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A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress.

ABSTRACT: The Atg4 cysteine proteases play crucial roles in the processing of Atg8 proteins during autophagy, but their regulation during cellular stress and differentiation remains poorly understood. We have found that two Atg4 family members--Atg4C and Atg4D--contain cryptic mitochondrial targeting sequences immediately downstream of their canonical (DEVD) caspase cleavage sites. Consequently, caspase-cleaved Atg4D (?N63 Atg4D) localizes to the mitochondrial matrix when expressed in mammalian cells, where it undergoes further processing to a ~42 kDa mitochondrial form. Interestingly, caspase cleavage is not needed for Atg4D mitochondrial import, because ~42 kDa mitochondrial Atg4D is observed in cells treated with caspase inhibitors and in cells expressing caspase-resistant Atg4D (DEVA(63)). Using HeLa cell lines stably expressing ?N63 Atg4D, we showed that mitochondrial Atg4D sensitizes cells to cell death in the presence of the mitochondrial uncoupler, CCCP, and that mitochondrial cristae are less extensive in these cells. We further showed that the organization of mitochondrial cristae is altered during the mitochondrial clearance phase in differentiating primary human erythroblasts stably expressing ?N63 Atg4D, and that these cells have elevated levels of mitochondrial reactive oxygen species (ROS) during late stages of erythropoiesis. Together these data suggest that the import of Atg4D during cellular stress and differentiation may play important roles in the regulation of mitochondrial physiology, ROS, mitophagy and cell viability.

SUBMITTER: Betin VM

PROVIDER: S-EPMC3405841 | biostudies-other | 2012 Apr

REPOSITORIES: biostudies-other

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A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress.

Betin Virginie M S VM MacVicar Thomas D B TD Parsons Stephen F SF Anstee David J DJ Lane Jon D JD

Autophagy 20120401 4

The Atg4 cysteine proteases play crucial roles in the processing of Atg8 proteins during autophagy, but their regulation during cellular stress and differentiation remains poorly understood. We have found that two Atg4 family members--Atg4C and Atg4D--contain cryptic mitochondrial targeting sequences immediately downstream of their canonical (DEVD) caspase cleavage sites. Consequently, caspase-cleaved Atg4D (ΔN63 Atg4D) localizes to the mitochondrial matrix when expressed in mammalian cells, whe ...[more]

PMID: 22441018

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Project description:Dengue virus infections, which have been reported in nearly 140 countries, pose a significant threat to human health. The genome of dengue virus encodes three structural and seven nonstructural (NS) proteins along with two untranslated regions, one each on both ends. Among them, dengue protease (NS3) plays a pivotal role in polyprotein processing and virus multiplication. NS3 is also known to regulate several host proteins to induce and maintain pathogenesis. Certain viral proteins are known to interact with mitochondrial membrane proteins and interfere with their functions, but the association of a virus-coded protein with the mitochondrial matrix is not known. In this report, by using in silico analysis, we show that NS3pro alone is capable of mitochondrial import; however, this is dependent on its innate mitochondrial transport signal (MTS). Transient-transfection and protein import studies confirm the import of NS3pro to the mitochondrial matrix. Similarly, NS3pro-helicase (amino acids 1 to 464 of NS3) also targets the mitochondria. Intriguingly, reduced levels of matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), were noticed in NS3pro-expressing, NS3pro-helicase-expressing, and virus-infected cells. Upon the use of purified components, GrpEL1 undergoes cleavage, and the cleavage sites have been mapped to KR81A and QR92S. Importantly, GrpEL1 levels are seriously compromised in severe dengue virus-infected clinical samples. Our studies provide novel insights into the import of NS3 into host mitochondria and identify a hitherto unknown factor, GrpEL1, as a cleavage target, thereby providing new avenues for dengue virus research and the design of potential therapeutics.IMPORTANCE Approximately 40% of the world's population is at risk of dengue virus infection. There is currently no specific drug or potential vaccine for these infections. Lack of complete understanding of the pathogenesis of the virus is one of the hurdles that must be overcome in developing antivirals for this virus infection. In the present study, we observed that the dengue virus-coded protease imports to the mitochondrial matrix, and our report is the first ever of a virus-coded protein, either animal or human, importing to the mitochondrial matrix. Our analysis indicates that the observed mitochondrial import is due to an inherited mitochondrial transport signal. We also show that matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), is also the substrate of dengue virus protease, as observed in vitro and ex vivo in virus-infected cells and dengue virus-infected clinical samples. Hence, our studies reveal an essential aspect of the pathogenesis of dengue virus infections, which may aid in developing antidengue therapeutics.

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A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress.

Publications

A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress.

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