73-Deoxychondropsin A: A specific inhibitor of resorption and osteosarcoma?

Long/Grigoriadis实验室之前的工作已经确定了73-脱氧软骨蛋白A(73-DOC)在骨生物学中的一个新角色。73-DOC已从大堡礁海绵I.ramosa中分离出来，并由Paul Long与澳大利亚海洋科学研究所(AIMS)合作提纯。73-DOC对哺乳动物的V-ATPase有很强的抑制活性。V-ATPase通过调节细胞内外的pH，在细胞的基本生理过程中发挥作用。此外，对于骨组织，V-ATPase在骨吸收破骨细胞中至关重要，因为骨吸收破骨细胞需要V-ATPase才能正常发挥功能。Long/Grigoriadis实验室的初步和尚未发表的结果已经确立了73-DOC在靶向破骨细胞中的独特作用，尽管由于V-ATPase蛋白质复合体的复杂性，73-DOC的确切靶点及其作用机制尚不清楚。本项目将研究73-DOC对破骨细胞吸收的具体影响，并将其扩展到破骨细胞参与肿瘤进展的骨癌模型。该项目的假设是，73-DOC通过靶向破骨细胞和骨肉瘤等肿瘤之间共享的V-ATPase亚基亚基，特异性地抑制破骨细胞的吸收，而不影响骨重建，因此应该能有效地抑制吸收和肿瘤的发病。总体而言，73-DOC给药对破骨细胞吸收和骨肉瘤的影响将采用表型、生化和蛋白质组学相结合的方法进行综合表征。具体目的：研究73-DOC对破骨细胞和骨肉瘤细胞表型和功能的影响。确定73-DOC是否通过靶向特定亚基异构体来抑制V-ATPase活性，并确定73-DOC介导的V-ATPase抑制的分子机制。评估73-DOC介导的抑制特定V-ATPase亚型是否是治疗骨溶解疾病和骨肉瘤的可行方法。工作计划将遵循从新的73-DOC化合物的纯化、体外分析造血干细胞向破骨细胞分化、功能吸收分析和表面轮廓测量法定量、骨肉瘤细胞生长和活性、细胞骨架分析/共聚焦成像、质谱学/蛋白质组学分析和体内肿瘤建模的逻辑进展。因此，该项目将为学生提供各种不同的和互补的技能，包括细胞生物学和哺乳动物细胞培养，分子分析(qPCR，RNAi策略)，蛋白质生物化学/质谱仪，共聚焦显微镜，以及肿瘤发展的体内模型，包括组织学和免疫定位分析。这些研究方法将确保在博士学位的时间范围内实现指定的目标。

Previous work by the Long/Grigoriadis labs have identified a novel role for 73-deoxychondropsin A (73-DOC) in bone biology. 73-DOC has been isolated from the Great Barrier Reef sponge, I. ramosa, and purified by Paul Long in collaboration with the Australian Institute of Marine Sciences (AIMS). 73-DOC exhibits potent inhibitory activity to mammalian V-ATPases. V-ATPases have a role in fundamental cellular physiology processes through regulation of intra/extracellular pH. Additionally, for bone tissue, V-ATPases are critically important in bone-resorbing osteoclasts which require V-ATPases for normal function. Preliminary and yet unpublished results from the Long/Grigoriadis labs have established a unique role for 73-DOC in targeting osteoclasts, although due to the complexity of the V-ATPase protein complex, the precise targets of 73-DOC and its mechanism of action are not yet known. This project will investigate the specific effects of 73-DOC on osteoclastic resorption and extend this to a bone cancer model in which osteoclasts are implicated in tumour progression. The hypothesis of this project is that 73-DOC specifically inhibits osteoclast resorption, without affecting bone remodeling by targeting V-ATPase subunit isoforms shared between osteoclasts and tumors such as osteosarcoma and thus should effectively inhibit resorption and tumor pathogenesis. Overall, the effects of 73-DOC administration on osteoclast resorption and osteosarcoma will be characterized comprehensively using a combined phenotypic, biochemical, and proteomic approach. Specific aims: Characterize the effect of 73-DOC administration on the phenotype and functioning of osteoclast and osteosarcoma cells. Determine whether 73-DOC inhibits V-ATPase activity by targeting specific subunit isoforms and determine the molecular mechanics of 73-DOC mediated V-ATPase inhibition. Assess whether 73-DOC mediated inhibition of specific V-ATPases isoforms is a viable approach to therapeutically managing osteolytic diseases and osteosarcoma.The plan of work will follow a logical progression from purification of new 73-DOC compound, in vitro assays on osteoclast differentiation from haematopoietic stem cells, functional resorption assays and quantification by surface profilometry, osteosarcoma cell growth and activity, cytoskeletal analysis/confocal imaging, mass spec/proteomics analysis, and in vivo tumour modelling. The project therefore will provide the student with a variety of diverse and complementary skills, including cell biology and mammalian cell culture, molecular analyses (qPCR, RNAi strategies), protein biochemistry/mass spec, confocal microscopy, and in vivo models of tumour development, including histological and immunolocalisation analyses. These research methods will ensure that the specified aims are met within the time-frame of the PhD.