Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid
Curation statements for this article:-
Curated by eLife
eLife assessment
This is a potentially important study that deals with the toxic effects of an intermediary in lipid degradation [trans-2-hexadecenal (t-2-hex)] in yeast through modification of mitochondrial protein import via the TOM complex. However, in the current version, the claims are incompletely supported by the data. Lacking is evidence that Tom40 is a direct target of the lipid derivative or causally implicated in the described consequences. Were such evidence forthcoming, the paper would be interesting to a broad audience of molecular and cell biologists.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Molecular Biology (eLife)
Abstract
Mitochondria mediated cell death is critically regulated by bioactive lipids derived from sphingolipid metabolism. The lipid aldehyde trans-2-hexadecenal (t-2-hex) induces mitochondrial dysfunction in a conserved manner from yeast to humans. Here we apply unbiased transcriptomic, functional genomics and chemoproteomic approaches in the yeast model to uncover the principal mechanisms and biological targets underlying this lipid-induced mitochondrial inhibition. We find that loss of Hfd1 fatty aldehyde dehydrogenase function efficiently sensitizes cells for t-2-hex inhibition and apoptotic cell death. Excess of t-2-hex causes a profound transcriptomic response with characteristic hallmarks of impaired mitochondrial protein import like activation of mitochondrial and cytosolic chaperones or proteasomal function and severe repression of translation. We confirm that t-2-hex stress induces rapid accumulation of mitochondrial pre-proteins and protein aggregates and subsequent activation of Hsf1- and Rpn4-dependent gene expression. By saturated transposon mutagenesis we find that t-2-hex tolerance requires an efficient heat shock response and specific mitochondrial and ER functions and that mutations in ribosome, protein and amino acid biogenesis are beneficial upon t-2-hex stress. We further show that genetic and pharmacological inhibition of protein translation causes t-2-hex resistance indicating that loss of proteostasis is the predominant consequence of the pro-apoptotic lipid. Hfd1 associates with the Tom70 subunit of the TOM complex and t-2-hex covalently lipidates the central Tom40 channel, which altogether indicates that transport of mitochondrial precursor proteins through the outer mitochondrial membrane is directly inhibited by the pro-apoptotic lipid and thus represents a hotspot for pro- and anti-apoptotic signaling.
Article activity feed
-
-
-
eLife assessment
This is a potentially important study that deals with the toxic effects of an intermediary in lipid degradation [trans-2-hexadecenal (t-2-hex)] in yeast through modification of mitochondrial protein import via the TOM complex. However, in the current version, the claims are incompletely supported by the data. Lacking is evidence that Tom40 is a direct target of the lipid derivative or causally implicated in the described consequences. Were such evidence forthcoming, the paper would be interesting to a broad audience of molecular and cell biologists.
-
Reviewer #1 (Public Review):
Summary:
Fita-Torró et al. study the toxic effects of the intermediary lipid degradation product trans-2-hexadecenal (t-2-hex) on yeast mitochondria and suggest a mechanism by which Hfd1 safeguards Tom40 from lipidation by t-2-hex and its consequences, such as mitochondrial protein import inhibition, cellular proteostasis deregulation, and stress-responses.
The authors aimed to dissect a mechanism for t-2-hex' apoptotic consequences in yeast and they suggest it is via lipidation of Tom40 but really under the tested conditions everything seems lipidated. Thus, it is unclear whether Tom40 is the crucial causal target. They also do not provide much biochemical experiments to investigate this phenomenon further functionally. Tom40 is one possible and perhaps, given the cellular consequences, a reasonable …Reviewer #1 (Public Review):
Summary:
Fita-Torró et al. study the toxic effects of the intermediary lipid degradation product trans-2-hexadecenal (t-2-hex) on yeast mitochondria and suggest a mechanism by which Hfd1 safeguards Tom40 from lipidation by t-2-hex and its consequences, such as mitochondrial protein import inhibition, cellular proteostasis deregulation, and stress-responses.
The authors aimed to dissect a mechanism for t-2-hex' apoptotic consequences in yeast and they suggest it is via lipidation of Tom40 but really under the tested conditions everything seems lipidated. Thus, it is unclear whether Tom40 is the crucial causal target. They also do not provide much biochemical experiments to investigate this phenomenon further functionally. Tom40 is one possible and perhaps, given the cellular consequences, a reasonable candidate but not validated beyond in vitro lipidation by exogenous t-2-hex.Strengths:
The effects of lipids and their metabolic intermediates on protein function are understudied thus the authors' research contributing to elucidating direct effects of a single lipid is appreciated. It is particularly unknown by which mechanism t-2-hex causes cell death in yeast. The authors elegantly use modulation of the levels of enzyme Hfd1 that endogenously catabolizes t-2-hex as an approach to studying t-2-hex stress. Understanding the cause and consequences of this stress is relevant for understanding fundamental regulation mechanisms, and also to human health since the human homolog of Hfd1, ALDH3A2, is mutated in Sjögren-Larsson Syndrome. The application of a variety of global transcriptomic, functional genomic, and chemoproteomic approaches to study t-2-hex stress targets in the yeast model is laudable.Weaknesses:
- The extent of the contribution of Tom40 lipidation to the general t-2-hex stress phenotype is unclear. Is Tom40 lipidation alone enough to cause the phenotype? An alteration of the cysteine residue in question could help answer this key question.
- It is unclear whether the exogenously applied amounts of t-2-hex (concentrations chosen between 25-200 uM) are physiologically relevant in yeast cells. For comparison, Chipuk et al. (2012) used at most 1 uM on mitochondria of human cells, while Jarugumilli et al. (2018) considered 25 uM a 'lower dose' on human cells. Since the authors saw responses below 10 uM (Fig. 3B) and at the lowest selected concentration of 25 uM (Fig. 8), why were no lower, likely more specific, concentrations applied for the global transcriptomic and chemoproteomic experiments? Key experiments have to be repeated with the lower concentrations.
- The amount of t-2-hex applied is especially important to consider in light of over 1300 proteins lipidated to an extent equal to or greater than Tom40 (Supp. Table 6). This chemoproteomic experiment (Fig. 8B, Supp. Table 6) is also weakened by the inclusion of only 2 replicates, thus precluding assessment of statistical significance. The selection of targets in Fig. 8B as "among the best hits" is neither immediately comprehensible nor further explained and represents at best cherry-picking. Further evidence based on statistical significance or validation by other means should be provided.
- The authors unfortunately also underuse the possible contribution of mass spectrometry technology to in addition determine the extent and localization of lipidation on a global scale (especially relevant since Cohen et al. (2020) suggest site-specific mechanisms).
- The general novelty of studying t-2-hex stress is lowered in light of existing literature in humans (see e. g. Chipuk et al., 2012; Cohen et al., 2020; Jarugumilli et al., 2018), and in yeast by the same authors (Manzanares-Estreder et al., 2017) and as the authors comment themselves, a significant part of the manuscript may represent rather a confirmation of the already described consequences of t-2-hex stress -
Reviewer #2 (Public Review):
This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress, and causes the accumulation of mitochondrial precursor proteins in the cytosol.
The data shown are of high quality and well controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. The last part of the study is less convincing. The authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hex-resistant TOM mutant was not identified. …Reviewer #2 (Public Review):
This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress, and causes the accumulation of mitochondrial precursor proteins in the cytosol.
The data shown are of high quality and well controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. The last part of the study is less convincing. The authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hex-resistant TOM mutant was not identified. Moreover, it is not clear whether the concentrations of t-2-hex in this study are physiological. This is, however, a critical aspect. The literature is full of studies claiming the toxic effects of compounds such as H2O2; even if such studies are technically sound, they are misleading if non-physiological concentrations of such compounds were used.
Nevertheless, this is an interesting study of high quality. A few specific aspects should be addressed. -
Reviewer #3 (Public Review):
Summary: The authors investigate the effect of the lipid aldehyde trans-2-hexadecenal (t-2-hex) in yeast using multiple omic analyses that show that a large range of cellular functions across all compartments are affected, e.g. transcriptomic changes affect 1/3 of all genes. The authors provide additional analyses, from which they built a model that mitochondrial protein import caused by modification of Tom40 is blocked.
Strengths: Global analyses (transcriptomic and functional genomics approach) to obtain an unbiased overview of changes upon t-2-hex treatment.
Weaknesses: It is not clear why the authors decided to focus on mitochondria, as only 30 genes assigned to the GO term "mitochondria" are increasing, and also the follow-up analyses using SATAY is not showing a predominance for mitochondrial proteins …
Reviewer #3 (Public Review):
Summary: The authors investigate the effect of the lipid aldehyde trans-2-hexadecenal (t-2-hex) in yeast using multiple omic analyses that show that a large range of cellular functions across all compartments are affected, e.g. transcriptomic changes affect 1/3 of all genes. The authors provide additional analyses, from which they built a model that mitochondrial protein import caused by modification of Tom40 is blocked.
Strengths: Global analyses (transcriptomic and functional genomics approach) to obtain an unbiased overview of changes upon t-2-hex treatment.
Weaknesses: It is not clear why the authors decided to focus on mitochondria, as only 30 genes assigned to the GO term "mitochondria" are increasing, and also the follow-up analyses using SATAY is not showing a predominance for mitochondrial proteins (only 4 genes are identified as hits). The provided additional experimental data do not support the main claims as neither protein import is investigated nor is there experimental evidence that lipidation of Tom40 occurs in vivo and impacts on protein translocation.
-
