SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

LC-MS/MS Proteoform Profiling Exposes Cytochrome c Peroxidase Self-Oxidation in Mitochondria and Functionally Important Hole Hopping from Its Heme.

LC-MS/MS profiling reveals that the proteoforms of cytochrome c peroxidase (Ccp1) isolated from respiring yeast mitochondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccp1 by H2O2 in the absence of its reducing substrate, ferrocytochrome c, gives rise to similar proteoforms, indicating uncoupling of Ccp1 oxidation and reduction in mitochondria. The oxidative modifications found in the Ccp1 proteoforms are consistent with radical transfer (hole hopping) from the heme along several chains of redox-active residues (Trp, Met, Tyr). These modifications delineate likely hole-hopping pathways to novel substrate-binding sites. Moreover, a decrease in recombinant Ccp1 oxidation by H2O2 in vitro in the presence of glutathione supports a protective role for hole hopping to this antioxidant. Isolation and characterization of extramitochondrial Ccp1 proteoforms reveals that hole hopping from the heme in these proteoforms results in selective oxidation of the proximal heme ligand (H175) and heme labilization. Previously, we demonstrated that this labilized heme is recruited for catalase maturation (Kathiresan, M.; Martins, D.; English, A. M. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17468-17473; DOI: 10.1073/pnas.1409692111 ). Following heme release, apoCcp1 exits mitochondria, yielding the extramitochondrial proteoforms that we characterize here. The targeting of Ccp1 for selective H175 oxidation may be linked to the phosphorylation status of Y153 close to the heme since pY153 is abundant in certain proteoforms. In sum, when insufficient electrons from ferrocytochrome c are available to Ccp1 in mitochondria, hole hopping from its heme expands its physiological functions. Specifically, we observe an unprecedented hole-hopping sequence for heme labilization and identify hole-hopping pathways from the heme to novel substrates and to glutathione at Ccp1's surface. Furthermore, our results underscore the power of proteoform profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.

LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2.

We recently reported that cytochrome c peroxidase (Ccp1) functions as a H2O2 sensor protein when H2O2 levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (CycII), determines whether Ccp1 acts as a H2O2 sensor or peroxidase. For H2O2 to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H2O2 in the absence of CycII to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H2O2 at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H2O2 shuts down heterolytic cleavage of H2O2 at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H2O2 signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O2 reactivity.

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

Cytochrome c peroxidase is a mitochondrial heme-based H2O2 sensor that modulates antioxidant defense.

Hydrogen peroxide (H2O2) is a key signaling molecule that also induces apoptosis. Thus, cells must rapidly sense and tightly control H2O2 levels. Well-characterized cellular responses to exogenous H2O2 involve oxidation of specific cytosolic protein-based thiols but sensing of H2O2 generated by mitochondrial respiration is less well described. Here we provide substantial biochemical evidence that the heme enzyme Ccp1 (cytochrome c peroxidase), which is targeted to the intermembrane space, functions primarily as a mitochondrial H2O2 sensing and signaling protein in Saccharomyces cerevisiae. Key evidence for a sensing role for Ccp1 is the significantly higher H2O2 accumulation in ccp1-null cells(ccp1Δ) vs ccp1(W191F) cells producing the catalytically inactive Ccp1(W191F) variant. In fact, intracellular H2O2 levels (ccp1Δ>wildtype >ccp1(W191F)) correlate inversely with the activity of the mitochondrial (and peroxisomal) heme catalase, Cta1 (ccp1Δwildtype >ccp1(W191F)) and ccp1Δ cells exhibit low superoxide levels. Notably, Ccp1(W191F) is a more persistent H2O2 signaling protein than wild-type Ccp1, and this enhanced mitochondrial H2O2 signaling decreases the mitochondrial fitness of ccp1(W191F) cells. However, these cells are fully protected from a bolus (0.4mM) of exogenous H2O2 added after 12h of growth, whereas the viability of ccp1Δ cells drops below 20%, which additionally associates Ccp1 with Yap1-dependent H2O2 signaling. Combined, our results strongly implicate Ccp1, independent of its peroxidase activity, in mitochondrial H2O2 sensing and signaling to maintain reactive oxygen species homeostasis.

Biochemical conservation and evolution of germacrene A oxidase in asteraceae.

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.