Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. ...Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.
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•Formate from mitochondrial serine metabolism protects cytosolic THF from degradation•Methotrexate causes degradation of cytosolic folate by a similar pathway•QDPR can reverse THF oxidation and preserve cytosolic folate levels•This role of QDPR can explain the folate deficiency in patients with QDPR mutations
Stratification of folate metabolism into distinct cytosolic and mitochondrial compartments enables repair of oxidative-stress-induced damage to folate metabolites while maintaining overall cellular pools of this nutrient.
The discovery of the phosphatidylinositol-3-kinase (PI3K) pathway was a major advance in understanding growth factor signaling. The high frequency of PI3K pathway mutations in many cancers has ...encouraged a new field targeting cancer driver mutations. Although there have been many successes, targeting PI3K itself has proven challenging, in part because of its multiple isoforms with distinct roles. Despite promising preclinical results, development of PI3K inhibitors as pharmacologic anticancer agents has been limited by modest single-agent efficacy and significant adverse effects. If we could overcome these limitations, PI3K inhibitors would be a powerful cancer-fighting tool. Data from phase III clinical trials yields insight into some of the problems with PI3K inhibitors. Recent advances have shed light on the mechanisms of tumor resistance to PI3K inhibitors via feedback pathways that cause elevated insulin levels that then activate the same PI3K pathways that are the targets of inhibition. Improving our understanding of the complex regulatory feedback pathways that activate in response to PI3K inhibition will reveal ways to increase the efficacy of PI3K inhibitors and reduce adverse effects, increasing the usefulness of this class as a treatment option for multiple cancer types. Display omitted
Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. ...PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P3. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling.
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•PIP4Ks suppress PIP5K activity and insulin-stimulated production of PI(3,4,5)P3•PIP5K inhibition is independent of PIP4K catalytic activity•PIP5K inhibition is dependent on the N-terminal motif VMLLPDD on PIP4K•PIP4Ks have distinct catalytic and structural roles regulating metabolism
PI(4,5)P2 is produced by both phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Wang et al. report an allosteric function of a conserved N-terminal motif of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3. This non-catalytic role has implications for the development of PIP4K targeted therapies.
Inactivation of the tumor suppressor lipid phosphatase INPP4B is common in triple-negative breast cancer (TNBC). We generated a genetically engineered TNBC mouse model deficient in
. We found a ...dose-dependent increase in tumor incidence in
homozygous and heterozygous knockout mice compared with wild-type (WT), supporting a role for
as a tumor suppressor in TNBC. Tumors derived from
knockout mice are enriched for AKT and MEK gene signatures. Consequently, mice with
deficiency are more sensitive to PI3K or MEK inhibitors compared with WT mice. Mechanistically, we found that
deficiency increases PI(3,4)P
levels in endocytic vesicles but not at the plasma membrane. Moreover,
loss delays degradation of EGFR and MET, while promoting recycling of receptor tyrosine kinases (RTK), thus enhancing the duration and amplitude of signaling output upon growth factor stimulation. Therefore,
inactivation in TNBC promotes tumorigenesis by modulating RTK recycling and signaling duration. SIGNIFICANCE: Inactivation of the lipid phosphatase
is frequent in TNBC. Using a genetically engineered mouse model, we show that INPP4B functions as a tumor suppressor in TNBC. INPP4B regulates RTK trafficking and degradation, such that loss of INPP4B prolongs both PI3K and ERK activation.
.
The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of ...potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.
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•Inhibitor THZ-P1-2 shows PI5P4K enzyme inhibition and target engagement in cells•THZ-P1-2 covalently targets unannotated cysteines outside the PI5P4K active site•AML/ALL cell lines are broadly sensitive to THZ-P1-2's covalent effects•PI5P4K inhibition causes autophagy disruption and upregulates TFEB signaling
PI5P4K, an understudied kinase family, is essential in various disease contexts. Sivakumaren et al. develop and characterize PI5P4K inhibitor THZ-P1-2, which targets unique cysteines, exhibits effects in biochemical and cellular assays, displays anticancer activity in leukemia cell lines, and causes defects in autophagy similar to PI5P4K gene knockdown or deletion.
The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel ...regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.
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•The Hippo kinases phosphorylate p110α at T1061, which inhibits its activity•Activation of MST1/2 with epinephrine impairs PI3K signaling and glycogen synthesis•Loss of MST1/2, or mutating T1061A, prevents the effects of epinephrine on glycogen synthesis
Lin et al. describe the finding that p110α, the catalytic subunit of PI3Kα, can be directly inhibited via phosphorylation by MST1, MST2, and other members of the Hippo kinase family. Stimulation of MST1/2 with epinephrine, forskolin, or fasting impedes hepatic PI3K activity and restricts glycogen synthesis.
Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase ...(AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1(-/-) DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in ...disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN, and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.PTPN2 (protein tyrosine phosphatase non-receptor type 2) and PTPN1 are attractive immuno-oncology targets, however, targeting PTPN2/N1 poses significant challenges. Here, the authors report the development of potent PTPN2/N1 heterobifunctional degraders and reveal biochemical and structural insights into the formation of ternary structures with cereblon E3 ligase by X-ray diffraction, cryo-EM and MD simulations.
Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response ...to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd) messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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