Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid ...peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation.
The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which ...nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.
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•Cells starve for cholesterol and iron under lysosomal dysfunction•Upon increased lysosomal pH, only iron addition enables cell proliferation•Iron does not restore lysosomal pH-related catabolic and signaling functions•Iron reverses other cellular processes related to depleted cellular iron
The lysosome is a multi-functional organelle whose low pH is required for cell viability. Weber et al. identified iron as necessary and sufficient for cell proliferation under lysosomal dysfunction. While iron addition uncouples lysosomal acidity from cell viability, iron chelation combined with lysosome-targeting compounds represents a potential cancer therapeutic strategy.
Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for DNA alkylation-induced necrosis, but its function and substrates remain unclear. Herein, we show ALKBH7 regulates ...dialdehyde metabolism, which impacts the cardiac response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we find no evidence ALKBH7 functions as a prolyl-hydroxylase, but we do find
mice have elevated glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Metabolic pathways related to the glycolytic by-product methylglyoxal (MGO) are rewired in
mice, along with elevated levels of MGO protein adducts. Despite greater glycative stress, hearts from
mice are protected against IR injury, in a manner blocked by GLO-1 inhibition. Integrating these observations, we propose ALKBH7 regulates glyoxal metabolism, and that protection against necrosis and cardiac IR injury bought on by ALKBH7 deficiency originates from the signaling response to elevated MGO stress.
Heme‐containing proteins have recently attracted increasing attention for their ability to promote synthetically valuable transformations not found in nature. Following the recent discovery that ...engineered variants of myoglobin can catalyze the direct conversion of organic azides into aldehydes, we investigated the azide oxidative deamination reactivity of a variety of hemoproteins featuring different heme coordination environments. Our studies show that although several heme‐containing enzymes possess basal activity in this reaction, an engineered variant of the bacterial cytochrome P450 CYP102A1 constitutes a particularly efficient biocatalyst for promoting this transformation, and it exhibits a broad substrate scope along with high catalytic activity (up to 11 300 turnovers), excellent chemoselectivity, and enhanced reactivity toward secondary alkyl azides to yield ketones. Mechanistic studies and Michaelis–Menten analyses provided insight into the mechanism of the reaction and the impact of active‐site mutations on the catalytic properties of the P450. Altogether, these studies demonstrate that engineered P450 variants represent promising biocatalysts for the synthesis of aryl aldehydes and ketones through the oxidative deamination of alkyl azides under mild reaction conditions.
Move azide: An engineered variant of CYP102A1 efficiently catalyzes the oxidative deamination of primary alkyl azides to yield aldehydes and exhibits a broad substrate scope along with high catalytic activity and excellent chemoselectivity. The enhanced reactivity of this enzyme also enables the conversion of selected secondary alkyl azides into ketones.
The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward ...improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product. Via P450-mediated chemoenzymatic synthesis, a broad panel of novel micheliolide analogs could thus be obtained to gain structure–activity insights into the effect of C2, C4, and C14 substitutions on the antileukemic activity of micheliolide, ultimately leading to the discovery of “micheliologs” with improved potency against acute myelogenic leukemia cells. These late-stage C–H functionalization routes could be further leveraged to generate a panel of affinity probes for conducting a comprehensive analysis of the protein targeting profile of micheliolide in leukemia cells via chemical proteomics analyses. These studies introduce new micheliolide-based antileukemic agents and shed new light onto the biomolecular targets and mechanism of action of micheliolide in leukemia cells. More broadly, this work showcases the value of the present P450-mediated C–H functionalization strategy for streamlining the late-stage diversification and elucidation of the biomolecular targets of a complex bioactive molecule.
The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural ...products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (Topt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.
Handling the heat: Consensus‐guided mutagenesis was applied to stabilize the reductase domain of the biotechnologically useful P450BM3 enzyme. The evolved variants exhibited improved robustness to thermal denaturation and increased catalytic performance at elevated temperatures.
Previously published digital autoradiography of 3H‐labeled capecitabine reveals a near‐uniform distribution of activity throughout a murine pancreatic model. This is in contrast both to 14C‐labeled ...gemcitabine, and established expectations, as the dense stroma of pancreatic cancer is understood to inhibit drug penetration. Capecitabine is a pro‐drug for 5 FU. The positioning of the radiolabel on capecitabine leaves open the possibility that much of the autoradiographic signal is generated by nontoxic compounds. Studies were performed on tumors derived via organoid culture from a murine KPC tumor. As before, we performed autoradiography comparing 3H capecitabine to the gemcitabine analog 18F‐FAC. The metabolism of capecitabine in this model was studied through LC–MS of tumor tissue. The autoradiographs confirmed that the 3H label from capecitabine was much more uniformly distributed through the tumor than the 18F from the gemcitabine analog. LC–MS revealed that approximately 75% of the molar mass of capecitabine had been converted into 5 FU or pre‐5 FU compounds. The remainder had been converted into nontoxic species. Therapeutically relevant capecitabine metabolites achieve a relatively even distribution in this pancreatic cancer model, in contrast to the gemcitabine analog 18F‐FAC. In a human xenograft model, (BxPC3), the 3H label from capecitabine was also uniformly spread across the tumor autoradiographs. However, at 2 h post‐administration the metabolism of capecitabine had proceeded further and the bulk of the agent was in the form of nontoxic species.
Penetration of capecitabine‐derived species in models of pancreatic cancer.
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The plant-derived sesquiterpene lactone parthenolide (PTL) was recently found to possess promising anticancer activity but elaboration of this natural product scaffold for ...optimization of its pharmacological properties has proven challenging via available chemical methods. In this work, P450-catalyzed C–H hydroxylation of positions C9 and C14 in PTL was coupled to carbamoylation chemistry to yield a panel of novel carbamate-based PTL analogs (‘parthenologs’). These compounds, along with a series of other C9- and C14-functionalized parthenologs obtained via O–H acylation, alkylation, and metal-catalyzed carbene insertion, were profiled for their cytotoxicity against a diverse panel of human cancer cell lines. These studies led to the discovery of several parthenologs with significantly improved anticancer activity (2–14-fold) compared to the parent molecule. Most interestingly, two PTL analogs with high cytotoxicity (LC50∼1–3μM) against T cell leukemia (Jurkat), mantle cell lymphoma (JeKo-1), and adenocarcinoma (HeLa) cells as well as a carbamate derivative with potent activity (LC50=0.6μM) against neuroblastoma cells (SK-N-MC) were obtained. In addition, these analyses resulted in the identification of parthenologs featuring both a broad spectrum and tumor cell-specific anticancer activity profile, thus providing valuable probes for the future investigation of biomolecular targets that can affect cell viability across multiple as well as specific types of human cancers. Altogether, these results highlight the potential of P450-mediated chemoenzymatic C–H functionalization toward tuning and improving the anticancer activity of the natural product parthenolide.
Stress-induced cleavage of transfer RNAs (tRNAs) into tRNA-derived fragments (tRFs) occurs across organisms from yeast to humans; yet, its mechanistic underpinnings and pathological consequences ...remain poorly defined. Small RNA profiling revealed increased abundance of a cysteine tRNA fragment (5'-tRF
) during breast cancer metastatic progression. 5'-tRF
was required for efficient breast cancer metastatic lung colonization and cancer cell survival. We identified Nucleolin as the direct binding partner of 5'-tRF
. 5'-tRF
promoted the oligomerization of Nucleolin and its bound metabolic transcripts Mthfd1l and Pafah1b1 into a higher-order transcript stabilizing ribonucleoprotein complex, which protected these transcripts from exonucleolytic degradation. Consistent with this, Mthfd1l and Pafah1b1 mediated pro-metastatic and metabolic effects downstream of 5'-tRF
-impacting folate, one-carbon, and phosphatidylcholine metabolism. Our findings reveal that a tRF can promote oligomerization of an RNA-binding protein into a transcript stabilizing ribonucleoprotein complex, thereby driving specific metabolic pathways underlying cancer progression.
Insect societies are tightly integrated, complex biological systems in which group-level properties arise from the interactions between individuals
. However, these interactions have not been studied ...systematically and therefore remain incompletely known. Here, using a reverse engineering approach, we reveal that unlike solitary insects, ant pupae extrude a secretion derived from the moulting fluid that is rich in nutrients, hormones and neuroactive substances. This secretion elicits parental care behaviour and is rapidly removed and consumed by the adults. This behaviour is crucial for pupal survival; if the secretion is not removed, pupae develop fungal infections and die. Analogous to mammalian milk, the secretion is also an important source of early larval nutrition, and young larvae exhibit stunted growth and decreased survival without access to the fluid. We show that this derived social function of the moulting fluid generalizes across the ants. This secretion thus forms the basis of a central and hitherto overlooked interaction network in ant societies, and constitutes a rare example of how a conserved developmental process can be co-opted to provide the mechanistic basis of social interactions. These results implicate moulting fluids in having a major role in the evolution of ant eusociality.
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