糖化清蛋白、高敏C反应蛋白与冠心病临界病变患者冠脉斑块形态学特征的关系及对功能性心肌缺血的预测研究

摘要 目的：分析糖化清蛋白、高敏C反应蛋白与冠心病临界病变患者冠脉斑块形态学特征的关系及对功能性心肌缺血的预测价值。方法：选择自2020年1月至2022年6月我院经冠脉造影确诊的165例冠心病临界病变患者作为研究对象,分为不稳定型心绞痛组和稳定型心绞痛组。检测两组血清糖化清蛋白、高敏C反应蛋白表达水平,使用靶血管造影检测冠脉斑块形态学指标,Pearson相关性分析血清糖化清蛋白、高敏C反应蛋白与冠脉斑块形态学指标的关系,通过ROC曲线下面积（AUC）评价血清糖化清蛋白联合高敏C反应蛋白对功能性心肌缺血的预测价值。结果：不稳定型心绞痛组血清糖化清蛋白、高敏C反应蛋白表达水平均高于稳定型心绞痛组（P＜0.05）;不稳定型心绞痛组最小管腔直径、最小管腔面积均小于稳定型心绞痛组,直径狭窄率、管腔面积狭窄率、斑块面积均大于稳定型心绞痛组（P＜0.05）;在165例冠心病临界病变患者中,发生冠脉易损斑块53例;易损斑块组血清糖化清蛋白、高敏C反应蛋白表达水平均高于非易损斑块组（P＜0.05）;经Pearson相关性分析,冠心病临界病变患者血清糖化清蛋白、高敏C反应蛋白表达水平均与最小管腔直径、最小管腔面积呈负相关,与直径狭窄率、管腔面积狭窄率、斑块面积呈正相关（P＜0.05）;经ROC曲线分析,血清糖化清蛋白联合高敏C反应蛋白预测冠心病临界病变患者发生功能性心肌缺血的AUC为0.910。结论：糖化清蛋白、高敏C反应蛋白与冠心病临界病变患者冠脉斑块形态学特征密切相关,有助于评估冠脉斑块易损性,联合预测功能性心肌缺血的效能较好,值得临床予以重视应用。     

血清ESM-1、VE-Cad、CC16水平与脓毒症并发急性呼吸窘迫综合征患者炎性因子及预后的关系研究

摘要 目的：探讨血清内皮细胞特异性分子-1（ESM-1）、血管内皮钙黏蛋白（VE-Cad）、Clara细胞分泌蛋白16（CC16）水平与脓毒症并发急性呼吸窘迫综合征（ARDS）患者炎性因子及预后的关系。方法：选取我院2018年10月～2020年1月收治的59例脓毒症并发ARDS患者为研究对象,记作观察组。另取同期于我院进行体检的60例健康志愿者作为对照组。比较两组血清ESM-1、VE-Cad、CC16及炎症因子水平,并以Pearson相关性分析血清ESM-1、VE-Cad、CC16与炎症因子的相关性。此外,将观察组患者按照预后分作死亡组27例和存活组32例,通过多因素Logistic回归分析脓毒症并发ARDS患者预后的影响因素。结果：观察组的血清ESM-1、VE-Cad、CC16水平均高于对照组（P＜0.05）。观察组的IL-6、CRP、PCT水平均高于对照组（P＜0.05）。Pearson相关性分析结果显示：脓毒症并发ARDS患者的血清ESM-1、VE-Cad、CC16水平与IL-6、CRP、PCT水平均呈正相关（P＜0.05）。多因素Logistic回归分析结果显示：年龄（较大）、入院24 h内APACHEⅡ评分（较高）以及血清ESM-1（升高）、VE-Cad（升高）、CC16（升高）、IL-6（升高）、CRP（升高）、PCT（升高）均是脓毒症并发ARDS患者死亡的危险因素,而氧合指数（升高）是患者死亡的保护因素（均P＜0.05）。结论：脓毒症并发ARDS患者血清ESM-1、VE-Cad、CC16水平存在明显升高,且与炎性因子以及预后密切相关,可作为脓毒症并发ARDS患者预后的辅助评估指标。     

血清同型半胱氨酸、叶酸、维生素B12水平与冠状动脉病变严重程度的相关性分析

摘要 目的：探讨血清同型半胱氨酸(homocysteine, Hcy)、叶酸、维生素B12水平与冠状动脉病变严重程度的相关性。方法：选取经冠脉造影检查确诊的稳定期冠心病患者220例为研究组,并以同期健康查体志愿者100例为对照组。检测和比较两组血清Hcy、叶酸、维生素B12和N -末端脑钠肽前体(N-terminal brain natriuretic peptide precursor,NT-proBNP)水平。研究组根据冠脉造影情况进行SYNTAX评分评价,通过心脏超声检查检测左室射血分数(left ventricular ejection fraction,LVEF),确定冠脉病变严重程度。比较研究组SYNTAX低分组(1～22分)、中分组(23～32分)和高分组(≥33分)患者上述各指标水平,并分析研究组血清Hcy、叶酸、维生素B12水平与其血清NT-proBNP 水平、SYNTAX评分和LVEF的关系。结果：与对照组比较,研究组血清Hcy和NT-proBNP水平升高而血清叶酸、维生素B12水平降低(P＜0.05)。研究组SYNTAX评分和LVEF分别为(28.76±6.58)分和(47.33±8.66)%,SYNTAX中分和高分患者血清Hcy和NT-proBNP水平高于SYNTAX低分患者而血清叶酸、维生素B12水平和LVEF则低于SYNTAX低分患者,SYNTAX高分患者血清Hcy和NT-proBNP水平高于SYNTAX中分患者而血清叶酸、维生素B12水平和LVEF则低于SYNTAX中分患者(P＜0.05)。Pearson线性相关分析结果显示研究组血清Hcy水平与其血清NT-proBNP 水平、SYNTAX评分均呈正相关(r=0.881,0.793,P＜0.05),与其LVEF则呈负相关(r=-0.876,P＜0.05);而其血清叶酸、维生素B12水平与其血清NT-proBNP 水平、SYNTAX评分均呈负相关(叶酸：r=-0.786,-0.825;维生素B12：r=-0.884,-0.818,P＜0.05),与其LVEF则呈正相关(r=0.893,0.859,P＜0.05)。结论：血清Hcy是冠心病的重要危险因素,其水平随着冠状动脉病变程度加重而升高;血清叶酸、维生素B12是冠心病的保护因素,其水平随着冠状动脉病变程度加重而降低。     

急性缺血性脑卒中患者血清RBP4、Lp-PLA2及MCP-1水平表达及其与患者病情及预后的关系研究

摘要 目的：探究急性缺血性脑卒中（AIS）患者血清视黄醇结合蛋白4（RBP4）、脂蛋白磷脂酶A2（Lp-PLA2）及单核细胞趋化蛋白-1（MCP-1）水平表达及其与患者病情及预后的关系。方法：选取2020年6月～2021年12月我院收治的130例初发AIS患者作为研究对象,根据美国国立卫生研究院卒中量表（NIHSS）评分分为轻度组、中度组、重度组,另取同期在我院体检的志愿者45例作为对照组。采用酶联免疫吸附测定（ELLSA）法检测并对比各组血清RBP4、Lp-PLA2、MCP-1水平差异。改良Rankin量表（MRS）评估AIS患者入院14 d预后,比较不同预后患者临床资料差异。采用Pearson相关系数分析血清RBP4、Lp-PLA2、MCP-1水平与NIHSS、MRS评分的相关性。受试者工作特征（ROC）曲线分析血清RBP4、Lp-PLA2、MCP-1对AIS患者预后的预测价值。结果：AIS患者的血清RBP4、Lp-PLA2、MCP-1水平均明显高于对照组,且随着病情程度的加重,血清RBP4、Lp-PLA2、MCP-1水平逐渐升高（P＜0.05）。预后不良组年龄、血清RBP4、Lp-PLA2、MCP-1水平以及NIHSS评分均明显高于预后良好组（P＜0.05）。AIS患者的血清RBP4、Lp-PLA2、MCP-1水平与NIHSS、MRS评分之间均呈正相关（P＜0.05）。血清RBP4、Lp-PLA2、MCP-1三项联合检测预测AIS患者预后的ROC曲线下面积为0.957,明显高于各指标单独检测的0.775、0.799、0.781。结论：AIS患者血清RBP4、Lp-PLA2、MCP-1水平显著升高,升高程度与患者病情及预后密切相关,三项指标联合检测对AIS患者预后具有良好的预测价值。     

血清CTRP4、CTRP5、CTRP6与2型糖尿病合并冠心病患者冠状动脉病变的关系研究

摘要 目的：探讨血清补体1q/肿瘤坏死因子相关蛋白（CTRP）4、CTRP5、CTRP6与2型糖尿病（T2DM）合并冠心病（CHD）患者冠状动脉病变的关系。方法：选取2021年3月～2021年7月我院收治的100例T2DM合并CHD患者为合并组,根据SYNTAX评分分为中重度病变组44例和轻度病变组56例,选取同期收治的100例单纯T2DM患者为T2DM组,另选取同期体检健康志愿者80名为对照组。采用酶联免疫吸附法检测血清CTRP4、CTRP5、CTRP6水平。采用Spearman相关性分析T2DM合并CHD患者SYNTAX评分与血清CTRP4、CTRP5、CTRP6水平的相关性,多因素Logistic回归分析T2DM合并CHD患者冠状动脉中重度病变的影响因素。结果：对照组、T2DM组、合并组血清CTRP4、CTRP5水平依次升高,CTRP6水平依次降低（P＜0.05）。Spearman相关性分析显示,T2DM合并CHD患者SYNTAX评分与血清CTRP4、CTRP5水平呈正相关（rs=0.820、0.833,P均＜0.001）,与CTRP6水平呈负相关（rs=-0.838,P＜0.001）。多因素Logistic回归分析显示,T2DM病程延长和糖化血红白蛋白（HbA1c）、CTRP4、CTRP5升高为T2DM合并CHD患者冠状动脉中重度病变的独立危险因素,CTRP6升高为独立保护因素（P＜0.05）。结论：血清CTRP4、CTRP5水平升高和CTRP6水平降低与T2DM合并CHD患者冠状动脉病变加重独立相关,可能成为T2DM合并CHD患者冠状动脉病变评估指标。     

多囊卵巢综合征患者血清IMA、HIF1α、Vaspin、IGF-1水平与性激素、糖脂代谢及胰岛素抵抗的关系研究

摘要 目的：探讨多囊卵巢综合征（PCOS）患者血清缺血修饰白蛋白（IMA）、缺氧诱导因子1α（HIF1α）、内脏脂肪特异性丝氨酸蛋白酶抑制剂（Vaspin）、胰岛素样生长因子-1（IGF-1）水平与性激素、糖脂代谢及胰岛素抵抗的相关性。方法：选择2017年1月至2019年12月我院收治的268例PCOS患者（PCOS组）,根据体质量指数（BMI）将PCOS患者分为超重/肥胖组（BMI≥24 kg/m²,161例）和正常体重组（18.5 kg/m2≤BMI＜24 kg/m²,107例）,另选择135例于妇科门诊体检的健康女性志愿者为对照组。检测血清IMA、HIF1α、Vaspin、IGF-1水平以及性激素、糖脂代谢、胰岛素抵抗指标,Pearson相关性分析其相关性。结果：PCOS组血清IMA、HIF1α、Vaspin、IGF-1、睾酮（T）、促黄体生成素(LH)、促卵泡激素（FSH）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）、空腹血糖（FPG）、餐后2小时血糖（2hPG）、空腹胰岛素（FINS）水平,胰岛素抵抗指数（HOMA-IR）均高于对照组（P＜0.001）。PCOS患者超重/肥胖组血清IMA、HIF1α、Vaspin、IGF-1、T、TC、LDL-C、FPG、FINS、2hPG水平,HOMA-IR均高于正常体重组（P＜0.001）。PCOS患者血清IMA、HIF1α水平与T、HOMA-IR呈正相关（P＜0.05）,血清Vaspin、IGF-1水平与TC、LDL-C、FPG、FINS、2hPG 、HOMA-IR呈正相关（P＜0.05）。结论：PCOS患者血清IMA、HIF-1α、Vaspin、IGF-1水平均升高,IMA、HIF-1α、Vaspin、IGF-1均与PCOS胰岛素抵抗有关。     

lp-PLA2、RBP与冠心病病变程度相关性及其疾病诱发的危险因素分析

摘要 目的：研究脂蛋白磷脂酶A2（lp-PLA2）、视黄醇结合蛋白（RBP）与冠心病（CAD）病变程度的相关性及冠心病病变发生的危险因素。方法：以2019年12月至2021年12月在本院诊治的冠心病患者140例作为研究对象,所有患者均进行冠状动脉造影检查,并根据冠状动脉侧支循环形成情况进行分级。所有患者都给予血清lp-PLA2、RBP检测并进行相关性、危险因素分析。结果：在140例患者中,冠状动脉侧支循环未形成40例（对照组）,冠状动脉侧支循环形成100例（研究组）,研究组中Ⅰ级40例,Ⅱ级38例,Ⅲ级22例。研究组的血清lp-PLA2、RBP含量都高于对照组（P<0.05）;不同分级患者的血清lp-PLA2、RBP含量对比也有明显差异（P<0.05）。在冠心病患者中,Spearsman相关分析显示血清lp-PLA2、RBP含量与侧支循环形成分级存在正相关性（P<0.05）。logistic回归分析显示血清lp-PLA2、RBP含量均为影响冠心病患者侧支循环形成分级的危险因素（P<0.05）。ROC曲线分析显示血清lp-PLA2、RBP含量预测冠心病患者侧支循环分级的曲线下面积为0.891、0.805。结论：随着冠状动脉侧支循环形成,冠心病患者的血清lp-PLA2、RBP含量明显增加,lp-PLA2、RBP与侧支循环形成分级存在相关性,也是影响侧支循环分级的危险因素,也可预测侧支循环分级状况。     

不同分期慢性肾脏病患者血清Klotho蛋白、FGF-23、RBP4、TWEAK水平与肾功能的相关性研究

摘要 目的：探究不同分期慢性肾脏病(CKD)患者血清Klotho蛋白、成纤维细胞生长因子-23(FGF-23)、视黄醇结合蛋白4(RBP4)、肿瘤坏死因子样弱凋亡诱导因子(TWEAK)水平与肾功能的相关性。方法：选择2018年2月至2020年2月我院诊治的80例CKD患者作为CKD组,选择同期在我院进行健康体检的80名健康者作为对照组。检测并比较对照组和CKD组以及不同CKD分期患者血清Klotho蛋白、FGF-23、RBP4、TWEAK水平及肾功能指标水平,采用Pearson相关性分析对血清Klotho蛋白、FGF-23、RBP4、TWEAK水平与肾功能指标的相关性进行分析。结果：与对照组相比,CKD组血清Klotho蛋白、TWEAK水平和肾小球滤过率（GFR）明显下降,而血清FGF-23、RBP4水平、血清肌酐、24尿蛋白定量和血清尿素氮水平明显升高（P<0.05）。Ⅰ～Ⅱ期患者的血清Klotho蛋白、TWEAK水平和GFR最高,其次为Ⅲ～Ⅳ期患者,Ⅴ期患者最低（P<0.05）。而Ⅰ～Ⅱ期患者血清FGF-23、RBP4水平、血清肌酐、24尿蛋白定量和血清尿素氮水平最低,其次为Ⅲ～Ⅳ期患者,Ⅴ期患者最高（P<0.05）。Klotho蛋白和TWEAK水平与血清肌酐、24尿蛋白定量和血清尿素氮水平呈负相关,与GFR呈正相关（P<0.05）。FGF-23和RBP4水平与血清肌酐、24尿蛋白定量和血清尿素氮水平呈正相关,与GFR呈负相关（P<0.05）。结论：CKD患者中血清Klotho蛋白、TWEAK水平以及肾功能下降,FGF-23、RBP4水平明显升高,且血清Klotho蛋白、TWEAK、FGF-23、RBP4水平与肾功能及CKD分期相关。     

一氧化氮、血清尾加压素Ⅱ与老年稳定型心绞痛患者冠脉粥样硬化斑块的关系及对功能性心肌缺血的预测

摘要 目的：探讨一氧化氮（NO）、血清尾加压素Ⅱ（UⅡ）与老年稳定型心绞痛患者冠脉粥样硬化斑块的关系及对功能性心肌缺血的预测。方法：选取我院2020年10月到2022年12月收治的120例老年稳定型心绞痛患者作为研究对象,回顾性分析所有患者CT造影诊断结果,依照动脉狭窄程度将患者分为不稳定斑块组（n=35）,稳定斑块组（n=46）和无斑块组（n=39）。对比三组患者NO、UⅡ表达水平,并分析其与老年稳定型心绞痛患者冠脉粥样硬化斑块的相关性。所有患者均采取保守治疗,将治疗后出现功能性心肌缺血的40例患者分为心肌缺血组,将其余80例患者分为非心肌缺血组,对比两组患者临床一般情况和NO、UⅡ,并分析NO、UⅡ对功能性心肌缺血的预测价值。结果：不稳定斑块组NO低于稳定斑块组和无斑块组、UⅡ水平高于稳定斑块组和无斑块组（P＜0.05）,且稳定斑块组与无斑块组对比差异显著（P＜0.05）;Spearman相关分析结果显示：NO、UⅡ与老年稳定型心绞痛患者冠脉粥样硬化斑块稳定程度具有相关性（P<0.05）;心肌缺血组和非心肌缺血组患者性别、年龄、BMI、合并糖尿病、高血压、左心室射血分数、高脂血症情况对比无明显差异（P＞0.05）,心肌缺血组和非心肌缺血组患者心功能分级、合并陈旧性心肌梗死、NO、UⅡ水平对比差异显著（P＜0.05）;最终logistic回归分析结果显示：NO、UⅡ升高是老年稳定型心绞痛患者功能性心肌缺血的独立影响因素（P＜0.05）。结论：NO、UⅡ与老年稳定型心绞痛患者冠脉粥样硬化斑块稳定程度具有明显关系,且通过NO、UⅡ水平可预测患者功能性心肌缺血的发生,因此临床上对于NO、UⅡ升高的老年稳定型心绞痛患者需及时调整治疗措施,进一步预防患者治疗后出现的功能性心肌缺血现象。     

免疫性不孕患者全血Cu、Zn、Se水平及与炎性因子相关性研究

摘要 目的：考察免疫性不孕患者全血微量元素铜（Cu）、锌（Zn）、硒（Se）水平和血清炎性因子白细胞介素（Interleukin,IL）-6、IL-8、肿瘤坏死因子-α(Tumor Necrosis Factor-α,TNF-α)水平,明确微量元素、炎性因子与免疫性不孕的关系。方法：回顾性选择40例免疫性不孕患者作为研究组,另以同期40名健康者作为对照。对两组全血微量元素Cu、Zn、Se进行检测,以酶联免疫法试剂盒检测血清IL-6、IL-8以及TNF-α,并对Cu、Zn、Se水平和IL-6、IL-8、TNF-α与免疫性不孕的相关性进行分析。结果：研究组的全血Cu水平显著高于对照组,而Zn、Se水平显著低于对照组(P＜0.05),即免疫性不孕患者全血微量元素Cu、Zn和Se水平异常,其中Cu异常增高,而Zn和Se异常降低;研究组患者的IL-6、IL-8和TNF-α均显著高于对照组健康女性(P＜0.05),即免疫性不孕患者血清存在炎性因子的异常增高;全血微量元素Cu、Zn、Se和血清炎性因子IL-6、IL-8、TNF-α均与免疫性不孕密切相关（P＜0.05)。结论：免疫性不孕患者全血微量元素Cu和Zn水平异常,血清炎性因子水平异常,微量元素和炎性因均与免疫性不孕密切相关。     

Protein misfolding, aggregation, and degradation in disease

Pathologies associated with protein misfolding have been observed in neurodegenerative diseases such as Alzheimer’s disease, metabolic diseases like phenylketonuria, and diseases affecting structural proteins like collagen or keratin. Misfolding of mutant proteins in these and many other diseases may result in premature degradation, formation of toxic aggregates, or incorporation of toxic conformations into structures. We review common traits of these diverse diseases under the unifying view of protein misfolding. The molecular pathogenesis is discussed in the context of protein quality control systems consisting of molecular chaperones and intracellular proteases that assist the folding and supervise the maintenance of the folded structure. Furthermore, genetic and environmental factors that may modify the severity of these diseases are underscored. The present article represents a partly revised and updated version of chapter 1 published earlier in volume 232 of the series Methods in Molecular Biology (Walker, J. M., ed., Humana Press, Totowa, NJ), Protein Misfolding and Disease: Principles and Protocols (Bross, P. & Gregersen, N., eds.), pp. 3–16 (2003).     

Protein design on computers. Five new proteins: Shpilka, Grendel, Fingerclasp, Leather, and Aida.

What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded β-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Fingerclasp, a dimer of interdigitating β–β–α units, the simplest variant of the “handshake” structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather—a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

NHERFs, NEP, MAGUKs, and more: interactions that regulate PTEN

This year marks the 10th anniversary of the discovery of the PTEN/MMAC1/TEP1 tumor suppressor gene (hereafter referred to as PTEN), one of the most commonly mutated genes in cancer. PTEN encodes a lipid phosphatase that dephosphorylates phosphoinositide-3,4,5-triphosphate (PIP(3)), thereby counteracting mitogenic signaling pathways driven by phosphoinositol-3-kinases (PI3K). By opposing PI3K signaling, PTEN inhibits the activation of the critical PI3K effector proteins Akt1-3 (also known as protein kinase B or PKB). Given its central role in antagonizing PI3K signaling, one might expect that like PI3K, the activity of the PTEN protein would be highly regulated by numerous protein/protein interactions. However, surprisingly little is known about such interactions. This fact, combined with the generally accepted notion that phosphatases are less exquisitely regulated than kinases, has led to the idea that PTEN may function in a relatively unregulated fashion. Here we review the identities and proposed functions of known PTEN-interacting proteins, and point out avenues of investigation that we hope may be fruitful in identifying important new mechanisms of PTEN regulation in mammalian cells.     

Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. "Deep etch" EM of purified COG revealed an approximately 37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Structures, basins, and energies: a deconstruction of the Protein Coil Library

Globular proteins adopt complex folds, composed of organized assemblies of alpha-helix and beta-sheet together with irregular regions that interconnect these scaffold elements. Here, we seek to parse the irregular regions into their structural constituents and to rationalize their formative energetics. Toward this end, we dissected the Protein Coil Library, a structural database of protein segments that are neither alpha-helix nor beta-strand, extracted from high-resolution protein structures. The backbone dihedral angles of residues from coil library segments are distributed indiscriminately across the phi,psi map, but when contoured, seven distinct basins emerge clearly. The structures and energetics associated with the two least-studied basins are the primary focus of this article. Specifically, the structural motifs associated with these basins were characterized in detail and then assessed in simple simulations designed to capture their energetic determinants. It is found that conformational constraints imposed by excluded volume and hydrogen bonding are sufficient to reproduce the observed ,psi distributions of these motifs; no additional energy terms are required. These three motifs in conjunction with alpha-helices, strands of beta-sheet, canonical beta-turns, and polyproline II conformers comprise approximately 90% of all protein structure.