PROTEIN INTERACTIONS AND FUNCTION OF IQ MOTIF-CONTAINING PROTEINS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。细胞中存在许多未知的含有IQ基序的蛋白质，其生物学功能未知。这些推测的钙调蛋白结合蛋白中的三个是ASPM(纺锤体初级小头症异常)、KIAA1023和KIAA0036。这些蛋白具有多个智商基序，在细胞中广泛表达，对有丝分裂和发育似乎很重要。ASPM最近被发现与人类的小头畸形症有关，并似乎与有丝分裂纺锤体相互作用，这是基于其与小鼠SHA1蛋白和果蝇ASP的同源性，后者也包含多个智商基序。ASPM是与人类原发性小头畸形症相关的六个基因座之一，似乎与发育中的神经发生有关。2005年，第二个基因KIAA0036被认为与囊性肾脏疾病或肾单位肾炎-5有关，后者会导致年轻人的慢性肾功能衰竭和视网膜色素变性。该项目完成的工作包括测量三个多智商基序基因在不同人类细胞系和成人组织中的mRNA表达。这些基因出现在几乎所有被检查的转化的人类细胞系中。这三个基因都在超过16个人类组织中高度表达，除了成人脑和骨骼肌，只有ASPM水平可以忽略不计。其他人的研究未能发现ASPM基因在成年小鼠大脑的不同区域表达，如嗅球、海马体和皮质。已知成人的这些区域中的一些经历了神经发生。我们早些时候在一些灵长类动物中发现了ASPM基因。与小鼠外显子18两个选定区域的序列比较表明，灵长类动物的ASPM没有表现出啮齿动物中发生的任何缺失。在测序的特定区域中，IQ基序在数量、位置和序列上都是相似的，这表明所有灵长类动物都有非常相似的蛋白质。这表明ASPM基因序列的差异，特别是智商区域的差异，并不是一些实验室所说的灵长类动物大脑皮质大小增加的原因。这一数据现在得到了其他实验室在一些灵长类动物中对ASPM基因的完整测序的支持。该实验室的一名博士前候选人还与俄勒冈大学合作，研究ASPM基因在成年斑马鱼和斑马鱼发育中的作用。据推测，ASPM在斑马鱼的大脑发育中起着重要作用。在胚胎发育过程中，使用吗啡反义寡核苷酸来敲除该基因，并研究其对脑组织形态和标志酶的影响。此外，还开发了一些特异的引物，用于对斑马鱼基因缺失区域进行测序，并用于制备用于原位杂交的RNA探针。我们还进行了生物信息学研究，以确定ASPM可能的微管结合区，并检测ASPM家族成员的多结构域结构。就物种分布而言，ASPM似乎存在于所有脊椎动物以及一些无脊椎动物和植物中，但在酵母中不存在。人们还致力于在细菌中表达这些蛋白质的不同区域(即IQ基序、SHA特异性区域和肌动蛋白结合区)，以检测这些区域的潜在结合伙伴和功能。虽然ASPM的两个区域已被克隆到Pet15b载体中，但该蛋白尚未得到表达。克隆和其他技术，如双杂交系统，正在进行研究，试图确定蛋白质的相互作用和这些基因的功能。相互作用研究对于了解这些蛋白质在有丝分裂和发育中的功能至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A number of unidentified IQ motif-containing proteins of unknown biological function occur in cells. Three of these putative calmodulin-binding proteins are ASPM (abnormal spindle primary microcephaly), KIAA1023 and KIAA0036. These proteins have multiple IQ motifs, are widely expressed in cells and appear important to mitosis and development. ASPM has recently been found to be associated with microcephaly in humans and appears to interact with the mitotic spindle based on its orthology to the mouse SHA1 protein and Drosophila ASP which also contain multiple IQ motifs. ASPM is one of six loci associated with primary microcephaly in humans and appears to be involved in developmental neurogenesis. In 2005, the second gene KIAA0036 was linked genetically to cystic kidney disease or nephronophthisis-5 that leads to chronic renal failure and retinitis pigmentosum in the young. Work completed on this project involves measurement of mRNA expression of the three multiple IQ-motif genes in different human cell lines and in adult tissues. These genes occur in nearly all transformed human cell lines examined. All three genes were highly expressed in over sixteen human tissues with the exception of adult brain and skeletal muscle where only ASPM levels were negligible. Studies by others failed to find expression of the ASPM gene in different regions of adult mouse brain such as olfactory bulb, hippocampus and cortex. Some of these regions in the adult are known to undergo neurogenesis. We earlier identified the ASPM gene in a number of primates. Comparisons with the mouse sequence in two selected regions of exon 18 indicated that ASPM in primates did not exhibit any of the deletions that occur in rodents. In the specific regions sequenced, the IQ motifs were similar in number, placement and sequence suggesting that all primates have very analogous proteins. This suggests that differences in the sequence of the ASPM gene, particularly in the IQ region are not responsible for the increase in the size of the cortex among primates as suggested by some laboratories. This data is now supported by the complete sequencing of the ASPM gene in a number of primates by other laboratories. A pre-doctoral candidate in this laboratory is also examining the role of the ASPM gene in adult and developing zebrafish in collaboration with the University of Oregon. It is hypothesized that ASPM is important in the development of the brain in zebrafish. Morpholino anti-sense oligonucleotides were used to knockdown the gene during embryogenesis and investigate the effects on morphology of the brain and marker enzymes. Additionally, a number of specific primers were developed for sequencing missing regions of the zebrafish gene and for the preparation of RNA probes for in situ hybridization. We have also conducted bioinformatic studies to characterize the putative microtubule binding region of ASPM and to examine the multidomain structure of ASPM family members. With regard to species distribution, ASPM appears to be present in all vertebrate and in some invertebrates and plants, but is absent in yeast. Efforts are also focused on expressing in bacteria the different regions of these proteins (i.e., IQ motifs, SHA-specific region, and actin-binding regions) to examine potential binding partners and functionality of these regions. Although two regions of ASPM have been cloned into the pet15b vector, expression of the protein has not been demonstrated. Cloning and other techniques such as the two-hybrid system are being examined in attempts to define protein interactions and the function of these genes. Interaction studies are critical to understanding the function of these proteins in mitosis and development.