A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins

Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.     

BI-1作为酵母双杂交系统诱饵的载体构建

目的 用人BI-1完整编码区构建酵母双杂交系统的诱饵蛋白载体,排除自身激活作用,并证实其能否在酵母细胞中的表达,验证它能否用于酵母双杂交系统。方法 以人正常肺组织cDNA为模板,扩增BI-1完整编码区,经EcoRI、SalI双酶切后克隆到酵母双杂交系统诱饵载体pGBKT7上,构建诱饵蛋白载体pGBKT7-BI-1,转化酵母细胞,通过β-半乳糖苷酶活性分析验证有无自激活,Western验证其表达。结果 DNA测序显示BI-1编码区内无突变,β-半乳糖苷酶活性分析显示转入pGBKT7-BI-1和阴性对照的酵母菌落没有激活报告基因LacZ,而阳性对照菌落呈现蓝色。结论 含有BI-1完整编码区的诱饵载体不存在自身激活作用,并能在酵母细胞中表达BI-1蛋白,能够应用于酵母双杂交系统筛选相互作用蛋白。     

Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus

The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system. Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins. The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells. The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes.     

Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1

Using yeast two-hybrid assays and a Nicotiana benthamiana cDNA library, we previously identified an N. benthamiana protein, NbPCIP1, that interacts with Potato virus X (PVX) coat protein (CP). We also previously determined that NbPCIP1 enhances PVX replication in plants. To determine the domains and/or amino acid residues required for PVX CP and NbPCIP1 interaction, here we used yeast two-hybrid and β-galactosidase filter assays to test the effects of deletion and site-directed mutations on the interaction. Truncation analysis revealed that the N-terminal region of PVX CP interacts with NbPCIP1. To identify which N-terminal region PVX CP amino acid(s) interact with NbPCIP1, we substituted the 12 charged amino acids on the PVX CP N-terminal region to alanine. Yeast two-hybrid, β-galactosidase filter, and bimolecular fluorescence complementation (BiFC) assays confirmed that ten of the 12 alanine-substituted mutations blocked the interaction with NbPCIP1. The results suggest that the N-terminal region of PVX CP including its helical structure is important for interaction with NbPCIP1.     

Maturation of wild-type and mutated frataxin by the mitochondrial processing peptidase

Frataxin is a mitochondrial protein deficient in Friedreich ataxia (FRDA) and which is associated with abnormal intramitochondrial iron handling. We identified the mitochondrial processing peptidase beta (MPPbeta) as a frataxin protein partner using the yeast two-hybrid assay. In in vitro assays, MPPbeta binds frataxin which is cleaved by the reconstituted MPP heterodimer. MPP cleavage of frataxin results in an intermediate form (amino acids 41-210) that is processed further to the mature form. In vitro and in vivo experiments suggest that two C- terminal missense mutations found in FRDA patients modulate interaction with MPPbeta, resulting in a slower maturation process at the normal cleavage site. The slower processing rate of frataxin carrying such missense mutations may therefore contribute to frataxin deficiency, in addition to an impairment of its function.      

A yeast assay for functional detection of mutations in the human cystathionine {beta}-synthase gene

Mutations in the human cystathionine ß-synthase (CBS)gene are known to cause homocystinuria and may also be a significantrisk factor for premature atherosclerosis. We have previouslyshown that the human CBS protein can substitute for the endogenousyeast CBS protein in Saccharomyces cerevisiae. We now show thatexpression of three different CBS mutants known to be associatedwith reduced enzyme activity in humans fail to complement growthin the yeast assay. in addition, we have used the yeast CBSassay to identity eight mutant CBS alleles in cell lines frompatients with CBS deficiency. These mutant alleles include twopreviously identified and five novel CBS mutations. Our resultsalso demonstrate that the yeast CBS assay can detect a largepercentage of individuals heterozygous for mutations in CBS.This system should be useful in determining the relationshipbetween CBS mutations and human disease.     

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。     

Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin.

Critical functions of hamartin and tuberin, encoded by the TSC1 and TSC2 genes, are likely to be closely linked. The proteins interact directly with one another and mutations affecting either gene result in the tuberous sclerosis phenotype. However, the regions of hamartin and tuberin that interact have not been well defined, and the relationship between their interaction and the pathogenesis of tuberous sclerosis has not been explored. To address these issues a series of hamartin and tuberin constructs were used to assay for interaction in the yeast two-hybrid system. Hamartin (amino acids 302-430) and tuberin (amino acids 1-418) interacted strongly with one another. A region of tuberin encoding a putative coiled-coil (amino acids 346-371) was necessary but not sufficient to mediate the interaction with hamartin, as more N-terminal residues were also required. A region of hamartin (amino acids 719-998) predicted to encode coiled-coils was capable of oligermerization but was not important for the interaction with tuberin. Subtle, non-truncating mutations identified in patients with tuberous sclerosis and located within the putative binding regions of hamartin (N198_F199delinsI;593-595delACT) or tuberin (G294E and I365del), abolished or dramatically reduced interaction of the proteins as assessed by yeast two-hybrid assays and by co-immunoprecipitation of the full-length proteins from Cos7 cells. In contrast, three non-pathogenic missense polymorphisms of tuberin (R261W, M286V, R367Q) in the same region as the disease-causing TSC2 mutations did not. These results indicate a requirement for interaction in critical growth suppressing functions of hamartin and tuberin.     

Screening human EST database for identification of candidate genes in respiratory chain deficiency

Disorders of mitochondrial oxidative phosphorylation (OXPHOS) are now recognized as major causes of human metabolic diseases and several mutations of mitochondrial and nuclear genes encoding respiratory chain components have been reported. Interestingly, mutations of nuclear genes involved in mitochondrial respiratory chain assembly, protein trafficking, and iron metabolism are also known to alter oxidative phosphorylation. While several hundred of these genes have been described in yeast, only a few nuclear genes have been hitherto identified in humans. Yeast gene databases present therefore an invaluable tool for identification of human homologues that should be regarded as candidate genes in OXPHOS diseases. In an attempt to identify the human counterparts of yeast genes, we developed a systematic comparison of yeast protein sequences to the GenBank dbEST database. Starting from 340 yeast protein sequences as templates, we searched the human dbEST counterparts using the BLAST similarity searching program and identified 102 groups of human EST likely to represent orthologues of yeast genes because of significant homology. This collection of human genes possibly related to mitochondrial OXPHOS may help identify nuclear genes responsible of mitochondrial disorders.     

ATPase 抑制因子1是与HCMV pUL23蛋白相作用的宿主蛋白分子-酵母双杂交技术筛选与鉴定

目的： 利用酵母双杂交技术筛选与人巨细胞病毒相互作用的宿主蛋白分子,为探讨人巨细胞病毒pUL23蛋白在HCMV生活周期中的作用机制提供依据。方法: 利用GAL4酵母双杂交系统筛选人胚肾cDNA文库,以获得与人巨细胞病毒pUL23蛋白相互作用的宿主蛋白分子,再通过回交试验和体外GST-pulldown试验验证两者之间的相互作用。结果: 酵母双杂交筛选得到宿主蛋白分子ATPase inhibitory factor 1（ATIF1）,回交试验和体外GST-pulldown试验再次确认ATIF1能够与人巨细胞病毒pUL23蛋白相互作用。结论: pUL23确实能够与ATIF1相互作用,它们之间的相互作用可能为研究pUL23在病毒生活周期发挥的功能提供依据。     

Assessment of human Nter and Cter BRCA1 mutations using growth and localization assays in yeast

A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.     

酵母双杂交筛选与单剪接型2.2 kb乙型肝炎病毒剪接特异性新蛋白相互作用的肝细胞蛋白

目的 筛选与单剪接型2.2 kb乙型肝炎病毒(HBV)剪接特异性新蛋白相互作用的肝细胞蛋白.方法 PCR扩增单剪接型2.2 kb HBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7,在证实TPss蛋白不具有自激活作用的前提下,以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白,进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用.结果 构建酵母双杂交诱饵载体pGBKT7-TPss,Western blot显示其在酵母中表达TPss蛋白.酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用,即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链.结论 TPss可与多种肝细胞蛋白相互作用.     

UV-induced mutagenesis of human p53: analysis using a double-selection method in yeast

Comparison of the mutation patterns of p53 in human tumors with those of selectable genes in model systems is a powerful approach to identify potential etiological factors for specific tumor types. Recently, we validated use of a yeast assay to permit direct determination of the mutation spectrum induced in human p53 by carcinogens that would reduce uncertainties inherent in comparing spectra induced in different target genes. Here, we describe modifications in the assay designed to facilitate screening for mutants and to permit intracellular exposure of the gene instead of in vitro treatment. This was accomplished by introducing growth-based selection for transactivation-deficient p53 mutants into yeast already possessing red/white colony color selection. This improved model system was able to detect cells harboring p53 mutations among cells with wild-type p53 at a frequency of 10(-4) or less. Additionally, UV light was used to verify that the majority of mutagenized cells with the appropriate phenotype on selective medium contained mutations in p53, not elsewhere in the genome. Sequence analysis of UV-induced mutations revealed that the nature of the mutations was similar to those obtained in previous studies of this mutagen. This system will prove useful in the determination of the ability of environmental agents to mutate the human p53 gene, and thus may contribute to hazard identification.     

Detection of protein truncating mutations in exons 1-14 of the APC gene using an in vivo fusion protein assay. Mutations in brief no. 214. Online

About 80% of the mutations identified to date in the Adenomatous Polyposis Coli (APC) gene have been found in the 5' half of the coding sequence, the vast majority of which (>95%) are nonsense or frameshift mutations that result in the loss of the carboxyl terminus of APC protein. Using a stop codon assay in yeast recently developed by others (Ishioka et al., 1997), we have screened the 5' half of the APC gene for mutations in 7 unrelated families affected with Familial Adenomatous Polyposis. The assay relies on the expression of a yeast reporter gene fused in frame to one of 3 contiguous segments of the APC open reading frame. Here we report on the detection by this assay of 5 germline mutations, 4 of which lie upstream of exon 15, where lesions appear to be sometimes difficult to detect by standard methods.     

Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system

Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.     

Two novel mutations of the AIRE protein affecting its homodimerization properties

We report two novel mutations, c.230T>C (p.F77S) and c.64_69del (p.V22_D23del) within the HSR domain of the AIRE protein in two patients of Italian descent affected by APECED. Both mutations were found in the compound heterozygous state respectively with c.994+5G>T and c.232T>A (p.W78R). With the two-hybrid assay in the yeast system we found that constructs containing the two mutations fail to interact with the wild-type protein. These findings indicate that both mutations negatively affected the homodimerization properties of the AIRE protein, thereby leading to a defective function.     

Klotho:与FPC蛋白相互作用的蛋白质分子

目的:利用酵母双杂交技术筛选与纤囊素相互作用的蛋白质,为进一步探讨纤囊素(FPC)在常染色体隐性遗传多囊肾病(ARPKD)发生、发展中的作用机制提供依据。方法:利用酵母双杂交系统以质粒pG-BKT7-FPC为"诱饵",在人类胚肾cDNA文库中筛选与FPC蛋白C末端相互作用宿主蛋白的基因,再通过一对一回交试验验证两者之间的相互作用。结果:酵母双杂交筛选得到相互作用的蛋白分子Klotho(后简称KL),回交试验再次确认KL能够与FPC蛋白相互作用。结论:FPC的C末端能够与KL相互作用,它们之间的相互作用可能为研究FPC在ARPKD发病中的功能及作用机制提供新途径。