附睾特异分泌蛋白Lipocalin13的原核表达和纯化及其与配体结合特性的分析

大鼠Lipocalin13(rLcn13)是最近发现的一个大鼠附睾特异分泌的lipocalin家族蛋白,与男性生殖密切相关。该工作分析了rLcn13的染色体定位和基因特征,用同源模建方法获得了rLcn13蛋白的三级结构,并构建了rLcn13蛋白的原核表达系统,对rLcn13蛋白进行了可溶表达和纯化,并研究了rLcn13蛋白的配体结合特性。结果表明:(1)rLcn13蛋白在原核表达系统中能实现可溶性表达,并具有良好的三级结构;(2)rLcn13蛋白的二级结构以β-折叠为主;(3)rLcn13蛋白具有较强的视黄酸结合能力,也能结合其它一些lipocalin蛋白的配体;(4)rLcn13蛋白在大鼠附睾中可能起着视黄酸结合和转运作用,并有可能参与免疫反应。该研究为rLcn13蛋白的三维结构解析及其生理功能以及其它lipocalin家族蛋白的结构与功能关系的研究奠定了基础。     

聚乙二醇单修饰的重组人粒细胞集落刺激因子突变体的纯化与分析

目的 获得高活性的聚乙二醇定点单修饰的产物PEG-rmhG-CSF-Cys176.方法 用聚乙二醇马来酰亚胺基和高活性的重组人粒细胞集落刺激因子突变体(rmhG-CSF)的半胱氨酸(Cys)基团特异性结合,并用离子交换层析分离纯化修饰产物,通过高效凝胶过滤色谱检测纯度,MTT法检测体外的生物活性.结果 蛋白纯度为97%,体外生物活性为0.8×10~8 IU·mg~(-1).结论 半胱氨酸反应性PEG和游离Cys的结合是在rmhG-CSF活性区以外的位置C末端,修饰后的蛋白活性基本未受影响.     

终止密码子位点及无义突变介导的mRNA降解途径抑制对抗早发性无义突变药物atalruen通读效率的影响

目的探讨终止密码子上下文结构和无义突变介导的m RNA降解途径(NMD)抑制对抗早发性无义突变(PTC)药物atalruen通读效率的影响。方法利用聚合酶链反应(PCR)定点突变的方法构建双荧光哺乳动物表达载体的早发性无义突变体p Ds Red-EGFPmtag-Y596X和p Ds Red-EGFPmtag-Y653X,转染哺乳动物细胞COS7,抗早发性无义突变药物atalruen,结合si RNA-upf1、放线菌酮(CHX)共同作用转染细胞,采用实时荧光定量PCR反应检测突变体上红色荧光蛋白(Ds Red)和绿色荧光蛋白(EGFP)的表达,测定通读效率。结果PTC突变体p Ds Red-EGFPmtag-Y653X对atalruen的促通读敏感性显著大于p Ds Red-EGFPmtag-Y596X;si RNAupf1和CHX抑制NMD对atalruen的促通读效率没有显著影响。结论 PTC位点的+4位核苷酸对于atalruen的促通读效率十分关键,该位置是胞嘧啶(C)的突变体敏感性要显著大于腺嘌呤(A);atalruen与常见促通读氨基糖苷类药物不同,不能协同NMD途径的抑制来促进早发性无义突变的通读。     

蝎镇痛活性肽AS的一个二硫键对镇痛活性的影响

目的研究蝎镇痛活性肽AS中二硫键C12-C62对其生物活性的影响。方法利用一步聚合酶链式反应(polymerase chain reaction,PCR)分别用Ser替代蝎镇痛活性肽AS中第12位、第62位及第12位和第62位的Cys,从而构建其3个突变体。采用金属离子螯合亲和层析方法对表达突变体蛋白进行纯化。采用小鼠扭体法测定3个突变体的镇痛活性。结果构建了3个突变体质粒,在BL21(DE3)中实现了可溶性表达。采用金属离子螯合层析方法获得了电泳纯样品。其镇痛活性明显低于Bmk AS(P<0.01)。结论3个突变体与rBmK AS镇痛活性有明显差异,二硫键C12-C62与蝎毒镇痛活性密切相关。     

特异靶向KRAS-G12C突变的抗肿瘤药物研究进展

RAS是肿瘤中突变最为广泛的癌基因,但是至今尚无针对RAS突变肿瘤的靶向治疗药物获批在临床使用.近年来,针对KRAS-G12C突变体的抑制剂研发进展迅猛,被认为是当前针对RAS突变肿瘤最具希望的突破方向.本综述围绕KRAS-G12C突变,重点介绍了针对半胱氨酸的共价抑制剂研发进展、联合用药策略和基于蛋白降解的蛋白水解靶...     

酵母单一亚单位NADH-泛醌氧化还原酶定点突变对酶活性及泛醌结合部位的影响

目的采用定点突变方法探讨突变体对酵母Ndil的NADH一泛醌氧化还原酶活性及泛醌结合部位的影响。方法定点突变方法将Ndil的His残基（His^193His^252、His^301）突变为丙氨酸,转染大肠杆菌后表述蛋白,测定光谱特性,NADH-泛醌氧化还原酶活性和米氏常数Km及最大反应速度Vmax。结果各突变体蛋白H193A、H252A、H301A吸收光谱显示均在383nm和448nm处出现两个FAD峰与野生型一甄各突变体酶活性显示,突变体对UQ。的Vmax均明显减小,但仅H301A对UQ1的Km增加约3倍,对UQ亲和力明显减小。各突变体蛋白对NADH的Km未见明显增大,但Vmax均出现明显减小。结论酵母NdiI中H301点突变可能是参与形成UQ结合位点所需的氨基酸残基之一,该位点突变影响了UQ与酶的结合及电子向UQ的传递。     

血清胱抑素C和同型半胱氨酸与急性脑梗死相关性分析

<正>脑梗死具有发病率高、再发率高、致残率高、死亡率高等特点,给家庭、社会带来了严重的负担和损失。早期发现脑梗死的发病因素及提前干预显得尤为最要。胱抑素C(cystatin C,Cys C)是为一种分子量相对较小的碱性蛋白质、胱氨酸蛋白酶抑制剂C,主要与半胱氨酸蛋白酶结合,抑制半胱氨酸蛋白     

奥拉西坦对急性脑出血患者Hcy、hs-CRP、尿酸、半胱氨酸蛋白酶抑制剂及血脂指标水平的影响

目的探讨奥拉西坦对急性脑出血患者同型半胱氨酸(Hcy)、超敏C-反应蛋白(hs-CRP)、尿酸(UA)、半胱氨酸蛋白酶抑制剂(Cys C)及血脂指标水平的影响。方法急性脑出血患者98例依据随机数字表法随机分为观察组49例与对照组49例。对照组采用常规治疗,观察组在对照组基础上结合奥拉西坦治疗。2组疗程均为2周。结果观察组治疗后NIHSS评分低于对照组,GCS评分和ADL评分高于对照组(P<0.05);观察组血清Hcy、hs-CRP、Cys C水平治疗后低于对照组,而UA水平高于对照组(P<0.05);观察组TG、TC、LDL-C水平治疗后低于对照组(P<0.05);2组均未见严重不良反应。结论奥拉西坦可通过降低急性脑出血患者Hcy、hs-CRP、Cys C水平,增加UA水平,及改善血脂异常发挥作用,从而改善患者症状。     

血清半胱氨酸蛋白酶抑制剂C检验在诊断高血压患者早期肾损伤中的价值探析

目的分析Cys C(血清半胱氨酸蛋白酶抑制剂C)检验在诊断高血压患者早期肾损伤中的价值。方法选取我院收治的159例原发性高血压疑似合并早期肾损伤患者为研究对象,根据99mTc-DTPA(99mTc-二乙三胺五醋酸)清除率为标准,将159例原发性高血压患者分为肾损伤组与对照组,肾损伤组63例,对照组96例,均进行Cys C检测,分析Cys C在原发性高血压早期肾损伤中的诊断价值。结果肾损伤组患者血清Cys C值显著高于对照组(P<0.05),有统计学意义;且早期肾损伤组血清Cys C>1.26 mg/L发生率显著高于对照组,有统计学意义(P<0.05)。结论血清半胱氨酸蛋白酶抑制剂C检验在高血压患者早期肾损伤中的诊断价值显著,值得推广。     

非糖基化尿激酶原的研究

目的：构建一种非糖基化、抗凝血酶激活的尿激酶原突变体，并观察其表达及活性情况。方法：使用PCR扩增的方法，将302位天冬酰胺（Asn302）突变为丙氨酸（Ala302），去掉糖基化位点；将156位精氨酸（Arg156）突变为赖氨酸（Lys156），去掉凝血酶作用位点。将突变体基因转染到哺乳动物细胞中。收取无血清培养上清．并用纤维蛋白板溶解圈法鉴定活性。结果：PCR产物经琼脂糖电泳鉴定分子质量略大于1200bp，与理论分子质量1296bp基本符合。转染成功的细胞株用于扩大培养。纤维蛋白板溶解圈法测定上清活性为295．58IU／mL。结论：非糖基化、抗凝血酶的尿激酶原突变体构建成功。转染后细胞生长良好，表达水平较高。     

Construction of a phage displayed library based on a lipocalin scaffold and preliminary screening of anticalins with specificity for the FcεRI-α receptor

The primers were designed according to the gene sequence of lipocalin protein family, and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR. The random mutation lipocalin library was constructed using phagemid expression vector. Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells, and the lipocalin secondary library binding to mast cells was obtained. Then the lipocalin secondary library was enriched and screened with FcεRI-α receptor protein as target molecule, and specific binding phages were eluted. After three rounds of screening, eight recombinant phage clones were randomly selected from elution clones of the third round. ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-α receptor of mast cells. These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-α receptor, and also lay the foundation for the development of biological small molecule drugs to treat IgE associated allergic diseases.     

Lipocalin‐2—The myth of its expression and function

Lipocalin‐2 is a functional biomarker for acute and chronic kidney diseases, heart failure and obesity‐related medical complications. It is rapidly induced in epithelial cells under stress conditions, but constitutively produced from pre‐adipocytes and mature adipocytes. Measuring the lipocalin‐2 levels represents an effective approach for risk prediction, patient stratification and disease management. Nevertheless, due to ligand‐binding, post‐translational modification and protein‐protein interaction, lipocalin‐2 exists as multiple variants that elicit different pathophysiological functions. To characterize the specific structure‐functional relationships of lipocalin‐2 variants is critical for the development of biomarker assays with sufficient precision and reliability. Moreover, identifying the pathological forms of lipocalin‐2 will provide new therapeutic targets and treatment approaches for obesity‐related complications.     

Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder,Paralichthys olivaceus

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon–intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two α-helices and eight β-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6 μg/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.     

Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder, Paralichthys olivaceus

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon–intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two α-helices and eight β-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6 μg/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.     

Tick histamine-binding proteins and related lipocalins: potential as therapeutic agents

Tick histamine-binding proteins bind histamine with high affinity and specificity. This is attained by a novel binding mechanism, whereby histamine is sequestered within a binding cavity of the lipocalin fold. The histamine binding proteins and related protein family members are currently under investigation as potential therapeutic agents for the treatment of various diseases, including conjunctivitis, allergic rhinitis, carcinoid syndrome and rheumatoid arthritis. While these proteins show great therapeutic potential, they are part of a diverse family of tick lipocalin proteins, some of which have been implicated in tick-host rejection and host pathogenesis. As such, the therapeutic mining of tick lipocalins should be considered within the framework of the rest of the family.     

Neutrophil gelatinase-associated lipocalin (NGAL) as a new biomarker for non--acute kidney injury (AKI) diseases

Neutrophil gelatinase-associated lipocalin, or NGAL, an acute phase protein, is part of the lipocalin family. NGAL is highly induced in inflammatory conditions and ischemia, and is a critical component of innate immunity to bacterial infection. Recently, NGAL has been proven as an emerging biomarker for predicting acute kidney injury (AKI). Meanwhile, numerous studies have also demonstrated that NGAL may be a potential biomarker for the diagnosis, prediction, prevention, and prognosis of non--AKI diseases such as chronic kidney diseases, vascular disorders, cancer, preeclampsia, and allergies. This article systematically reviews the clinical utilities of NGAL as a new biomarker for non--AKI diseases.     

Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales

Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT.     

Effect of pioglitazone on endotoxin-induced decreases in hepatic drug-metabolizing enzyme activity and expression of CYP3A2 and CYP2C11

It has been reported that peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands ameliorate the expression of inducible nitric oxide synthase (iNOS) by endotoxin. In the present study, we investigated the effect of pioglitazone, a potent PPAR-gamma ligand, on the endotoxin-induced reduction of hepatic drug-metabolizing enzyme activity and on the down-regulation of the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP2C11 proteins in rats. Endotoxin (1 mg/kg) significantly decreased hepatic drug-metabolizing enzyme activity in vivo, as represented by the systemic clearance of antipyrine and protein levels of CYP3A2 and CYP2C11 24 h after intraperitoneal injection. Pretreatment with pioglitazone (10 mg/kg, 4 times at 10-min intervals) significantly protected the endotoxin-induced decreases in the systemic clearance of antipyrine and protein levels of CYP3A2, but not CYP2C11, with no biochemical and histopathological changes in the liver. Pioglitazone alone had no effect on the systemic clearance of antipyrine and protein levels of CYP3A2 or CYP2C11. Pioglitazone significantly protected endotoxin-induced overexpression of iNOS in the liver, but not the overproduction of nitric oxide (NO) in plasma. It is unlikely that the protective effect of pioglitazone against endotoxin-induced decreases in the hepatic drug-metabolizing enzyme activity and protein levels of CYP3A2 in the liver is due to the inhibition of the overproduction of NO.