Cellular models of microvillus inclusion disease

描述（由申请人提供）： 我们已经建立了微绒毛包涵体病的细胞模型。微绒毛包涵体病（MVID或MID~ MIM#251850）的特征是肠上皮细胞中微绒毛的丢失和微绒毛包涵体的出现。 这导致肠道吸收不足，在没有肠外营养（TPN）的情况下迅速导致死亡。 在编码分子马达肌球蛋白Vb的基因中，只记录了与这种疾病有关的基因。大多数突变导致功能性肌球蛋白Vb蛋白的丧失，而一部分突变是可能改变运动功能的错义突变。我们已经产生了一种肠上皮细胞系（Caco-2细胞），其中肌球蛋白Vb水平<5%， 野生型，并且当它们被极化时不能再维持微绒毛。这将作为微绒毛包涵体疾病的一个子集的细胞培养模型，并可用作肌球蛋白Vb功能的细胞测定。我们将描述MVID引起的错义突变对肌球蛋白肌球蛋白Vb马达动力学的影响。基于生化表征的结果，我们将创建许多新的Caco-2细胞系，其在近零（敲低）背景中表达代表性突变肌球蛋白Vb蛋白，从而创建MVID的新细胞模型。在这些细胞系分化后，我们将研究肌球蛋白Vb错义突变对微绒毛发育/维持的影响。 这些细胞系的建立将为未来的研究奠定基础，以了解肌球蛋白Vb在顶膜再循环中的作用，并更好地了解肌球蛋白Vb在顶膜再循环中的作用。 MVID的发病机制。 公共卫生相关性：编码非常规肌球蛋白（肌球蛋白Vb）的基因中的许多突变已被发现导致微绒毛包涵体病（MVID）。该项目旨在创建MVID的细胞模型，这将允许对肌球蛋白Vb突变的影响进行足够详细的研究，以描述发生的病理过程。这是制定未来治疗干预策略的第一步。

DESCRIPTION (provided by applicant): We have developed a cellular model of microvillus inclusion disease. Microvillus inclusion disease (MVID or MID~ MIM#251850) is characterized by the loss of microvilli and the appearance of microvillus inclusion bodies in intestinal epithelial cells. This results in a lss of intestinal absorption, rapidly leading to death without parenteral nutrition (TPN). Te only documented gene involved in this disease in the gene encoding the molecular motor, myosin Vb. The majority of the mutations result in loss of functional myosin Vb protein, while a subset of the mutations are missense mutations that likely alter motor function. We have generated a line of intestinal epithelial cells (Caco-2 cells) in which myosin Vb levels are <5% of wild type and can no longer maintain microvilli when they are polarized. This will serve as a cell culture model for a subset of microvillus inclusion disease, and can be used as a cellular assay for myosin Vb function. We will characterize the impact of MVID-causing missense mutations on the kinetics of the myosin myosin Vb motor. Based on the outcome of the biochemical characterizations, we will create a number of new lines of Caco-2 cells that express representative mutant myosin Vb proteins in the near null (knock down) background, thus creating new cellular models of MVID. Upon differentiation of these cells lines, we will examine the impact the myosin Vb missense mutations on microvilli development/maintenance. The creation of these lines will set the stage for future studies to understand the role of myosin Vb in apical membrane recycling as well as provide a better understanding of the pathogenesis of MVID. PUBLIC HEALTH RELEVANCE: A number of mutations in the gene that codes for the unconventional myosin, myosin Vb, have been found to cause microvillus inclusion disease (MVID). This project seeks to create cell models of the MVID that will allow studies on the effects of the mutations in myosin Vb in sufficient detail to delineate the pathological processes that takes place. This is the first step toward developing strategies for future therapeutic interventions.