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Characterizing the role of OCIAD1 in the proteolytic processing of holocytochrome c1 and CIII2 assembly

ABSTRACT: To characterize the nature of the cytochrome c1 (CYC1) processivity defect in native CIII2 assemblies we identified CYC1 peptides using mass spectrometry analysis of blue native (BN)-PAGE. Gel slices ranging from ~600-900kDa and containing CIII2 assemblies were excised from U2OS control cells, U2OS OCIAD1 knockdown cells, and U2OS OCIAD1 knockdown cells rescued with wildtype OCIAD1. Gel slices were then digested and analyzed by mass spectrometry.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Maxence Le Vasseur

LAB HEAD: Jodi Nunnari

PROVIDER: PXD025576 | Pride | 2021-05-18

REPOSITORIES: Pride

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			Action	DRS
	191118_DIAMAX_05_Slot1-58_1_454.d.zip	Other
	191118_DIAMAX_G10_Slot1-62_1_459.d.zip	Other
	191118_DIAMAX_G11_Slot1-45_1_439.d.zip	Other
	191118_DIAMAX_G12_Slot1-57_1_453.d.zip	Other
	191118_DIAMAX_G13_Slot1-69_1_467.d.zip	Other

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Project description:We describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on sequential DNA digestion with a pair of methylation-blocked and methylation-tolerant neoschizomeric restriction enzymes SmaI/XmaI followed by end repair and ultra-deep sequencing. DREAM provides information on 160,000 unique CpG sites of which 39,000 are in CpG islands, and 33,000 are at transcription start sites (-1 kb to +1 kb) of 13,139 RefSeq genes. We compared DNA methylation values in white blood cells from 4 healthy individuals and found them to be remarkably uniform. Interindividual differences >30% were observed only at 227 of 28,331 (0.8%) of autosomal CCCGGG sites covered by 100+ sequencing reads. Similarly, differences at only 59 sites were observed between the cord and adult blood. Conserved methylation patterns in healthy blood cells contrasted with extensive changes affecting 18-40% of CpG sites in leukemia. The method is cost effective, quantitative (r2=0.93 when compared to bisulfite pyrosequencing), reproducible (r2=0.997), and can detect differences >25% with false positive rate <0.001. Accurate analysis of changes in DNA methylation will be useful in quantifying epigenetic effects of environment and nutrition, correlating developmental epigenetic variation with phenotypes, understanding epigenetics of cancer and chronic diseases, measuring the effects of drugs on DNA methylation or deriving new biological insights into mammalian genomes. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the DNA methylation profiles of healthy white blood cells from cord blood and adult blood, acute myeloid leukemia bone marrow, and two leukemia cell lines (HEL and K562). In this approach, genomic DNA is sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represent methylation status of the CCCGGG sites. These end sequences are analyzed by next-generation sequencing, and thereafter the methylation status at individual CCCGGG sites across the genome can be determined.

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Characterizing the role of OCIAD1 in the proteolytic processing of holocytochrome c1 and CIII2 assembly

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