝癌细胞HepG2增殖和凋亡的影响

目的研究没药甾酮对人肝癌细胞HepG2增殖和凋亡的影响。方法以正常人肝细胞L-02作为对照,采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐法观察不同浓度没药甾酮（5～100μmol/L）对人肝癌细胞HepG2和L-02细胞增殖的影响并观察细胞形态的变化;应用流式细胞术检测细胞周期变化和凋亡发生。结果不同浓度没药甾酮均可显著抑制人肝癌细胞HepG2生长,并呈时间、剂量依赖性,最大抑制率可达81.9%±1.92%（100μmol/L）;没药甾酮可使G0/G1期细胞比例增多,G2/M期细胞比例下降,可将细胞阻滞于G0/G1期;没药甾酮诱导人肝癌细胞HepG2发生凋亡,50μmol/L和75μmol/L没药甾酮早期细胞凋亡率分别为24.91%±2.41%、53.03%±2.28%,与对照组相比,差异均具有统计学意义（P〈0.05）。结论没药甾酮可抑制人肝癌细胞HepG2增殖并诱导凋亡,其作用可能与干扰细胞周期有关。     

HH胶囊体外抗乙型肝炎病毒的作用及其机制

目的:研究HH胶囊体外抗乙型肝炎病毒的作用及其对抗病毒蛋白2'5'-寡腺苷酸合成酶(2'5'-Oligoadenylate Synthetase,2'5'-OAS)、RAN依赖蛋白激酶(RAN-dependent protein kinase,PKR)的影响.方法:HepG2.2.15是目前最常用的乙型肝炎病毒感染的体外实验模型,将细胞随机分为空白对照组、阳性对照组(3TC)、不同浓度的HH胶囊组.用CCK-8检测药物细胞毒性,酶联免疫法检测乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量聚合酶链反应法检测乙型肝炎病毒脱氧核糖核酸(HBV DNA);用Western blot、荧光定量PCR方法分别检测细胞内抗病毒蛋白2'5'-OAS、PKR及其mRNA水平.结果:HH胶囊的TC50是2.11g/L、312.00 mg/L、1 56.00 mg/L、78.00 mg/L、39.00 mg/L HH胶囊均可以降低细胞外HBeAg(0.285±0.007,0.462±0.008,0.565±0.009.0.733±0.008 vs1.334±0.007)和HbsAg(0.834±0.008.1.021±0.011.1.347±0.017.1.548±0.015±2.593±0.008)水平,3 12.00 mg/L、156.00 mg/LHH胶囊均可以减低细胞内乙型肝炎病毒DNA[(3.423±0.110)×109copies/L,(3.640±0.082)×109copies/L vs (6.857±0.060)×109copies/L]、细胞外乙型肝炎病毒DNAf6 547±87、7 710±62 vs 24 300±200).312.00 mg/LHH胶囊可以增加细胞内OAS mRNA(0.885±0.038 vs 0.688±0.068)、PKR mRNA(0.139±0.06 vs 0.058+0.005)表达及其OAS、PKR蛋白水平.结论:HH胶囊具有良好的抗乙型肝炎病毒作用,推测可能与增加细胞内OAS、PKR及其mRNA水平有关.     

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.     

In vivo protein stabilization based on fragment complementation and a split GFP system

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.     

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.     

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.     

Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm

Thermodynamic stabilities are pivotal for understanding structure–function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure–stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.     

Folding without charges

Surface charges of proteins have in several cases been found to function as “structural gatekeepers,” which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.     

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.     

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.     

Pancreatic stone protein – sepsis and the riddles of the exocrine pancreas

Pancreatic stone protein (PSP), discovered in the 1970ies, was first associated with stone formation during chronic pancreatitis. Later, the same protein was independently detected in islet preparations and named regenerating protein 1 (REG1). Additional isoforms of PSP, including pancreatitis-associated protein (PAP), belong to the same protein family. Although the names indicate a potential function in stone formation or islet regeneration, involvements in cellular processes were only suggestive and never unequivocally proven. We established an association between PSP levels in patient blood samples and the development of sepsis. In this review, written in connection with receiving the Lifetime Achievement Award of the European Pancreatic Club, the evolution of the sepsis aspect of PSP is described. We conclude that the true functional properties of this fascinating pancreatic protein still remain an enigma.     

Computing protein stabilities from their chain lengths

New amino acid sequences of proteins are being learned at a rapid rate, thanks to modern genomics. The native structures and functions of those proteins can often be inferred using bioinformatics methods. We show here that it is also possible to infer the stabilities and thermal folding properties of proteins, given only simple genomics information: the chain length and the numbers of charged side chains. In particular, our model predicts ΔH(T), ΔS(T), ΔC_p, and ΔF(T) —the folding enthalpy, entropy, heat capacity, and free energy—as functions of temperature T; the denaturant m values in guanidine and urea; the pH-temperature-salt phase diagrams, and the energy of confinement F(s) of the protein inside a cavity of radius s. All combinations of these phase equilibria can also then be computed from that information. As one illustration, we compute the pH and salt conditions that would denature a protein inside a small confined cavity. Because the model is analytical, it is computationally efficient enough that it could be used to automatically annotate whole proteomes with protein stability information.