Genome-wide association studies (GWASs) of serum metabolites have the potential to uncover genes that influence human metabolism. Here, we combined an integrative genetic analysis that associates ...serum metabolites to membrane transporters with a coessentiality map of metabolic genes. This analysis revealed a connection between feline leukemia virus subgroup C cellular receptor 1 (FLVCR1) and phosphocholine, a downstream metabolite of choline metabolism. Loss of FLVCR1 in human cells strongly impairs choline metabolism due to the inhibition of choline import. Consistently, CRISPR-based genetic screens identified phospholipid synthesis and salvage machinery as synthetic lethal with FLVCR1 loss. Cells and mice lacking FLVCR1 exhibit structural defects in mitochondria and upregulate integrated stress response (ISR) through heme-regulated inhibitor (HRI) kinase. Finally, Flvcr1 knockout mice are embryonic lethal, which is partially rescued by choline supplementation. Altogether, our findings propose FLVCR1 as a major choline transporter in mammals and provide a platform to discover substrates for unknown metabolite transporters.
Display omitted
•Identification of metabolite transporter associations from GWAS of serum metabolites•FLVCR1 is required for choline uptake and metabolism•Loss of FLVCR1 impairs mitochondrial function leading to the activation of ISRmt•Flvcr1-mediated choline transport is necessary for murine embryogenesis
Kenny et al. query serum metabolite genome-wide association study (GWAS) data to identify an association between choline metabolism and feline leukemia virus subgroup C cellular receptor 1 (FLVCR1). Metabolite tracing, radioactive uptake experiments, and CRISPR screens demonstrate that FLVCR1 is the major plasma-membrane choline transporter. Highlighting the importance of choline metabolism, FLVCR1 loss severely compromises cellular and organismal physiology.
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of ...metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
The multidomain, catalytically self-sufficient cytochrome P450 BM-3 from Bacillus megaterium (P450
) 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 P450
. 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 P450
variants indicated that the introduced mutations increased the thermal stability of the FAD-binding domain and that the optimal temperature (T
) 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 P450
variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.
Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis ...is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.
Display omitted
Parthenolide is a naturally occurring terpene with promising anticancer properties, particularly in the context of acute myeloid leukemia (AML). Optimization of this natural product ...has been challenged by limited opportunities for the late-stage functionalization of this molecule without affecting the pharmacologically important α-methylene-γ-lactone moiety. Here, we report the further development and application of a chemoenzymatic strategy to afford a series of new analogs of parthenolide functionalized at the aliphatic positions C9 and C14. Several of these compounds were determined to be able to kill leukemia cells and patient-derived primary AML specimens with improved activity compared to parthenolide, exhibiting LC50 values in the low micromolar range. These studies demonstrate that different O–H functionalization chemistries can be applied to elaborate the parthenolide scaffold and that modifications at the C9 or C14 position can effectively enhance the antileukemic properties of this natural product. The C9-functionalized analogs 22a and 25b were identified as the most interesting compounds in terms of antileukemic potency and selectivity toward AML versus healthy blood cells.
Selective late-stage oxidation of ubiquitous C-H bonds in complex molecules is an important reaction for late-stage diversification of biologically active natural products. Cytochrome P450s are ...capable of selectively activating C(sp 3)-H bonds in a wide array of small molecules and complex natural products. This thesis will describe the design and application of engineered P450 enzymes for the late-stage functionalization of biologically active sesquiterpenoids. First, we report the development and application of a chemoenzymatic strategy to afford hydroxylation of parthenolide at the aliphatic positions C9(S) and C14 in high yields. P450-catalyzed C–H hydroxylation of positions C9 and C14 in parthenolide was coupled to various O-H functionalization chemistries to yield a panel of novel parthenolide analogs ('parthenologs'). These compounds were profiled for their cytotoxicity against a diverse panel of human cancer cell lines leading to the discovery of several parthenologs with significantly improved anticancer activity (2–14-fold) in comparison to the parent molecule. Next, we developed a fingerprinting tool for predicting P450 activity and regio-selectivity using the sesquiterpene lactone micheliolide as a model substrate. We implemented a three-tier strategy to evolve fine-tuned P450 catalysts for the regio-selective hydroxylation of micheliolide at the aliphatic C2 and C14 sites. The C2(R)- and C14-hydroxy-micheliolide 'intermediates' provided access to a panel of semisynthetic micheliolide derivatives through O-H-functionalization chemistries. We were able to enhance the anti-leukemic activity of MCL up to 9-fold in patient-derived primary AML cells. We also synthesized a panel of biotinylated MCL derivatives to probe the biochemical mechanisms of this natural product and found that merging the information derived from each of these probes provided a preliminary understanding of the MCL-targeted proteome. Furthermore, we applied osteoclast-targeting nanoparticles for the delivery of micheliolide-based anti-leukemic agents directly to the bone marrow. We investigated the drug-micelle interactions necessary for improving drug loading capacity and drug release and developed a novel micheliolide analog, namely MCL-64, as a lead for in vivo studies due to its high anti-leukemic activity (∼2μM), loading capacity (∼12%) and controlled release from nanoparticles in vitro (t1/2 ≈28 hours). Nanoparticles loaded with MCL-64 reduced the load of leukemia stem cells in an animal model of blast crisis chronic myelogenous leukemia, in low dosages (7.5 mg/kg). Lastly, we used consensus-guided mutagenesis to enhance the thermal stability of the reductase domain of P450BM3 to improving its synthetic utility as an oxidation catalyst. Recombination of six stabilizing mutations generated two thermostable variants featuring up to ten-fold longer half-lives at 50°C and increased catalytic performance at elevated temperatures.
Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The ...mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients’ primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.
Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the ...nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.
PDF
