| Summary: | 碩士 === 國立暨南國際大學 === 應用化學系 === 106 === Proteins, acting as downstream products of genes, are the key molecules that practically control most vital cellular functions. A protein must fold into its correct three-dimensional structure, before it executes its functions. Although many aspects of folding are intrinsic to the biophysical properties of the protein itself, the process is quite complex and susceptible to errors, particularly in cancer cells and aged neural tissues. Once proteins are folded incorrectly, they inhibit cell proliferation, promote cell death and thereby induce many human diseases if the misfolded proteins are left uncorrected. When proteasome is impaired, ubiquitinated proteins are accumulated and then bound by the deacetylase HDAC6. The HDAC6-ubiquitinated protein complex are then transported by Dynein along microtubule to the cell center, where aggresome is formed to cage and quarantine those toxic unfolded proteins. However, rare studies focus on the aggresome structure. To understand the detailed structure of aggresome, the study employes various microscopic methods to analyse the organelle. We found that aggresome is a huge and well-organized organelle with Vimentin caged outside and HDAC6 attached to it interiorly, and it takes around 22 hours to build the cone-like structure with a pointed end upward and an open end downeard. Such a giant architecture likely requires tremendous amount of de novo protein synthesis. Hence, the study focuses itself on testing the idea by examing the role of a transcription factor NF-кB in the process of aggresome formation. We found NF-кB was targeted to nucleus and aggresome when MG132 was added to induce aggresome formation in A549 cells, which effectively generate a hugh aggresome upon the exposure to the proteasome inhibitor MG132. Knockdown of NF-кB impaired aggresome formation and disturbed subsequent targeting of autophagolysosome. The rapid nuclear targeting of NF-кB suggests that NF-кB is likely regulated by upstream factors with quick reponse to MG132. To our surprise, numerous NF-кB upstream regulators were found, including HDAC6, PRMT5, HURP, Ajuba and IкB, regulating the protein level or the nuclear targeting of NF-кB during aggresome formation. Finally, we defined stable microtubule, a subset of microtubule with acetylated or detyrosinated tubulin, as the downstream target of NF-кB. Reduction of acetylated and detyrosinated tubulin decreased aggresome size and impaired ubiquitin, autophagosome amd lysosome targeting to aggresome. In conclusion, the study manifests itself that NF-кB, under the regulation of numerous factors, participates in aggresome construction partially by upregulating stable microtubule to recruit autophagolysosome and subsequently clear the caged ubiquitinated misfolded proteins, which are highly toxic to cells.
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