PERK 1,2 . By contrast, the effects of CDDO on CHOP were not blunted by any individual EIF2α kinase deficiency (Extended Data Fig. 1g) , possibly owing to the limited specificity of related compounds 11 . The comparative interrogation of CHOP regulators following three distinct cellular insults allowed us to differentiate global regulators of CHOP biology (Extended Data Fig. 1h-m) from such selectively operating in the context of CCCP-induced mitochondrial depolarization (Extended Data Figs. 2-4). In particular, stringent filtering for genes that prominently scored with CCCP, but not TM or CDDO, highlighted the transcriptional regulators TAF4 and GABPB1, glycolysis factors SLC2A1 and TPI1, and RNA binding proteins RBM27 and CLUH (KIAA0664). Moreover, the signature contained the mitochondrial proteins ATP5IF1 (ATPIF1) and OMA1. Most strikingly, it revealed a strong requirement for HRI (EIF2AK1) and the scarcely studied protein DELE1 (KIAA0141) (Fig. 1a, Extended Data Fig. 5a-b). Cellular dynamics of DELE1Given the scant knowledge on DELE1 and the unexpected involvement of HRI, we first sought to validate their requirement in a panel of cell systems including non-transformed cells, and indeed could confirm their importance in all cases (Extended Data Fig. 5c). Furthermore, CHOP induction also depended on DELE1 and HRI for other types of mitochondrial stress, including inhibition of complex V (oligomycin), TRAP1 (GTPP), and genetic ablation of LONP1 (Extended Data Fig. 5d-f). Failure to induce CHOP after stimulation with CCCP was preceded by a defect in EIF2α phosphorylation in HRI-or DELE1-deficient cells, suggesting that like HRI, DELE1 operates upstream of this event (Extended Data Fig. 5g). Strikingly, expression of HRI in DELE1 knockout cells was able to partially restore CHOP induction, whereas DELE1 expression in HRI-deficient cells was unproductive (Fig. 2a, Extended Data Fig. 6a-b). This indicated that DELE1 requires HRI to trigger CHOP but not vice versa, suggesting that DELE1 may act upstream of both EIF2α and HRI. Given that DELE1 is a mitochondrial protein 12 (Extended Data Fig. 6c), whereas HRI resides in the cytoplasm, we next wondered whether the activity of DELE1 towards HRI might be regulated by its localization. To test this hypothesis, we investigated if artificially rerouting DELE1 to the cytosol would bypass the need for a mitochondrial insult to provoke CHOP expression. Indeed, expression of a DELE1 mutant lacking the mitochondrial targeting sequence (DELE1 ∆MTS ) yielded a predominantly cytoplasmic protein that readily induced CHOP expression independently of CCCP (Fig. 2b-c, Extended Data Fig. 6d-e). This constitutively active version of DELE1 still required HRI to induce CHOP, underscoring its likely role as an activator of HRI. Based on these findings, we asked whether the activity of wild-type DELE1 might be regulated via a similar mechanism. Indeed, while DELE1 localized to mitochondria in unperturbed cells, it could be detected in the cytosol upon CCCP treatment (Fig. 2d). We did not ob...

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.

Mitochondrial fidelity is a key determinant of longevity and was found to be perturbed in a multitude of disease contexts ranging from neurodegeneration to heart failure. Tight homeostatic control of the mitochondrial proteome is a crucial aspect of mitochondrial function, which is severely complicated by the evolutionary origin and resulting peculiarities of the organelle. This is, on one hand, reflected by a range of basal quality control factors such as mitochondria-resident chaperones and proteases, that assist in import and folding of precursors as well as removal of aggregated proteins. On the other hand, stress causes the activation of several additional mechanisms that counteract any damage that may threaten mitochondrial function. Countermeasures depend on the location and intensity of the stress and on a range of factors that are equipped to sense and signal the nature of the encountered perturbation. Defective mitochondrial import activates mechanisms that combat the accumulation of precursors in the cytosol and the import pore. To resolve proteotoxic stress in the organelle interior, mitochondria depend on nuclear transcriptional programs, such as the mitochondrial unfolded protein response and the integrated stress response. If organelle damage is too severe, mitochondria signal for their own destruction in a process termed mitophagy, thereby preventing further harm to the mitochondrial network and allowing the cell to salvage their biological building blocks. Here, we provide an overview of how different types and intensities of stress activate distinct pathways aimed at preserving mitochondrial fidelity.

cytosine DnA bases can be methylated by DnA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. to dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation. DNA methylation plays critical roles in the epigenetic regulation of gene expression and genome stability in mammals 1. During mammalian development, methylated cytosine (5mC) serves as a critical epigenetic barrier to guide cell fate decisions and restrict developmental potential 2. Genomic 5mC patterns are established by the de novo DNA methyltransferases DNMT3A and DNMT3B and maintained through subsequent cell divisions by DNMT1 3. The mitotic inheritance of 5mC constitutes a form of epigenetic memory enabling the long term maintenance of cell identity. Extinguishing such memory requires extensive epigenetic reprogramming and is key for the acquisition of naive pluripotency (i.e. the capacity of cells to contribute to all lineages in the embryo) during development 4. In mammals, genome-wide erasure of 5mC accompanies the restoration of developmental potential following fertilization, reaching a nadir in the naive pluripotent inner cell mass (ICM) of the pre-implantation blastocyst 5-7. In turn, the transition from a naive pluripotent state to one "primed" for lineage commitment upon implantation coincides with the establishment of global DNA methylation patterns 8-10. The cellular landscape of 5mC can be altered by the inhibition of maintenance DNA methylation and/ or via the action of the Ten-eleven Translocation (TET) family of dioxygenases 11. The three mammalian