PROTEIN INTERACTIONS AND FUNCTION OF IQ MOTIF-CONTAINING PROTEINS

含有 IQ 基序的蛋白质的蛋白质相互作用和功能

• 批准号: 7336024
• 负责人: ALLEN R RHOADS
• 金额: $ 5.13万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7336024
• 关键词: PROTEIN; INTERACTIONS; FUNCTION; IQ; MOTIF

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。细胞中存在许多生物功能未知的含有 IQ 基序的蛋白质。其中三种推定的钙调蛋白结合蛋白是 ASPM（异常纺锤体原发性小头畸形）、KIAA1023 和 KIAA0036。 这些蛋白质具有多个 IQ 基序，在细胞中广泛表达，并且对有丝分裂和发育很重要。最近发现 ASPM 与人类小头畸形有关，并且基于其与小鼠 SHA1 蛋白和果蝇 ASP 的直系同源性，ASPM 似乎与有丝分裂纺锤体相互作用，后者也包含多个 IQ 基序。 ASPM 是与人类原发性小头畸形相关的六个基因座之一，似乎与发育神经发生有关。 2005 年，第二个基因 KIAA0036 与囊性肾病或肾痨 5 存在遗传关联，后者会导致年轻人慢性肾功能衰竭和色素性视网膜炎。 该项目完成的工作包括测量不同人类细胞系和成体组织中三种多重 IQ 基序基因的 mRNA 表达。这些基因几乎存在于所有被检测的转化人类细胞系中。所有这三个基因在超过 16 个人体组织中均高度表达，但成人大脑和骨骼肌除外，其中 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（如嗅球、海马和皮质）中表达。已知成人的这些区域中的一些区域会经历神经发生。我们之前在许多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，灵长类动物中的 ASPM 没有表现出啮齿类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、位置和序列上相似，表明所有灵长类动物都具有非常相似的蛋白质。这表明 ASPM 基因序列的差异，尤其是智商区域的差异，并不是像一些实验室所认为的那样导致灵长类动物皮层尺寸增大的原因。该数据现在得到了其他实验室对许多灵长类动物 ASPM 基因的完整测序的支持。 该实验室的一名博士生也在与俄勒冈大学合作研究 ASPM 基因在成年斑马鱼和发育中斑马鱼中的作用。据推测 ASPM 对斑马鱼大脑的发育很重要。吗啉代反义寡核苷酸用于在胚胎发生过程中敲低该基因，并研究其对大脑形态和标记酶的影响。此外，还开发了许多特异性引物，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来表征 ASPM 的推定微管结合区域，并检查 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。 我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域），以检查这些区域的潜在结合伙伴和功能。尽管 ASPM 的两个区域已被克隆到 pet15b 载体中，但该蛋白的表达尚未得到证实。克隆和其他技术（例如双杂交系统）正在接受检查，试图定义蛋白质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。