With early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme's key kinetic obstacle—how to maintain catalytic speed on a cooler Earth—by exploiting transition-state heat capacity. Tracing the evolution of enzyme activity and stability from the hot-start toward modern hyperthermophilic, mesophilic, and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level, refutes the debated activity/stability trade-off, and suggests that the catalytic speed of adenylate kinase is an evolutionary driver for organismal fitness.

Triple-negative breast cancer (TNBC) is traditionally considered a glycolytic tumor with a poor prognosis while lacking targeted therapies. Here we show that high expression of dihydrolipoamide S-succinyltransferase (DLST), a tricarboxylic acid (TCA) cycle enzyme, predicts poor overall and recurrence-free survival among TNBC patients. DLST depletion suppresses growth and induces death in subsets of human TNBC cell lines, which are capable of utilizing glutamine anaplerosis. Metabolomics profiling reveals significant changes in the TCA cycle and reactive oxygen species (ROS) related pathways for sensitive but not resistant TNBC cells. Consequently, DLST depletion in sensitive TNBC cells increases ROS levels while N-acetyl-L-cysteine partially rescues cell growth. Importantly, suppression of the TCA cycle through DLST depletion or CPI-613, a drug currently in clinical trials for treating other cancers, decreases the burden and invasion of these TNBC. Together, our data demonstrate differential TCA-cycle usage in TNBC and provide therapeutic implications for the DLST-dependent subsets.

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ADP-ribosylation is a reversible post-translational modification where an ADP-ribose moiety is covalently attached to amino acid side-chains of target proteins either as mono-ADP-ribose (MARylation or MAR) or poly-ADP-ribose chains (PARylation or PAR) by a class of enzymes called ADP-ribosyltransferases (ARTs). Although ADP-ribosylation is best known for its nuclear roles, ADP-ribosylation of extra nuclear proteins is increasingly recognized as a key regulatory strategy across cellular compartments. ADP-ribosylation of mitochondrial proteins, in particular, has been widely reported, even though the extent to which ADP-ribosylation of specific proteins regulates mitochondrial functions is unclear and the exact nature of mitochondrial ART enzymes is debated.Here, we have identified Neuralized-like protein 4 (NEURL4) as a mitochondrial ART enzyme and profiled the NEURL4-dependent ADP-ribosylome in mitochondrial extracts from Hela cells by LC-MS/MS, using isobaric tandem mass tag (TMT) labeling for relative quantification. Comparison of WT and NEURL4-KO cells generated by CRISPR/Cas9 genome editing revealed that most ART activity associated with mitochondria is lost in absence of NEURL4. Putative NEURL4 targets include numerous mitochondrial proteins previously shown to be ADP-ribosylated. In particular, we show that NEURL4 enzymatic activity is required for the regulation of mtDNA integrity via poly-ADP-ribosylation of mitochondrial specific Ligase III (mtLIG3), the rate-limiting enzyme for mitochondrial DNA (mtDNA) Base Excision Repair (BER).Collectively, our studies reveal that NEURL4 acts as the main mitochondrial ART enzyme under physiological conditions and provide novel insights in the regulation of mitochondria homeostasis through ADP-ribosylation.