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Determining the composition of unfolded barnase aggregates

ABSTRACT: We showed the phase separation or aggregation of globular proteins is dependent on the protein stability and the sticker composition of the unfolded protein using barnase, a model protein. Here, we determined the composition of barnase aggregates in cells to investigate how the stability and sequence grammar of the unfolded barnase influences interactions with the endogenous proteome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yoon Hee Choi

LAB HEAD: Danny Hatters

PROVIDER: PXD033716 | Pride | 2022-07-12

REPOSITORIES: Pride

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			Action	DRS
	210702_Yoon_YHC-2569_1.raw	Raw
	210702_Yoon_YHC-2569_2.raw	Raw
	210702_Yoon_YHC-2569_3.raw	Raw
	210702_Yoon_YHC-2569_4.raw	Raw
	210702_Yoon_YHC-2569_5.raw	Raw

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Project description:Inadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER. Microarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-time PCR analyses confirmed that four of these factors, VEGF, FGF2, angiogenin and IL-8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGF regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGF promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGF expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGF gene, although its contribution to VEGF transcription appeared to be fairly modest. We also found that VEGF mRNA stability is increased in response to UPR activation, via activation of the AMP and p38MAP kinases, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGF protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGF expression at multiple levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer. We used microarrays to perform genome wide expression analysis in Daoy medulloblastoma cells to identify gene profiles that change in response to thapsigargin treatment which causes induction of the unfolded protein response.

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Determining the composition of unfolded barnase aggregates

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