慢性牙周炎伴咬合创伤患者龈沟液Sclerostin、SLIT3与牙周临床指标和RANKL-OPG系统的相关性研究

摘要 目的：探讨慢性牙周炎伴咬合创伤患者龈沟液骨硬化蛋白（Sclerostin）、神经轴突导向因子3（SLIT3）与牙周临床指标和核因子-κb受体活化因子配体（RANKL）-骨保护蛋白（OPG）系统的相关性。方法：选择2021年1月至2023年1月我院收治的123例慢性牙周炎伴咬合创伤患者（创伤组）和101例不伴咬合创伤的慢性牙周炎患者（对照组）。治疗前,检测龈沟液中Sclerostin、SLIT3、RANKL、OPG水平,计算RANKL/OPG比值,评估临床牙周指标出血指数（SBI）、菌斑指数（PLI）、探诊深度（PD）、附着丧失（AL）。分析Sclerostin、SLIT3与牙周临床指标、RANKL、OPG、RANKL/OPG之间的相关性。结果：创伤组治疗前龈沟液中Sclerostin、SLIT3、RANKL水平,RANKL/OPG,PLI、SBI、AL、PD高于对照组（P＜0.05）,OPG低于对照组（P＜0.05）。创伤组治疗前龈沟液中Sclerostin、SLIT3与PLI、SBI、AL、PD、RANKL、RANKL/OPG呈正相关（P＜0.05）,与OPG呈负相关（P＜0.05）。结论：慢性牙周炎伴咬合创伤患者龈沟液中Sclerostin、SLIT3水平显著增高,且与牙周临床指标异常、RANKL/OPG增加有关。     

牙周基础治疗对慢性牙周炎伴冠心病患者血清炎症因子、龈下菌群和龈沟液中自噬因子的影响

摘要 目的：观察牙周基础治疗对慢性牙周炎伴冠心病患者血清炎症因子、龈下菌群和龈沟液中自噬因子的影响。方法：选择成都市第三人民医院口腔科2020年3月~2022年2月期间收治的慢性牙周炎伴冠心病患者83例。按照随机数字表法将患者分为对照组（n=41）和研究组（n=42）,对照组接受常规内科维持治疗,研究组在对照组的基础上接受牙周基础治疗。对比两组牙周临床指标、血清炎症因子、龈下菌群、龈沟液中自噬因子的变化情况。结果：治疗3个月后,研究组牙龈指数（GI）、牙周附着丧失（AL）、牙周袋深度（PD）均低于对照组（P<0.05）。治疗3个月后,研究组C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）、白介素（IL）-1β、IL-6、IL-8、IL-17、IL-18低于对照组,IL-10高于对照组（P<0.05）。治疗3个月后,研究组牙龈卟啉单胞菌、伴放线菌嗜血菌、中间普雷沃菌、福赛斯坦纳菌、齿垢密螺旋体相对含量均低于对照组（P<0.05）。治疗3个月后,研究组微管相关蛋白1轻链3（LC3）、Beclin-1均低于对照组（P<0.05）。结论：牙周基础治疗可有效调节慢性牙周炎伴冠心病患者血清炎症因子水平,改善龈下菌群,降低龈沟液中自噬因子水平。     

二甲胺四环素联合茶多酚对早期种植体周围软组织炎患者牙周指标、炎性因子以及龈沟液RANKL、Shh水平的影响

摘要 目的：探讨二甲胺四环素联合茶多酚对早期种植体周围软组织炎患者牙周指标、炎性因子以及龈沟液核因子-?资?茁受体活化因子配体（RANKL）、音猬因子（Shh）水平的影响。方法：将106例于我院就诊的早期种植体周围软组织炎患者纳入研究,以信封抽签法分为对照组（n=53,二甲胺四环素治疗）和研究组（n=53,二甲胺四环素联合茶多酚治疗）。对比两组患者治疗前、治疗4周后的牙周指标、炎性因子、疗效以及龈沟液RANKL、Shh水平,记录两组治疗期间不良反应情况。结果：治疗4周后研究组临床总有效率90.57%（48/53）高于对照组73.58%（39/53）（P<0.05）。两组不良反应发生率对比无差异（P>0.05）。两组治疗4周后改良菌斑指数（mPLI）、改良龈沟出血指数（mSBI）、种植体周袋探诊深度（PPD）和血清白介素-1β（IL-1β）、单核细胞趋化蛋白-1(MCP-1)水平以及龈沟液RANKL、Shh水平均下降,且研究组低于对照组（P<0.05）。结论：二甲胺四环素联合茶多酚治疗早期种植体周围软组织炎患者,疗效显著,可有效改善患者牙周情况,降低患者血清IL-1β、MCP-1水平以及龈沟液RANKL、Shh水平,安全可靠。     

龈沟液CRP、IL-17、RANKL与种植体周围炎患者牙周临床指标的相关性分析及其联合检测对治疗后预后的评估价值

摘要 目的：分析龈沟液C反应蛋白（CRP）、白细胞介素-17（IL-17）及核因子-kB受体活化因子配体（RANKL）与种植体周围炎（PI）患者牙周临床指标的相关性及探讨其联合检测对治疗后预后的评估价值。方法：选取2018年1月～2022年1月我院收治的80例PI患者,记作炎症组。另取同期种植体健康患者80例作为健康组。检测并比较两组龈沟液CRP、IL-17及RANKL水平,牙周临床指标。并以Pearson相关性分析龈沟液CRP、IL-17及RANKL与牙周临床指标的相关性。此外,将所有PI患者按照急性生理学与慢性健康状况评分Ⅱ（APACHE Ⅱ）评分的差异分为预后良好组27例与预后不良组53例,比较两组龈沟液CRP、IL-17及RANKL水平。通过受试者工作特征（ROC）曲线分析上述三项指标联合预测PI患者治疗后预后不良的效能。结果：炎症组龈沟液CRP、IL-17及RANKL水平均高于健康组（均P＜0.05）。炎症组改良牙龈指数（mGI）、改良龈沟出血指数（mSBI）评分以及探诊深度（PD）均高于健康组（均P＜0.05）。经Pearson相关性分析发现,龈沟液CRP、IL-17及RANKL水平与mGI、mSBI评分及PD均呈正相关关系（均P＜0.05）。预后不良组龈沟液CRP、IL-17及RANKL水平均高于预后良好组（均P＜0.05）。经ROC曲线分析发现,龈沟液CRP、IL-17及RANKL联合预测PI患者治疗后预后不良的曲线下面积（0.95CI）为0.851（0.752～0.944）,优于上述三项指标单独预测。结论：龈沟液CRP、IL-17及RANKL与PI患者mGI、mSBI评分以及PD均密切相关,检测三指标有助于评估患者病情及治疗后预后。     

慢性牙周炎患者牙龈卟啉单胞菌感染与血清HMGB1、IL-1β、IL-6以及牙周临床指标的相关性研究

摘要 目的：研究慢性牙周炎患者牙龈卟啉单胞菌（Pg）感染与血清高迁移率族蛋白1（HMGB1）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）以及牙周临床指标的相关性。方法：将2019年1月～2020年12月在长沙市第三医院口腔科接受诊治的300例慢性牙周炎患者纳入研究,按照慢性牙周炎严重程度分成轻度组169例、中度组92例、重度组39例。对所有受试者进行Pg检测,同时检测血清HMGB1、IL-1β、IL-6水平和牙周临床指标水平。比较各组上述各项指标的差异,并以Pearson相关性分析慢性牙周炎患者Pg感染与血清HMGB1、IL-1β、IL-6水平以及牙周临床指标的相关性。结果：重度组和中度组的Pg感染阳性率、Pg感染浓度均高于轻度组,且重度组上述指标高于中度组（P＜0.05）。重度组和中度组的血清HMGB1、IL-1β、IL-6水平均高于轻度组,且重度组上述指标高于中度组（P＜0.05）。重度组和中度组的探诊深度（PD）、龈沟出血指数（SBI）以及附着丧失（AL）均高于轻度组,且重度组上述指标高于中度组（P＜0.05）。Pearson相关性分析显示：慢性牙周炎患者的Pg感染浓度与血清HMGB1、IL-1β、IL-6水平以及PD、SBI、AL均呈正相关（P＜0.05)。结论：慢性牙周炎患者的Pg感染浓度与血清HMGB1、IL-1β、IL-6水平及牙周健康状况密切相关,早期明确Pg感染浓度以及检测上述血清学指标水平,可能对抑制病程进展以及提高治疗效果具有指导意义。     

2型糖尿病伴慢性牙周炎患者龈沟液Omentin-1、MMP-9、OPG/RANKL比值与牙周指标、氧化应激和NLRP3炎症小体的关系

摘要 目的：探讨2型糖尿病（T2DM）伴慢性牙周炎（CP）患者龈沟液网膜素-1（Omentin-1）、基质金属蛋白酶-9（MMP-9）、骨保护素（OPG）/细胞核因子κB受体活化因子配体（RANKL）比值与牙周指标、氧化应激和核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎症小体的关系。方法：选择2020年6月至2022年6月首都医科大学附属北京康复医院收治的73例T2DM患者（T2DM组）,77例CP患者（CP组）,83例T2DM伴CP患者（T2DM伴CP组）。检测所有患者龈沟液中Omentin-1、MMP-9、OPG/RANKL比值,分析其与牙周指标、氧化应激和NLRP3炎症小体相关分子信使核糖核酸（mRNA）表达的相关性。结果：T2DM伴CP组龈沟液中Omentin-1,OPG/RANKL比值、总抗氧化能力（TAC）、超氧化物歧化酶（SOD）低于T2DM组和CP组（P＜0.05）,MMP-9、丙二醛（MDA）、NLRP3mRNA、凋亡相关斑点样蛋白（ASC）mRNA、半胱氨酸蛋白酶-1（caspase-1）mRNA表达以及出血指数（SBI）、菌斑指数（PLI）、牙周袋探诊深度（PD）、附着丧失（AL）高于T2DM组和CP组（P＜0.05）。T2DM伴CP患者龈沟液中Omentin-1、OPG/RANKL比值与TAC、SOD呈正相关（P＜0.05）,与MDA、NLRP3mRNA、ASC mRNA、caspase-1mRNA表达以及PLI、SBI、AL、PD呈负相关（P＜0.05）,MMP-9与TAC、SOD呈负相关（P＜0.05）,与MDA、NLRP3mRNA、ASC mRNA、caspase-1mRNA表达以及PLI、SBI、AL、PD呈正相关（P＜0.05）。结论：T2DM伴CP患者龈沟液中Omentin-1水平、OPG/ RANKL比值降低,MMP-9水平升高,与牙周组织破坏加重、氧化应激、NLRP3炎症小体激活有关。     

活动期溃疡性结肠炎血清S100A2、S100A12与炎症因子的相关性分析及对复发的预测价值

摘要 目的：探讨活动期溃疡性结肠炎（UC）血清S100钙结合蛋白（S100）A2、S100A12与炎症因子的相关性分析及对复发的预测价值。方法：选取2019年1月～2021年1月我院收治的102例活动期UC患者为活动期组,根据2年后是否复发分为复发组和未复发组,另选取同期来院复查的50名缓解期UC患者为缓解期组,50名体检健康者为对照组。检测并比较三组血清S100A2、S100A12与炎症因子[C反应蛋白（CRP）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）]水平。采用Pearson/Spearman相关性分析活动期UC患者血清S100A2、S100A12与炎症因子水平的相关性,单因素及多因素Logistic回归分析活动期UC复发的影响因素,受试者工作特征曲线分析血清S100A2、S100A12水平对活动期UC复发的预测价值。结果：对照组、缓解期组、活动期组血清S100A2、S100A12、CRP、IL-6、TNF-α水平依次升高（P＜0.05）。活动期UC患者血清S100A2、S100A12与CRP、IL-6、TNF-α水平均呈正相关（P均＜0.001）。随访2年,102例活动期UC患者复发率为56.86%（58/102）。单因素分析显示,病情严重程度、CRP、IL-6、TNF-α、S100A2、S100A12为活动期UC患者复发的影响因素（P＜0.05）。多因素Logistic回归分析显示,病情严重程度为重度和CRP、IL-6、TNF-α、S100A2、S100A12升高为活动期UC患者复发的独立危险因素（P＜0.05）。血清S100A2、S100A12水平单独和联合预测活动期UC复发的曲线下面积分别为0.782、0.784、0.876,两指标联合预测活动期UC患者复发的预测价值大于各指标单独预测。结论：活动期UC患者血清S100A2、S100A12与炎症因子水平和复发密切相关,血清S100A2、S100A12联合预测活动期UC患者复发的价值较高。     

不同正畸治疗措施对正畸患者牙齿移动距离和龈沟液TNF-α、IL-2、HGF及PTX3的水平的影响

摘要 目的：探讨不同正畸治疗对正畸患者牙齿移动距离以及龈沟液中肿瘤坏死因子-α（TNF-α）、白介素-2（IL-2）、肝细胞生长因子（HGF）、正五聚蛋白3（PTX3）水平的影响。方法：选择2016年7月至2019年12月我院收治的288例错颌畸形患者,按随机数字表法将患者分为四组,每组各72例。所有患者均采用方丝弓矫治器治疗,分别给予50 g（A组）、100 g （B组）、150 g（C组）、250 g（D组）的远中正畸力。观察四组正畸后1 d、3 d、1周、2周、3周、4周牙齿移动距离,以及正畸前、后1 d、3 d、1周、2周、3周、4周龈沟液中TNF-α、IL-2、HGF、PTX3水平变化和组间差异。结果：四组患者正畸后1 d、3 d、1周、2周、3周、4周牙位移距离先延长后缩短,然后再次延长后逐渐恢复基线水平（P＜0.05）。四组患者正畸后1 d、3 d、1周、2周、3周、4周TNF-α、IL-2、HGF、PTX3出现先升高后降低,再次升高后逐渐恢复基线水平趋势（P＜0.05）,组间牙齿位移距离、TNF-α、IL-2、HGF、PTX3与时间存在交互效应（P＜0.05）,B组正畸后1 d、3 d、1周、2周、3周、4周位移距离均大于A组（P＜0.05）,C组、D组与A组比较均无统计学差异（P＞0.05）。D组正畸后1 d、3 d、1周、2周、3周、4周龈沟液TNF-α、IL-2、HGF、PTX3水平均高于A、B、C组（P＜0.05）,C组高于A组和B组（P＜0.05）,A组与B组比较均无统计学差异（P＞0.05）。结论：100 g正畸力可促使牙齿移动,保持正畸过程中适当的细胞因子水平,减少对牙周组织的破坏,有利于牙周组织恢复。     

慢性牙周炎合并2型糖尿病患者龈沟液Sirtuin-1、Sirtuin-6的变化及临床价值研究

摘要 目的：探讨慢性牙周炎（CP）合并2型糖尿病（T2DM）患者龈沟液沉默信息调节因子-1（Sirtuin-1）、Sirtuin-6的变化和临床价值。方法：选择2020年3月至2023年3月中国人民解放军联勤保障部队第九七〇医院收治的147例CP合并T2DM患者（T2DM组）,128例单纯CP患者（CP组）和121例健康体检者（对照组）。根据牙周检查结果将T2DM组患者分为轻度组（n=49）、中度组（n=67）、重度组（n=31）。检测受试者龈沟液中Sirtuin-1、Sirtuin-6水平以及外周血单核细胞核苷酸结合寡聚化结构域样受体热蛋白结构域亚家族成员3（NLRP3）信使核糖核酸（mRNA）、程序性细胞死亡相关斑点样蛋白（ASC）mRNA、半胱氨酸蛋白酶1（Caspase-1）mRNA表达,并评估牙周临床指标。Pearson分析CP合并T2DM患者龈沟液Sirtuin-1、Sirtuin-6水平与牙周临床指标、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达的相关性。受试者工作特征（ROC）曲线分析龈沟液Sirtuin-1、Sirtuin-6诊断CP合并T2DM的价值。结果：T2DM组龈沟液Sirtuin-1、Sirtuin-6水平低于CP组和对照组（P＜0.05）,出血指数（SBI）、牙周袋探诊深度（PD）、牙龈指数（GI）、菌斑指数（PLI）、附着丧失（AL）、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于CP组和对照组（P＜0.05）。CP组龈沟液Sirtuin-1、Sirtuin-6水平低于和对照组（P＜0.05）,GI、SBI、PLI、PD、AL、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于对照组（P＜0.05）。重度组龈沟液Sirtuin-1、Sirtuin-6水平低于中度组和轻度组（P＜0.05）,GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于中度组和轻度组（P＜0.05）。中度组龈沟液Sirtuin-1、Sirtuin-6水平低于轻度组（P＜0.05）,GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于轻度组（P＜0.05）。CP合并T2DM患者龈沟液Sirtuin-1、 Sirtuin-6水平与GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达均呈负相关（P＜0.05）。龈沟液Sirtuin-1、 Sirtuin-6诊断CP合并T2DM的曲线下面积（AUC）为0.787、0.806,联合诊断AUC为0.912,高于单独诊断。结论：CP合并T2DM患者龈沟液中Sirtuin-1、Sirtuin-6水平降低,且与牙周组织破坏程度加重、NLRP3炎症小体激活有关。龈沟液Sirtuin-1联合Sirtuin-6在CP合并T2DM诊断中具有较高价值。     

龈沟液miR-155、miR-223表达水平与慢性牙周炎伴2型糖尿病患者牙周临床指标、口腔龈下菌群以及Th17/Treg失衡的相关性分析

摘要 目的：探讨龈沟液miR-155、miR-223表达水平与慢性牙周炎伴2型糖尿病（T2DM）患者牙周临床指标、口腔龈下菌群以及外周血辅助性T细胞17（Th17）/调节性T细胞（Treg）失衡的相关性。方法：选择2018年1月至2022年1月安徽理工大学第一附属医院口腔科收治的86例慢性牙周炎患者,根据是否伴T2DM将患者分为慢性牙周炎伴T2DM组15例和单纯慢性牙周炎组71例,另选择65例健康体检志愿者为对照组。检测龈沟液miR-155、miR-223表达水平,口腔龈下菌群以及外周血Th17细胞占比、Treg细胞占比、血清白细胞介素（IL）-17、转化生长因子-β（TGF-β）水平。Pearson相关分析龈沟液miR-155、miR-223表达水平与牙周临床指标、口腔龈下菌群、外周血Th17/Treg以及血清IL-17、TGF-β的相关性。结果：慢性牙周炎伴T2DM组、单纯慢性牙周炎组龈沟液miR-155、miR-223表达水平高于对照组（P＜0.05）,且慢性牙周炎伴T2DM组高于单纯慢性牙周炎组（P＜0.05）。慢性牙周炎伴T2DM组龈沟出血指数（SBI）、菌斑指数（PLI）、探诊深度（PD）、附着丧失（AL）、牙龈卟啉单胞菌、二氧化碳噬纤维菌、中间普氏菌、变黑普氏菌数量、外周血Th17细胞占比、Th17/Treg比值、血清IL-17水平高于单纯慢性牙周炎组（P＜0.05）,外周血Treg细胞占比低于单纯慢性牙周炎组（P＜0.05）。龈沟液miR-155、miR-223表达水平与PLI、SBI、AL、PD、牙龈卟啉单胞菌、二氧化碳噬纤维菌、中间普氏菌、变黑普氏菌数量、外周血Th17细胞占比、Th17/Treg比值、血清IL-17水平呈正相关（P＜0.05）,与外周血Treg细胞占比呈负相关（P＜0.05）。结论：慢性牙周炎伴T2DM患者龈沟液中miR-155、miR-233表达均上调,且与牙周组织破坏程度、龈下菌群紊乱和Th17/Treg失衡有关。     

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.     

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.     

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.     

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.     

Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations

Native proteins are marginally stable. Low thermodynamic stability may actually be advantageous, although the accumulation of neutral, destabilizing mutations may have also contributed to it. In any case, once marginal stability has been reached, it appears plausible that mutations at non-constrained positions become fixed in the course of evolution (due to random drift) with frequencies that roughly reflect the mutation effects on stability ("pseudo-equilibrium hypothesis"). We have found that all glutamate-->aspartate mutations in wild-type Escherichia coli thioredoxin are destabilizing, as well as most of the aspartate-->glutamate mutations. Furthermore, the effect of these mutations on thioredoxin thermodynamic stability shows a robust correlation with the frequencies of occurrence of the involved residues in several-hundred sequence alignments derived from a BLAST search. These results provide direct and quantitative experimental evidence for the pseudo-equilibrium hypothesis and should have general consequences for the interpretation of mutation effects on protein stability, as they suggest that residue environments in proteins may be optimized for stabilizing interactions to a remarkable degree of specificity. We also provide evidence that such stabilizing interactions may be detected in sequence alignments, and briefly discuss the implications of this possibility for the derivation of structural information (on native and denatured states) from comparative sequence analyses.     

Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide

In previous studies designed to increase the primary structure symmetry within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) a combination of five mutations were accommodated, resulting in structure, stability and folding kinetic properties similar to wild-type (despite the symmetric constraint upon the set of core residues). A sixth mutation in the core, involving a highly conserved Met residue at position 67, appeared intolerant to substitution. Structural analysis suggested that the local packing environment of position 67 involved two regions of apparent insertions that distorted the tertiary structure symmetry inherent in the beta-trefoil architecture. It was postulated that a symmetric constraint upon the primary structure within the core could only be achieved after these insertions had been deleted (concomitantly increasing the tertiary structure symmetry). The deletion of these insertions is now shown to permit mutation of position 67, thereby increasing the primary structure symmetry relationship within the core. Furthermore, despite the imposed symmetric constraint upon both the primary and tertiary structure, the resulting mutant form of FGF-1 is substantially more stable. The apparent inserted regions are shown to be associated with heparin-binding functionality; however, despite a marked reduction in heparin-binding affinity the mutant form of FGF-1 is surprisingly approximately 70 times more potent in 3T3 fibroblast mitogenic assays. The results support the hypothesis that primary structure symmetry within a symmetric protein superfold represents a possible solution, rather than a constraint, to achieving a foldable polypeptide.     

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.