Human telomerase processively adds telomeric repeats (dGGTTAG) to chromosome 3'-ends to maintain telomere length. While mostly absent in somatic cells, telomerase is aberrantly upregulated in most tumor cells to sustain...Human telomerase processively adds telomeric repeats (dGGTTAG) to chromosome 3'-ends to maintain telomere length. While mostly absent in somatic cells, telomerase is aberrantly upregulated in most tumor cells to sustain cellular immortality, making it a promising oncology target. However, to date there are no reported structures of human telomerase with inhibitor, impeding structure-based drug design and optimization. We report nine cryo-electron microscopy structures of human telomerase with and without BIBR1532, a highly selective small-molecule telomerase inhibitor. Unexpectedly, BIBR1532 binds a previously unknown pocket between TERT finger and palm. BIBR1532 inhibits each step but disproportionately affects the rate-limiting first step of telomere repeat nucleotide addition. The structures reveal a rigid finger that explains telomerase's slow rate and low fidelity. Our study provides insights into telomerase catalytic mechanism and its inhibition by BIBR1532, explains why prior BIBR derivatives did not improve potency and suggests a rational approach for design of small-molecule telomerase inhibitors.
Small, nonpolar cyclic peptides can both bind challenging targets and cross cell membranes, making them attractive for addressing currently undruggable targets such as many protein-protein interactions (PPIs). However, d...Small, nonpolar cyclic peptides can both bind challenging targets and cross cell membranes, making them attractive for addressing currently undruggable targets such as many protein-protein interactions (PPIs). However, developing such compounds de novo without prior information about lead structures such as natural ligands or fragments remains a notable challenge. Here we show that functional screening of structurally highly diverse cyclic peptide libraries synthesized at nanomole scale allows identification of sub-kDa inhibitors of a PPI. By screening 15,360 fully random cyclic peptides, we were able to identify an inhibitor of the E3 ligase adaptor Keap1 and its substrate Nrf2. Optimization by rapid design-build-test cycles produced a membrane-permeable compound active in live cells. This study demonstrates that large, diverse cyclic peptide libraries can enable the discovery of cell-permeable PPI inhibitors from the ground up, providing a way to harness the powerful modality of small cyclic peptides to address often difficult-to-target intracellular interactions.
Dynamic protein complex assembly is critical for regulating various biological processes. Proximity labeling (PL), best represented by the ascorbate peroxidase APEX2, allows these molecular events to be captured in livin...Dynamic protein complex assembly is critical for regulating various biological processes. Proximity labeling (PL), best represented by the ascorbate peroxidase APEX2, allows these molecular events to be captured in living cells in a spatiotemporal manner. However, the hydrogen peroxide (HO) dependence of APEX2 has hindered its application in sensitive living systems. Here we introduce ROProx, a radical- and oxygen-driven photoreactive PL technology that leverages the chemically evolved biotin-naphthylamine probe BN2, which has strong binding affinity for APEX2, and the unexpected tyrosyl radicals in APEX2. ROProx labels dynamic cytosolic protein complexes in living cells within seconds, with a range of 10 nm, and is precisely controlled by mild blue light irradiation without HO. Additionally, we apply ROProx to explore the phosphotyrosine-dependent GRB2 interactome in living mice by simply injecting BN2 for 5 minutes. ROProx should, therefore, open broad opportunities for PL chemical evolution and applications in other living systems.
Interferon regulatory factor 4 (IRF4) is an oncogenic transcription factor (TF) in several hematological malignancies. To date, no pharmacological agents have been developed specifically for IRF4 due to the challenging n...Interferon regulatory factor 4 (IRF4) is an oncogenic transcription factor (TF) in several hematological malignancies. To date, no pharmacological agents have been developed specifically for IRF4 due to the challenging nature of targeting TFs. Here we first identified (S)-H1, a binder of IRF4, by targeting the SPI1-IRF4 interaction on IRF4's interferon association domain via high-throughput screening. Next, we successfully turned our binder into dIRF4-2, a first-in-class proteolysis-targeting chimera of IRF4, by linking (S)-H1 to E3 ligase ligands of cereblon. dIRF4-2 can induce highly selective proteasomal degradation of IRF4 and has strong cytotoxic effects in all multiple myeloma lines evaluated in vitro. Our study showcases methodology to effectively target the IRF family of TFs and illustrates how to convert an inert binder into a powerful chemical probe for studying the functions of important oncoproteins that are structurally difficult to target.
Hu Z, Lv W, Wen T
… +25 more, Sun J, Ji G, Ru S, Sun X, Wang Z, Ren H, Zhang Y, Chen P, Zhou L, Wu S, Jia X, Liu S, Zhu Z, Xu J, Liu T, Feng Y, Li G, Ding Y, Wang Y, Luo Y, Liu C, Lu Z, Ren Z, Duan SZ, Xu D
Understanding of the metabolic determinants influencing immunotherapy responsiveness remains limited. Here we performed a multiomics analysis of tumor biopsies from patients with hepatocellular carcinoma (HCC) treated wi...Understanding of the metabolic determinants influencing immunotherapy responsiveness remains limited. Here we performed a multiomics analysis of tumor biopsies from patients with hepatocellular carcinoma (HCC) treated with immune checkpoint blockade (ICB) and revealed that heightened expression of OXCT1, a rate-limiting enzyme in ketone body metabolism, was negatively correlated with ICB efficacy, whereas its metabolic substrate, β-hydroxybutyrate (BHB), displayed an opposite effect. Mechanistically, glucose deprivation in HCC cells promotes AMPK-mediated OXCT1 S113 phosphorylation, which exposes the nuclear localization sequence of OXCT1 to trigger its nuclear translocation. Nucleus-translocated OXCT1 associates with IRF1 to locally consume BHB and suppress histone H3K9 BHB at the major histocompatibility complex class I (MHC-I) and chemokine gene loci, leading to repressed transcription of these immune genes. Targeting the AMPK-OXCT1-IRF1 axis sensitizes tumor cells to ICB upon ketogenic diet. These findings reveal a mechanism by which a non-canonical function of nuclear OXCT1 coordinates the interplay between ketone body metabolic reprogramming and immunotherapy responsiveness.
Historically, glycomics has lagged behind other omics fields, owing to analytical challenges. However, recent technological advances are rapidly transforming the field, enabling a growing number of large-scale studies to...Historically, glycomics has lagged behind other omics fields, owing to analytical challenges. However, recent technological advances are rapidly transforming the field, enabling a growing number of large-scale studies to harness the wealth of information encoded by glycans. In this Review, we provide an overview of the current state of large-scale glycomics and its role in understanding the functional importance of glycans. We discuss the main challenges in high-throughput glycoanalytical methods and highlight recent progress, with a particular emphasis on standardization and reproducibility. Special attention is given to the necessity of large, well-designed and ideally longitudinal and multicohort studies that use rigorous statistical approaches to uncover robust biological associations. Finally, we conclude with selected applications and perspectives, emphasizing the potential of large-scale glycomics to drive biomarker discovery and enhance the understanding of glycan-mediated biology.
Enzymatic depolymerization has the potential to contribute to nylon waste recycling. However, the implementation of a viable industrial process still lags far behind progress in enzymatic polyester recycling. Here we rev...Enzymatic depolymerization has the potential to contribute to nylon waste recycling. However, the implementation of a viable industrial process still lags far behind progress in enzymatic polyester recycling. Here we review the current biocatalytic nylon recycling landscape, highlighting biochemical, structural, and materials science barriers that currently limit depolymerization extents. We detail efforts to identify, engineer, and characterize new nylon depolymerases, where currently even the best biocatalysts rarely exceed ~1 wt% conversion of solid polymer to products. Based on our analyses, we suggest that limited substrate accessibility due to substrate crystallinity and hydrogen bonding, rather than intrinsic enzyme inefficiency or instability, is the primary bottleneck to enhanced depolymerization. Guided by successes in polyester enzymatic recycling, we outline a research roadmap combining nylonase engineering with polymer pretreatment, chemo-enzymatic cascades, and process analyses to accelerate development of viable enzymatic nylon recycling processes.
All organisms have thioredoxin reductase (TR) or glutathione reductase (GR), the only enzymes that use reduced nicotinamide adenine dinucleotide phosphate to reduce cytosolic disulfides into thiols, thereby powering deox...All organisms have thioredoxin reductase (TR) or glutathione reductase (GR), the only enzymes that use reduced nicotinamide adenine dinucleotide phosphate to reduce cytosolic disulfides into thiols, thereby powering deoxyribonucleotide biosynthesis, elimination of oxidants, oxidative damage repair and reduction of the disulfide nutrient cystine into the thiol amino acid cysteine. Hence, TR/GR-null bacteria or yeast are inviable; yet, remarkably, mice with TR/GR-null livers thrive, in part by synthesizing life-sustaining cysteine through alternative pathways that evolved in metazoans. Although TR/GR-null livers generate some of their cysteine through the serine transsulfuration pathway, we here show that most cysteine in TR/GR-null livers comes from a pathway in which pyridoxal-phosphate-dependent cleavage of a carbon-sulfur bond in cystine generates cysteine persulfide, which decomposes nonenzymatically into cysteine. This potent yet previously unrecognized pathway is regulated by cellular levels of sulfur metabolites and represents a potent cytoprotective response that might be induced in most mammalian cells under conditions that chronically elevate cytosolic cystine levels.
Mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient sensor that integrates diverse inputs to regulate protein translation and cell growth. While mTORC1 is activated on the lysosome in the classical model, it...Mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient sensor that integrates diverse inputs to regulate protein translation and cell growth. While mTORC1 is activated on the lysosome in the classical model, it has become increasingly clear that this multifaceted signaling complex is active at various subcellular locations, such as the nucleus. However, what specific functions mTORC1 serves at these locations and how its signaling is compartmentalized are unclear. To interrogate subcellular pools of mTORC1, we developed TerminaTOR, a genetically encodable inhibitor of mTORC1 that can be targeted to specific subcellular locations. When TerminaTOR is directed to the lysosome, it inhibits canonical lysosomal mTORC1 and induces autophagy. Furthermore, TerminaTOR targeted to the nucleus specifically inhibits nuclear mTORC1, uncovering noncanonical roles of nuclear mTORC1 in regulating the transcription of CCAAT motif-containing genes. Thus, mTORC1 exhibits functional spatial compartmentalization and TerminaTOR serves as a powerful tool for unraveling spatially regulated functions of mTORC1 across different scales.