Regulation of Myosin V Interaction with Cargo

描述（由申请人提供）：细胞器运动是细胞分裂和细胞分化的标志。在每种细胞类型中，每种类型的细胞器的定位、数量和形态被修改以实现特定的细胞功能。细胞器的长距离运动发生在微管上，而细胞器在其最终目的地的定位依赖于基于肌动蛋白的马达。肌球蛋白V马达在所有真核生物中基于肌动蛋白的运动中起关键作用，并且肌球蛋白V功能的破坏导致人类疾病。例如，基于肌球蛋白Va的转运的部分缺陷引起Griscelli综合征，其特征在于神经和色素沉着缺陷。肌球蛋白Vb的部分缺陷引起微绒毛包涵体病，其特征在于婴儿期危及生命的腹泻。目前，对于这两种疾病都没有有效的药物治疗。确定肌球蛋白V的转运是如何实现的，可能会为在正确的时间和地点治疗这些疾病提供重要的新见解。球尾域（GTD）的肌球蛋白V重视其货物通过细胞器特异性衔接蛋白。GTD和衔接蛋白的调节有助于指定货物附着。我们确定了一个高分辨率的结构的GTD的Myo2，酿酒酵母肌球蛋白V电机，并确定了两个不同的货物结合区，一个需要结合到酵母液泡，其他需要结合到分泌囊泡。我们发现液泡结合位点与一种新的蛋白质Vac17相互作用。分泌囊泡结合位点直接附着于Rab GT3。重要的是，我们发现Rab GT3结合位点在人肌球蛋白Va和肌球蛋白Vb中是保守的。因此，我们在酵母中的研究揭示了人肌球蛋白V马达上的Rab GT3结合位点。酵母Myo2和人肌球蛋白Va和Vb的货物结合结构域的一般保守性，以及Rab GTP酶直接作用于附着肌的事实。我们的主要目标是：1）确定单个Myo2接头蛋白的结合是否增强或抑制其他接头蛋白的结合。2)鉴定和表征调节Rab GTP酶与Myo2 GTD连接的蛋白质。3)确定调节肌球蛋白V从货物中分离的机制。 公共卫生相关性：肌球蛋白V马达细胞内细胞器运输对正常细胞功能和动物生理学至关重要。基于肌球蛋白V的转运缺陷导致选定的人类疾病，包括神经障碍。我们的总体目标是确定肌球蛋白V为基础的运输调节机制。

DESCRIPTION (provided by applicant): Organelle movement is a hallmark of cell division and cellular differentiation. In every cell-type, the localization, number and morphology of each type of organelle is modified to achieve specific cellular functions. Long-range movement of organelles occurs on microtubules, while positioning of organelles at their final destination relies on actin-based motors. Myosin V motors play a critical role in actin based movement in all eukaryotes, and disruption of myosin V function causes disease in humans. For example, partial defects in myosin Va based transport cause Griscelli's syndrome, characterized by neurological and pigmentation defects. Partial defects in myosin Vb cause microvillus inclusion disease, characterized by infantile, life threatening diarrhea. Currently there are no effective drug-based treatments for either disease. Determination of how myosin V based transport is achieved may provide important new insights into treating theseetach from cargoes at the correct time and place. The globular tail domain (GTD) of myosin V attaches to its cargoes through organelle-specific adaptor proteins. Regulation of the GTD and adaptor proteins contributes to specifying cargo attachment. We determined a high-resolution structure of the GTD of Myo2, a Saccharomyces cerevisiae myosin V motor, and identified two distinct cargo binding regions; one required for binding to the yeast vacuole, the other required for binding to secretory vesicles. We found that the vacuole binding site interacts with a novel protein, Vac17. The secretory vesicle binding site attaches directly to a Rab GTPase. Importantly, we found that the Rab GTPase binding site is conserved in human myosin Va and myosin Vb. Thus, our studies in yeast revealed the Rab GTPase binding site on human myosin V motors. The general conservation of the cargo binding domains of yeast Myo2 and human myosin Va and Vb, and the fact that Rab GTPases act directly to attach carns. Our major goals are to: 1) Determine whether the binding of an individual Myo2 adaptor protein enhances or inhibits binding of other adaptor proteins. 2) Identify and characterize proteins that regulate the attachment of Rab GTPases to the GTD of Myo2. 3) Determine mechanisms that regulate the detachment of myosin V from cargoes. PUBLIC HEALTH RELEVANCE: Intracellular transport of organelles by myosin V motors is crucial to normal cellular function, and animal physiology. Defects in myosin V based transport cause selected human diseases including neurological disorders. Our overall goal is to determine the mechanisms that regulate for myosin V-based transport.