Molecular and Physiological Function of the Tubby Gene Family

描述（由申请人提供）：我们描述了一个小的基因家族，像宝宝一样的蛋白质（TULP）。其创始成员tubby （TUB）的突变导致小鼠肥胖、视网膜和耳蜗变性的三方表型，这与在Alstrom或Bardet-Biedl综合征患者中观察到的表型相似，后者是由纤毛相关蛋白突变引起的。胖乎乎的老鼠的迟发、缓慢发展的肥胖反映了人类成熟型肥胖的常见形式。TUB基因的多态性已被证明与肥胖有关，而TULP1基因的突变会导致人类视网膜色素变性和利伯氏先天性黑朦。此外，Tulp3的零突变会导致小鼠胚胎死亡。tubby基因家族在高等真核生物的进化过程中具有很强的保守性，当它们发生突变时观察到相关疾病，这表明tulp在表达它们的细胞的正常功能中起着重要作用。目前，TULPs的分子功能尚不清楚。然而，tubby和Tulp1突变小鼠视网膜中含有视紫红质囊泡的积累，以及与参与细胞内运输的基因和蛋白质的相互作用，表明TULPs在这一过程中发挥了作用。为了开始了解TUB的细胞功能，我们将在转基因小鼠中进行实验，在细胞内运输标记物的共定位研究中，使用标记的TUB蛋白的高分辨率成像来测试TUB是否在细胞运输中发挥作用。我们还将使用酵母双杂交分析和免疫沉淀来鉴定与TUB直接相互作用的蛋白质。这些研究将确定TUB参与的途径。与缺乏对TULPs细胞功能的了解类似，对TULPs的破坏如何导致观察到的疾病表型的了解也有限。为了测试胖老鼠的肥胖是由于其在中枢神经系统或脂肪细胞中的破坏，还是两者的结合，我们将分析组织特异性靶向突变，使这两个部位的TUB失活。我们将测试胖胖的老鼠对棕色脂肪有慢性交感神经激活的假设，从而限制它们的肥胖。我们将通过产生一个他莫昔芬诱导的TULP3等位基因来验证TULP3突变会导致成年动物肥胖和视网膜疾病的假设。最后，通过位置克隆，我们将鉴定出胖墩突变的基因修饰符，可以防止胖墩老鼠肥胖。这一系列实验将为研究tulp的作用途径和洞察其细胞功能提供切入点，从而揭示其作用机制。

DESCRIPTION (provided by applicant): We have described a small gene family, the tubby like proteins (TULP). Mutations in its founding member, tubby (TUB), cause a tripartite phenotype of obesity, retinal and cochlear degeneration in mice, which is similar to the phenotypes observed in individuals with Alstrom or Bardet-Biedl syndromes which are caused by mutations in ciliary associated proteins. The late onset, slowly progressing obesity in tubby mice is reflective of common forms of maturity onset human obesity. Polymorphisms in the TUB gene have been shown to be associated with obesity, and mutations in TULP1 cause retinitis pigmentosa and Leber's Congenital Amaurosis in human populations. Furthermore, a null mutation for Tulp3 leads to embryonic lethality in mice. A strong conservation of the tubby gene family through evolution in higher eukaryotes and the associated diseases observed when they are mutated, indicate that TULPs play an important role in the normal function of the cells in which they are expressed. Currently, the molecular function of TULPs is not known. However, accumulation of rhodopsin containing vesicles in the retina of tubby and Tulp1 mutant mice, and interaction with genes and proteins involved in intracellular trafficking, suggest a role for TULPs in this process. To begin to understand the cellular function of TUB, we will carry out experiments in genetically modified mice that will test whether TUB plays a role in cellular transport using high resolution imaging of labeled TUB protein in co-localization studies with markers of intracellular trafficking. We will also use yeast two hybrid analysis and immunoprecipitation to identify proteins that directly interact with TUB. These studies will define pathways in which TUB participates. Similar to the lack of knowledge about the cellular function of TULPs, there is also limited knowledge about how disruptions in TULPs lead to the observed disease phenotypes. To test whether the obesity in tubby mice is a result of its disruption in the CNS or in adipocytes or a combination of both, we will analyze tissue specific targeted mutations that inactivate TUB at both of these sites individually. We will test the hypothesis that tubby mice have chronic sympathetic activation to brown fat, thus limiting their obesity. We will test the hypothesis that mutations of TULP3 will lead to obesity and retinal disease in the adult animal by generating a tamoxifen inducible allele of TULP3. And finally, by positional cloning we will identify a genetic modifier of the tubby mutation that can prevent obesity in tubby mice. This suite of experiments will provide entry points into the pathways in which TULPs act and insight into their cellular function and, thereby, reveal their mechanisms of action. PUBLIC HEALTH RELEVANCE: Our long term goal is to understand the role that tubby like proteins (TULPs) play in the cell and how defects in these proteins and in the primary cilium, a cellular structure that TULPs are associated with, lead to dysfunction within several organ systems and the organism. This project will further our understanding of how intracellular trafficking associated with the primary cilium contributes to obesity, a connection that has only recently been recognized with the analyses of Bardet-Biedl and Alstrom syndromes. Since these syndromes comprise diseases that are common in the general population such as obesity, type 2 diabetes, and blindness, understanding TULP function may also contribute to a better understanding of the common forms of these diseases.