RAB GTP BINDING PROTEINS AND CELL GROWTH

Rab蛋白包含哺乳动物细胞中最大的Ras相关GTP酶组。 尽管已知这些蛋白质中有几种调节特定的囊泡运输途径，但许多蛋白质尚未具有指定的功能。 目前的研究已经揭示了一种这样的Rab蛋白Rab24表现出与其他家族成员不同的性质：例如，Rab24具有非常低的内在GT3活性，不能被类异戊二烯脂质有效地修饰，并且与已知的Rab载体蛋白弱缔合。 当一个假定的显性负（核苷酸结合缺陷）Rab24突变体在培养的细胞中表达，它似乎诱导一个惊人的阵列的自噬空泡和核包涵体。 基于这些初步观察，已经发展了以下假设：（a）Rab24在哺乳动物细胞中正常地在自噬空泡的形成或成熟中起作用。 (b)Rab24的突变形式触发自噬细胞死亡级联，其涉及降解空泡的积累及其与细胞核的最终融合。 拟议研究的中心目标是检验这些假设。为此，重组Rab24的生化特性将被定义，Rab24的表达，核苷酸状态和定位将被研究与肿瘤细胞模型中的自噬相关（目的I）。遗传和生物化学策略将用于鉴定和表征新型Rab24效应物或载体蛋白（目的2）。免疫细胞化学和生物化学方法将用于进一步确定Rab24突变体诱导的空泡样结构的性质（目的3）。 结构域交换和定点诱变将用于区分哪些结构特征使得Rab24在Rab家族中是独特的，当以核苷酸缺陷形式表达时能够诱导自噬反应（目的4）。 最后，新的细胞可渗透的融合多肽将被用来测试的可能性，合成肽模拟独特的Rab24序列元素可能被用来触发转化细胞中的自噬死亡。 拟议的研究将提供有关Rab24的生物学功能和调节细胞巨自噬的机制知之甚少的新信息。 最终，这些见解可以揭示恶性细胞治疗干预的新靶点。

Rab proteins comprise the largest group of Ras-related GTPases in mammalian cells. Although several of these proteins are known to regulate specific vesicular trafficking pathways, many do not yet have an assigned function. Current studies have revealed that one such Rab protein, Rab24, exhibits properties distinct from other family members: For example, Rab24 has a very low intrinsic GTPase activity, is not efficiently modified by isoprenoid lipids and associates weakly with known Rab carrier proteins. When a putative dominant-negative (nucleotide-binding deficient) Rab24 mutant was expressed in cultured cells, it appeared to induce a striking array of autophagic vacuoles and nuclear inclusion bodies. Based on these preliminary observations, the following hypotheses have been developed: (a) Rab24 normally functions in the formation or maturation of autophagic vacuoles in mammalian cells. (b) Mutant forms of Rab24 trigger an autophagic cell death cascade that involves accumulation of degradative vacuoles and their ultimate fusion with the nucleus. The central objective of the proposed studies is to test these hypotheses. Toward this end, the biochemical properties of recombinant Rab24 will be defined, and the expression, nucleotide state, and localization of Rab24 will be studied in relation to autophagy in tumor cell models (Aim I). Genetic and biochemical strategies will be used to identify and characterize novel Rab24 effectors or carrier proteins (Aim 2). immunocytochemical and biochemical methods will be used to further define the nature of the vacuole-like structures induced by the Rab24 mutants (Aim 3). Domain-swapping and site-directed mutagenesis will be used to distinguish which structural features make Rab24 unique among the Rab family in being able to induce an autophagic response when expressed in the nucleotide-deficient form (Aim 4). Finally, novel cell-permeable fusion polypeptides will be used to test the possibility that synthetic peptides mimicking unique Rab24 sequence elements might be used to trigger autophagic death in transformed cells. The proposed studies will provide new information about the biological function of Rab24 and the poorly understood mechanisms that regulate cellular macroautophagy. Ultimately, these insights could reveal new targets for therapeutic intervention in malignant cells.