Dataset Information

Proteomics effects of HSP60 deficiency in HEK cells, using an inducible transgenic HSP60 ATPase deficient mutant.

ABSTRACT: To investigate the immediate effect of HSP60 dysfunction on mitochondrial functions, we induced the expression of a dominant-negative ATPase-deficient HSP60 mutant (HSP60-D423A) in HEK293 cells using tetracycline . Incorporation of HSP60-D423A subunits into HSP60 heptamer rings leads to dysfunction of the chaperonin complex. We titrated tetracycline levels to have a biologically relevant cellular model that can be monitored over time. Expression of the HSP60-D432A protein resulted in significantly reduced cell counts at 72 hours induction compared to uninduced and HSP60-WT co-expressing cells. To study the effects of onset of HSP60 deficiency, we monitored the proteomics changes using TMT based proteomics.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Kidney Cell

SUBMITTER: Johan Palmfeldt

LAB HEAD: Johan Palmfeldt

PROVIDER: PXD048918 | Pride | 2025-05-06

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Cagla_TMT-HEK-PD3.mzML	Mzml
	Cagla_TMT-HEK-PD3.mzid.gz	Mzid
	X2102-Cagla-TMT-HEK-2-excl.raw	Raw
	X2102-Cagla-TMT-HEK-2.raw	Raw
	X2104-Cagla-TMT-HEK-3-excl.raw	Raw

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Publications

HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis.

Cömert Cagla C Kjær-Sørensen Kasper K Hansen Jakob J Carlsen Jasper J Just Jesper J Meaney Brandon F BF Østergaard Elsebet E Luo Yonglun Y Oxvig Claus C Schmidt-Laursen Lisbeth L Palmfeldt Johan J Fernandez-Guerra Paula P Bross Peter P

Molecular metabolism 20240814

<h4>Objective</h4>Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized.<h4>Meth ...[more]

PMID: 39147275

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Project description:RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. Gene array analysis shows that RPF-1 transcription factor modify the transcriptome of HEK293 cells by regulating a genetic program endowed with developmental programs Comparison of HEK293 cells induced for RPF-1 expression (Tet-) over uninduced control (Tet+) and Mock cells. RPF-1 cDNA was inserted into the pTRE2puro vector to generate stable Tet-inducible KEK293 transfectants. Upon Tet removal from the medium, HEK/RPF-1 transfectants expressed the transcription factor RPF-1. Cells were then seeded in 10 cm dishes, cultured for 48 h either in complete medium supplemented with Tet (+) or in Tet-depleted medium (-), harvested, and spun down before RNA isolation. Comparative analysis between HEK/RPF-1 induced (Tet-) and uninduced (Tet+) transfectants allowed us to identify genes transcriptionally regulated by RPF-1. Residual effects on gene expression due to Tet supplementation into culture media was ruled out by comparison with the Mock transfectant.