人PIRH2b与ARF4相互作用的初步研究

目的 利用酵母双杂交技术筛选PIRH2b的相互作用蛋白。方法 以PIRH2b为诱饵蛋白,利用酵母双杂交技术筛选人胎肝cDNA文库,用GST—pull down验证PIRH2b与ARF4在体外的相互作用,并用绿色荧光蛋白标记PIRH2b,红色荧光蛋白标记ARF4,观察两者在肝癌细胞株Hep3B中的亚细胞定位。结果利用酵母双杂交筛选到一个能与PIRH2b相互作用的蛋白ARF4,GST—pull down验证了两者在体外的相互作用,荧光标记共定位结果显示两个蛋白共定位于Hep3B细胞的核周区域。结论首次发现并证实了PIRH2b与ARF4的相互作用,PIRH2b对ARF4的功能可能有重要影响。     

心肌特异性新蛋白激酶p93和peroxiredoxin 3的相互作用

为了明确p93在心血管系统中的生物学功能及在信号传导通路中的作用,采用酵母双杂交技术,选择p93 N端第101～372位氨基酸构建诱饵质粒,筛选成人心脏cDNA文库,寻找与之相互作用的蛋白质,结果得到蛋白peroxiredoxin 3（PRX 3）.采用体外结合实验和免疫共沉淀等方法,证实了原核和真核表达的p93确实可与PRX3相互作用,通过双重荧光共定位,为二者的相互作用提供了定位依据.研究提示p93可能参与心肌细胞中与PRX3有关的各种生理病理过程,如生长、分化、发育、凋亡、氧应激等.     

环指蛋白RING1与A型核纤层蛋白的细胞内相互作用

环指蛋白RING1能结合DNA并抑制基因的转录.采用酵母双杂交方法从人骨骼肌文库中筛选出了与A型核纤层蛋白(lamin A)结合的RING1蛋白,回复杂交酵母能在缺陷培养基上生长.RING1与绿色荧光蛋白融合载体转染HEK293细胞,激光共聚焦显微观察发现RING1能与带红色荧光蛋白的lamin A蛋白在细胞核周围共定位.免疫共沉淀结果证明RING1与lamin A能够相互作用.结果证明了一个新的lamin A结合蛋白,为揭示lamin A影响基因表达乃至细胞衰老提供了依据.     

禽传染性支气管炎病毒Nsp2蛋白与宿主蛋白eIF2α的互作鉴定

Nsp2蛋白是冠状病毒的非结构蛋白,在病毒早期感染中具有重要作用。【目的】为初步筛选可能与禽传染性支气管炎病毒(avian infectious bronchitis virus,IBV) Nsp2蛋白互作的宿主蛋白,鉴定Nsp2蛋白与真核翻译起始因子2α亚基(eIF2α)的相互作用。【方法】以pCAGGs-Flag-Nsp2和pCAGGs-Flag载体转染后的鸡胚肾(CEK)细胞为研究对象,利用免疫共沉淀(Co-IP)和液相色谱-串联质谱(LC-MS/MS)技术筛选出可能与IBVNsp2蛋白发生互作的宿主蛋白eIF2α,通过免疫共沉淀和间接免疫荧光试验进一步验证二者相互作用。【结果】经免疫共沉淀与质谱分析后筛选到97个可能与Nsp2蛋白互作的宿主蛋白,其中宿主抗病毒反应的关键蛋白eIF2α与Nsp2蛋白的相互作用通过免疫共沉淀和间接免疫荧光试验,表明二者存在直接互作关系,并共定位于细胞质中;此外,Nsp2蛋白表达和IBV感染都能显著提高宿主内源性eIF2α的转录水平。【结论】利用免疫共沉淀联合质谱技术筛选到CEK细胞中存在的97种可能与IBV Nsp2互作的候选蛋白,利用免疫共沉淀与...     

酵母双杂交系统筛选GATA-1相互作用蛋白质及功能验证

目的 利用酵母双杂交技术从人脑cDNA文库中筛选与人GATA-1相互作用的蛋白质.方法 从人K562细胞中扩增出全长GATA1基因,设计引物将其3段截断体亚克隆入酵母表达载体pDBLeu中,转化至AH109感受态酵母中,利用酵母双杂交技术筛选人脑cDNA文库中与其相互作用的蛋白质,阳性克隆通过回转及免疫共沉淀试验进行验证,利用3xGATA荧光素酶报告基因对相互作用蛋白质进行功能验证.结果 成功构建出酵母诱饵蛋白表达质粒pDBLeu-GATA1（1）,pDBLeu-GATA1（2）,pDBLeu-GATA1（3）,筛到34个阳性克隆,用生物信息学分析及回转验证得到5个与GATA-1相互作用的候选蛋白,通过免疫共沉淀试验进一步验证,获得3个蛋白质能与GATA-1相互作用,分别是ECSIT,EFEMP1和GPS2.荧光素酶试验表明这3个蛋白质均能对GATA1的转录活性产生影响,证实它们之间的相互作用具有影响GATA1转录的功能.结论 应用酵母双杂交技术及免疫共沉淀试验,从人脑cDNA文库中成功获得3个与GATA-1相互作用并对其转录活性具有调节作用的蛋白质,为研究GATA1蛋白质的功能提供了新的线索.     

结肠腺瘤性息肉病蛋白通过与SMAP/KAP3相互作用与微管结合

结肠腺瘤性息肉病基因(adenomatous polyposis coli,APC)的突变导致家族性结肠息肉腺瘤病和散发性结肠癌,APC基因编码一个具有多个结构域、多种磷酸化状态的大分子蛋白质.APC蛋白可通过C段直接或间接与微管结合,同时还可以通过中段与微管结合,但其结合的机制目前还不清楚.为进一步研究APC与其他蛋白质的相互作用,利用酵母双杂交技术运用APC中段（1 500 bp～4 800 bp）构建诱饵质粒,筛选人胎脑cDNA文库,得到一个与APC相互作用的蛋白SMAP/KAP3,SMAP/KAP3是驱动蛋白KIF3A/3B的相关蛋白.通过免疫共沉淀和双色免疫荧光共定位的方法,证实了APC与SMAP/KAP3在体内的相互作用,提示APC可能通过SMAP/KAP3-KIF3A/B参与沿微管的运动.     

PML coiled-coil结构域与RAN结合蛋白9相互作用的验证

目的验证前髓细胞性白血病的卷曲螺旋结构域（PML—C）和RAN结合蛋白9（RANBP9）之间的相互作用。方法构建分别表达诱饵蛋白PML—C和靶蛋白RANBP9的载体pGBKT7-PML-C和pACT2-RANBP9,然后转人酵母AH109,培养3～5d后对其是否有胞内相互作用进行检测。将目的片断PML-C和RANBP9再次构建于真核生物表达载体pCMV—HA和pCMV—myc里,然后共转染人胚肾293细胞里（HEK293）,最后对其是否有体外相互作用通过免疫共沉淀和免疫印迹进行分析。结果在共转化了质粒pGBKT7-PML—C和pACT2-RANBP9的AH109酵母平板里观察到蓝色菌落生长。用抗HA多克隆抗体对共转染过重组质粒的HEK293细胞的蛋白提取物进行免疫共沉淀,再用抗myc单克隆抗体作为一抗进行免疫印迹,最终检测出融合蛋白myc—RANBP9条带。结论酵母双杂交实验验证了PML—C和RANBP9之间存在胞内相互作用,同时免疫共沉淀实验也从体外验证了它们之间的相互作用。     

RIOK3促进了caspase-10对PAK2的酶解激活

RIO3为非典型激酶RIO家族的成员之一,仅在多细胞真核生物中出现,为研究RIOK3蛋白的功能,以其作为诱饵蛋白对成人肝cDNA文库进行酵母双杂交筛选,得到其相互作用蛋白PAK2,并通过细胞内免疫共沉淀和免疫荧光共定位实验验证了该相互作用。实时定量PCR和免疫印迹检测结果显示,RIOK3能够在蛋白水平上降低PAK2表达量。通过CCK-8和细胞凋亡检测,发现二者共表达可以抑制增殖并促进凋亡,并且这一促凋亡效应可以被caspase-10的无酶活最短剪切本caspase-10G所抑制。实验结果显示,RIOK3可能促进了caspase-10对PAK2的酶解,在PAK2的酶解激活途径中发挥重要作用。     

转录因子AP-2β与SLP-2的蛋白相互作用

AP-2β是转录因子AP-2家族中的重要一员.AP-2是一个与哺乳动物的发育、细胞生长、分化、凋亡和肿瘤发生都有密切联系的重要的转录因子家族.最近的研究表明,AP-2能通过与其他蛋白的相互作用来调控AP-2下游基因的转录活性.采用免疫共沉淀结合质谱鉴定的方法筛选与AP-2β相互作用的蛋白.结果筛选到Stomatin like protein2(SLP-2)蛋白可能与AP-2β相互作用.免疫共沉淀实验证实外源过表达和内源的SLP-2蛋白都能与AP-2β蛋白相互作用.而且,细胞免疫荧光实验发现外源表达的Myc-AP-2β蛋白与内源的SLP-2蛋白在MCF-7细胞的胞质中有共定位.此外,免疫印记实验结果表明过表达SLP-2能上调AP-2β的蛋白水平,这就表明SLP-2与AP-2β相互作用后,SLP-2可能使AP-2β蛋白更稳定.这些研究结果为进一步研究这两个基因的功能提供了新的切入点.     

发热伴血小板减少综合征病毒核蛋白特异结合宿主细胞60kD SSA/Ro

为了初步探索发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)核蛋白(nucleoprotein,NP)对宿主细胞免疫功能的影响。将SFTSV NP和NSs蛋白的编码基因插入真核表达载体VR1012中,通过免疫共沉淀(IP)、SDS-PAGE、质谱检测及蛋白质免疫印迹等方法寻找宿主细胞中与NP相互作用,同时又与免疫功能有关的蛋白质分子,并利用细胞免疫荧光方法检测SFTSV NP与该分子在细胞中的共定位情况。IP和质谱检测结果显示NP能与免疫功能相关的60kD SSA/Ro蛋白发生特异性结合,细胞免疫荧光试验进一步显示NP与60kD SSA/Ro蛋白在细胞质中存在共定位。提示SFTSV可能通过其核蛋白与免疫相关分子60kD SSA/Ro蛋白发生特异性结合,从而引起机体一系列的免疫反应和临床症状。     

Identification of the key LMO2-binding determinants on Ldb1

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.     

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). We have determined a high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.     

Identification and Characterization of MT-1X as a Novel FHL3-Binding Partner

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.