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In vivo structure-function analysis and redox interactomes of Leishmania tarentolae Erv

ABSTRACT: Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other excavates lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here we used the CRISPR-Cas9 system to generate a set of homozygous tagged and untagged point and deletion mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using SILAC and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrates with twin Cx3C- or twin Cx9C-motifs were absent in both redox interactomes, we identified a small set of alternative interaction candidates with potential redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in non-opisthokonts.

INSTRUMENT(S): TripleTOF 5600, Orbitrap Fusion

ORGANISM(S): Leishmania Tarentolae

DISEASE(S): Leishmaniasis

SUBMITTER: David Zimmer

LAB HEAD: Marcel Deponte

PROVIDER: PXD027121 | Pride | 2021-07-09

REPOSITORIES: Pride

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			Action	DRS
	20190626TTFSSA148dpG001.wiff	Wiff
	20190626TTFSSA148dpG001.wiff.scan	Wiff
	20190626TTFSSA148dpG002.wiff	Wiff
	20190626TTFSSA148dpG002.wiff.scan	Wiff
	20190626TTFSSA148dpG003.wiff	Wiff

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Project description:Background: Prion diseases such as bovine spongiform encephalopathies (BSE) are transmissible neurodegenerative diseases which are presumably caused by an infectious conformational isoform of the cellular prion protein. Previous work has provided evidence that in murine prion disease the endogenous retrovirus (ERV) expression is altered in the brain. To determine if prion-induced changes in ERV expression are a general phenomenon we used a non-human primate model for prion disease. Results: Cynomolgus macaques (Macaca fasicularis) were infected intracerebrally with BSE-positive brain stem material from cattle and allowed to develop prion disease. Brain tissue from the basis pontis and vermis cerebelli of the six animals and the same regions from four healthy controls were subjected to ERV expression profiling using a retrovirus-specific microarray and quantitative real-time PCR. We could show that Class I gammaretroviruses HERV-E4-1, ERV-9, and MacERV-4 increase expression in BSE-infected macaques. In a second approach, we analysed ERV-K-(HML-2) RNA and protein expression in extracts from the same cynomolgus macaques. Here we found a significant downregulation of both, the macaque ERV-K-(HML-2) Gag protein and RNA in the frontal/parietal cortex of BSE-infected macaques. Conclusions: We provide evidence that dysregulation of ERVs in response to BSE-infection can be detected on both, the RNA and the protein level. To our knowledge, this is the first report on the differential expression of ERV-derived structural proteins in prion disorders. Our findings suggest that endogenous retroviruses may induce or exacerbate the pathological consequences of prion-associated neurodegeneration. Cynomolgus macaques (Macaca fasicularis) were infected intracerebrally with BSE-positive brain stem material from cattle and allowed to develop prion disease. Brain tissue from the basis pontis and vermis cerebelli of the six animals and the same regions from four healthy controls were subjected to ERV expression profiling using a retrovirus-specific microarray and quantitative real-time PCR. In a second approach, ERV-K-(HML-2) RNA and protein expression was analysed in extracts from the same cynomolgus macaques.

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In vivo structure-function analysis and redox interactomes of Leishmania tarentolae Erv

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