Biology subjects

Hu, W.

Publications and source records attributed to Hu, W..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Proguanil and chlorhexidine augment the antibacterial activities of clarithromycin and rifampicin against Acinetobacter baumannii

The emergence of Acinetobacter baumannii infections as a significant healthcare concern in hospital settings, coupled with their association with poorer clinical outcomes, has prompted extensive investigation into novel therapeutic agents and innovative treatment strategies. Proguanil and chlorhexidine, both categorized as biguanide compounds, have displayed clinical efficacy as antimalarial and topical antibacterial agents, respectively. In this study, we conducted an investigation to assess the effectiveness of combining proguanil and chlorhexidine with clarithromycin or rifampicin against both laboratory strains and clinical isolates of A. baumannii. The combination therapy demonstrated rapid bactericidal activity against planktonic multidrug-resistant A. baumannii, exhibiting efficacy in eradicating mature biofilms and impeding the development of antibiotic resistance in vitro. Additionally, when administered in conjunction with clarithromycin or rifampicin, proguanil enhanced the survival rate of mice afflicted with intraperitoneal A. baumannii infections, and chlorhexidine expedited wound healing in mice with skin infections. These findings are likely attributable to the disruption of A. baumannii cell membrane integrity by proguanil and chlorhexidine, resulting in heightened membrane permeability and enhanced intracellular accumulation of clarithromycin and rifampicin. Overall, this study underscores the potential of employing proguanil and chlorhexidine in combination with specific antibiotics to effectively combat A. baumannii infections and improve treatment outcomes in clinically challenging scenarios. IMPORTANCEA. baumannii has emerged as a globally significant nosocomial pathogen due to its remarkable ability to acquire antibiotic resistance and develop biofilms on both biotic and abiotic surfaces. Recent research has demonstrated that the antidiabetic drug metformin has a potentiation effect on doxycycline and minocycline against certain multidrug-resistant bacterial pathogens, suggesting the potential of this biguanide agent as a novel tetracyclines adjuvant. In this study, we provide evidence showing that the combination of proguanil and chlorhexidine with clarithromycin or rifampicin exhibits rapid bactericidal activities against both planktonic cells and mature biofilms of A. baumannii, the capacity to inhibit the development of antibiotic resistance and improvement of the treatment outcomes in A. baumannii-infected mice. Given the advantages of repurposing non-antibiotic drugs as antibiotic adjuvants, proguanil and chlorhexidine show promise as adjuvants of specific antibiotics in combating clinically significant pathogenic A. baumannii.

microbiology↗

bioRxiv2023

A conserved and druggable pocket in class B G protein coupled receptors for orally active small molecule agonists

Class B G protein-coupled receptors (GPCRs), including glucagon-like receptor 1 (GLP-1R) and parathyroid hormone receptor 1 (PTH1R), are peptide hormone receptors and important drug targets. Injectable peptide drugs targeting class B GPCRs have been developed for the treatment of many diseases, including type 2 diabetes, obesity, and osteoporosis, but orally available small molecule drugs are hotly pursued in the field, especially small molecule agonists of GLP-1R and PTH1R. Here we report the first high-resolution structure of the human PTH1R in complex with the stimulatory G protein (Gs) and a small molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the interface of PTH1R and Gs. The binding site of PCO371 is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. Residues that make up the PCO371 binding pocket are mostly conserved in class B GPCRs and a single mutation in PTH type 2 receptor (PTH2R) and two residue mutations in GLP-1R convert these receptors to respond to PCO371 activation. Functional assays reveal that PCO371 is a G-protein biased agonist that is defective in promoting PTH1R-mediated arrestin signaling. Together, these results uncover a distinct binding site for designing small molecule agonists for PTH1R and possible other members of class B GPCRs and define a receptor conformation that is only specific for G protein activation but not arrestin signaling. These insights should facilitate the design of distinct types of class B GPCR small molecule agonists for various therapeutic indications.

biochemistry↗

bioRxiv2023

Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

BackgroundBone morphogenetic protein type II receptor (BMPR2) gene mutation accounts for 80% of patients with heritable pulmonary artery hypertension (HPAH), and female mutation carriers have significantly higher penetrance rate than males. The inducement of HPAH penetrance and the mechanism of sex differential penetrance are still elusive. MethodsWe infected or transfected pulmonary artery smooth muscle cells (PASMCs) with shBMPR2 lentivirus or siBMPR2 to simulate the pathologic condition of BMPR2 heterozygous mutation and treated them with progesterone. The HPAH patient-derived induced pluripotent stem cells (iPSCs) were induced into vascular smooth muscle cells (VSMCs) to further verify the cellular phenotype. Wild-type flox+/- female mice and SM22-cre BMPR2 flox+/- female mice (CKO mice) were administered with 1-month progesterone, and their phenotype of PAH was evaluated by right heart catheterization and histopathological examination. ResultsProgesterone promoted the proliferation of PASMCs with BMPR2-knockdownby activating ERK pathway via progesterone receptor (PGR). Activated ERK not only upregulated the phosphorylation and elevation of cMYC, but also induced the transcription of endothelin (EDN1) by promoting the nuclear entry of c-JUN and combination on the its promoter region. Similar results were confirmed by iPSCs-VSMCs experiment. CKO mice developed PAH spontaneously and had increased expression of EDN1, which was further aggravated by exogenous progesterone. ConclusionsProgesterone might be an inducement of HPAH penetrance caused by BMPR2 mutation, accounting for sex differential penetrance.

molecular biology↗

bioRxiv2023

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

genetics↗

bioRxiv2023

CD99 Promotes Self-renewal in Hematopoietic Stem Cells and Leukemia Stem Cells by Regulating Protein Synthesis

Blood production is sustained by hematopoietic stem cells (HSCs), which are typically the only blood cells capable of long-term self-renewal. Acute myeloid leukemia (AML) is initiated by aberrantly self-renewing malignant stem cells termed leukemia stem cells (LSCs). HSCs exhibit and depend on low levels of protein synthesis to self-renew. However, the mechanisms by which HSCs regulate protein synthesis to maintain their capacity for self-renewal in the setting of proliferative stress and leukemogenesis remain unknown. Here we show CD99, a cell surface protein upregulated in LSCs, is required for self-renewal of proliferating HSCs and LSCs. We found that loss of CD99 in HSCs and LSCs leads to increased protein synthesis and that their self-renewal capacity can be restored by translation inhibition. These data demonstrate a functional role for CD99 in constraining protein synthesis, which may promote the clonal expansion of HSCs and LSCs that leads to AML. Furthermore, they show that similar to HSCs, LSCs depend on regulated protein synthesis.

cancer biology↗

bioRxiv2023

Structural basis of peptide recognition and activation of endothelin receptors

Endothelin system comprises three endogenous 21-amino-acid peptide ligands endothelin-1, -2, and -3 (ET-1/2/3), and two G protein-coupled receptor (GPCR) subtypes--endothelin receptor A (ETAR) and B (ETBR). Since ET-1, the first endothelin, was identified in 1988 as one of the most potent endothelial cell-derived vasoconstrictor peptides with long-lasting actions, the endothelin system has attracted extensive attention due to its critical role in vasoregulation and close relevance in cardiovascular-related diseases. Here we present three cryo-electron microscopy structures of ETAR and ETBR bound to ET-1 and ETBR bound to the selective peptide IRL1620. These structures reveal a highly conserved recognition mode of ET-1 and characterize the ligand selectivity by ETRs. They also present several conformation features of the active ETRs, thus revealing a specific activation mechanism. Together, these findings deepen our understanding of endothelin system regulation and offer an opportunity to design selective drugs targeting specific ETR subtypes.

molecular biology↗

bioRxiv2023

TGFB1 Induces Fetal Reprogramming and Enhances Intestinal Regeneration

The adult gut epithelium has a remarkable ability to recover from damage. To achieve cellular therapies aimed at restoring and/or replacing defective gastrointestinal tissue, it is important to understand the natural mechanisms of tissue regeneration. We employed a combination of high throughput sequencing approaches, mouse genetic models, and murine and human organoid models, and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. Depletion of macrophages or genetic disruption of TGFB-signaling significantly impaired the regenerative response following irradiation. Murine intestinal regeneration is also characterized by a process where a fetal transcriptional signature is induced during repair. In organoid culture, TGFB1-treatment was necessary and sufficient to induce a transcriptomic shift to the fetal-like/regenerative state. The regenerative response was enhanced by the function of mesenchymal cells, which are also primed for regeneration by TGFB1. Mechanistically, integration of ATAC-seq, scRNA-seq, and ChIP-seq suggest that a regenerative YAP-SOX9 transcriptional circuit is activated in epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for the application of the TGFB-induced regenerative circuit in cellular therapy. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/523825v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@147f3b7org.highwire.dtl.DTLVardef@d1a64dorg.highwire.dtl.DTLVardef@1fd0dc0org.highwire.dtl.DTLVardef@91f7d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Integrated transcriptome and lineage analyses reveal novel catecholaminergic cardiomyocytes contributing to the cardiac conduction system in murine heart

Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using DbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using DbhCFP reporter and DbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed that Dbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using DbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2-tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh+-CMs. HighlightsO_LIStereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. C_LIO_LIEstablished the transcriptional landscape of cell types populating the developing murine heart. C_LIO_LIRevealed previously unreported catecholaminergic cardiomyocyte populations contributing to the developing and mature murine cardiac conduction system. C_LI

cell biology↗

bioRxiv2022

Genetic Dissection of Mutual Interference between Two Consecutively Learned Tasks in Drosophila

Animals can continuously learn different tasks to adapt to changing environments and therefore have strategies to effectively cope with inter-task interference, including both proactive interference (Pro-I) and retroactive interference (Retro-I). Many biological mechanisms are known to contribute to learning, memory, and forgetting for a single task, however, mechanisms involved only when learning sequential different tasks are relatively poorly understood. Here, we dissect the respective molecular mechanisms of Pro-I and Retro-I between two consecutive associative learning tasks in Drosophila. Pro-I is more sensitive to inter-task interval (ITI) than Retro-I. They occur together at short ITI (<20 min), while only Retro-I remains significant at ITI beyond 20 min. Acutely overexpressing Corkscrew (CSW), an evolutionarily conserved protein tyrosine phosphatase SHP2, in mushroom body (MB) neurons reduces Pro-I, whereas acute knockdown of CSW exacerbates Pro-I. Such function of CSW is further found to rely on the {gamma} subset of MB neurons and the downstream Raf/MAPK pathway. In contrast, manipulating CSW does not affect Retro-I as well as a single learning task. Interestingly, manipulation of Rac1, a molecule that regulates Retro-I, does not affect Pro-I. Thus, our findings suggest that learning different tasks consecutively triggers distinct molecular mechanisms to tune proactive and retroactive interference.

animal behavior and cognition↗

bioRxiv2022

Development and validation of PainFace, a software platform that simplifies and standardizes mouse grimace analyses

Facial grimaces are now commonly used to quantify spontaneous pain in mice and other mammalian species, but scoring remains subjective and relies on humans with very different levels of proficiency. Here, we developed a Mouse Grimace Scale (MGS) for black-coated (C57BL/6) mice consisting of four facial action units (orbitals, nose, ears, whiskers). We used this scale to generate ground truth data from over 70,000 images of black mice in different settings. With this large data set, we developed a deep neural network and cloud-based software platform called PainFace (http://painface.net) that accurately scores facial grimaces of black mice on a 0-8 scale. PainFace generates over two orders of magnitude more MGS data than humans can realistically achieve, and at superhuman speed. By analyzing the frequency distribution of grimace scores, we found that mice spent >7x more time in a high grimace state following laparotomy surgery relative to sham surgery controls. The analgesic carprofen reduced the amount of time animals spent in this high grimace state after surgery. Specific facial action unit score combinations were overrepresented following laparotomy surgery, suggesting that characteristic facial expressions are associated with a high grimace state. We performed validation experiments in two labs located in different countries to demonstrate reproducibility of the PainFace platform. To further enhance rigor and reproducibility, we will invite pain researchers to beta test PainFace and then incorporate their feedback into the software and manuscript prior to peer review. While this study is focused on mice, PainFace was designed to simplify, standardize, and scale up grimace analyses with many other mammalian species, including humans.

neuroscience↗

bioRxiv2022

The thermodynamic state of aerobic glycolytic flux plays a critical role in stabilizing aerobic glycolytic flux of cancer cells

Inhibition of aerobic glycolysis (AG) is a strategy for cancer treatment, but its clinical translation has not been successful, suggesting that the mechanism underlying the inhibition of AG is not completely understood. We introduce thermodynamic state of AG (TSAG) into AG flux control, which refers the actual change of Gibbs free energy ({Delta}G) of reactions along the pathway. By using PKM2 knockdown and knockout models, we found that TSAG is resistant to PKM2 perturbation and it can effectively counteract the inhibitory effect of PKM2 perturbation on AG flux. Moreover, we deciphered the interrelationship between TSAG, AG rate, intermediate concentrations, and PK activity, revealed that TSAG is a system property of AG independent of cancer cells pathophysiological features and it plays important roles in stabilizing AG flux responding to perturbation. We propose that both inhibition of glycolytic enzyme and disruption of TSAG are required for effective AG inhibition.

cancer biology↗

bioRxiv2022

Molecular atlas reveals the tri-sectional spinning mechanism of spider dragline silk

We performed the first molecular atlas of natural spider dragline silk production using genome assembly for the golden orb-web spider Trichonephila clavata and multiomics defining for the segmented major ampullate (Ma) gland: Tail, Sac, and Duct. We uncovered a hierarchical biosynthesis of spidroins, organic acids, lipids, and chitin in the sectionalized Ma gland dedicated to fine silk constitution. The ordered secretion of spidroins was achieved by the synergetic regulation of epigenetic and ceRNA signatures for genomic group-distributed spidroin genes. Single-cellular and spatial RNA profiling identified ten cell types with partitioned functional division determining the tri-sectional organization of the Ma gland. Convergent evolution and genetic manipulation analyses further validated that this tri-sectional architecture of the silk gland was analogous in silk-spinning animals and inextricably linked with silk formation. Our study provided multiple levels of data that significantly expand the knowledge of spider dragline silk generation and may eventually benefit spider-inspired fiber innovations.

genomics↗

bioRxiv2022

Single-base precision design of CRISPR-Cas13b enables systematic silencing of oncogenic fusions

Precision oncology programs can rapidly identify oncogenic gene fusions in individual patients1-3. However, despite their established oncogenic status, the vast majority of gene fusions remain undruggable due to the lack of specific inhibitory molecules4, 5. Here, we establish PspCas13b, a poorly characterized programmable RNA nuclease, as a versatile tool to silence various oncogenic fusion transcripts. Our Single-Base Tiled crRNA screens (SiBTil), unbiased computational analysis, and comprehensive spacer-target mutagenesis revealed key determinants of PspCas13b activity. De novo design of crRNAs harbouring basepaired or mismatched guanosine bases at key spacer positions greatly enhances the silencing efficacy of otherwise inefficient crRNAs, expanding the targeting spectrum of this enzyme. We also reveal the interface between mismatch tolerance and intolerance, which unlocks an unexpected single-base precision targeting capability of this RNA nuclease. Notably, our de novo design principles enable potent and selective silencing of various gene fusion transcripts and their downstream oncogenic networks, without off-targeting of non-translocated variants that share extensive sequence homology. We demonstrate that PspCas13b targeting the breakpoint of fusion transcripts enables efficient suppression of ancestral and single-nucleotide mutants (e.g. BCR-ABL1 T315I) that often drive clinical cancer relapse. Collectively, this study provides new design principles for PspCas13b programming to specifically recognise and degrade any undruggable fusion oncogenic transcript, thus providing a new conceptual framework for personalized oncology.

molecular biology↗

bioRxiv2022

A non-lemniscal thalamic interface connecting alerting sensory cues to internal states in mice

Alterations in internal states, such as elevated arousal level and increased anxiety or fear, triggered by alerting environmental cues are required for behavioral state transitions promoting survival. However, the specific brain region that plays an interfacing role between alerting stimuli and internal states remains to be identified. Here, we report that the medial sector of the auditory thalamus (ATm), which consists of a group of non-lemniscal thalamic nuclei, can fulfill this function. VGluT2-expressing ATm (ATmVGluT2+) neurons receive direct and strong inputs from both visual and auditory midbrain regions, and project to multiple downstream structures critically involved in brain state regulation. Their activity was correlated with, and indispensable for, both blue light- and sound-induced NREM sleep-to-Wake transition, and their arousing effects were mainly mediated by, but not limited to, the temporal association cortices. ATmVGluT2+ neuron activation in awake behaving mice induced pupil dilation and behavioral responses suggestive of anxiety. Blocking the neurotransmitter release of ATmVGluT2+ neurons receiving auditory inputs selectively abolished loud noise-triggered escape behavior but not locomotion. Thus, the ATm is an interface in mouse brain that can transform alerting environmental cues into internal arousal and emotional state alterations that promote survival.

neuroscience↗

bioRxiv2022

The N-terminal subunit of vitellogenin in planthopper eggs and saliva acts as a reliable elicitor that induces defenses in rice

Vitellogenins are essential for the development and fecundity of insects, but these proteins may also betray them, as we show here. We found that the small N-terminal subunit of vitellogenins of the planthopper Nilaparvata lugens (NlVgN) triggers strong defense responses in rice plants when it enters the plant during feeding or oviposition by the insect. The defenses induced by NlVgN in plants not only decreased the hatching rate of N. lugens eggs, but also induced volatile emissions in rice plants, which rendered them attractive to a common egg parasitoid. VgN of other planthoppers were found to trigger the same defense responses in rice. We further show that VgN deposited during planthopper feeding compared to during oviposition induces a somewhat different response, probably targeting the appropriate developmental stage of the insect. The key importance of VgN for planthopper performance precludes possible evolutionary adaptions to prevent detection by rice plants.

ecology↗

bioRxiv2022

Glutamine is essential for overcoming theimmunosuppressive microenvironment in malignant salivary gland tumors

RationaleImmunosuppression in the tumor microenvironment (TME) is key to the pathogenesis of solid tumors. Tumor cell-intrinsic autophagy is critical for sustaining both tumor cell metabolism and survival. However, the role of autophagy in the host immune system that allows cancer cells to escape immune destruction remains poorly understood. Here, we determined if attenuated host autophagy is sufficient to induce tumor rejection through reinforced adaptive immunity. Furthermore, we determined whether dietary glutamine supplementation, mimicking attenuated host autophagy, is capable of promoting antitumor immunity. MethodsA syngeneic orthotopic tumor model in Atg5+/+ and Atg5flox/flox mice was established to determine the impact of host autophagy on the antitumor effects against mouse malignant salivary gland tumors (MSTs). Multiple cohorts of immunocompetent mice were used for oncoimmunology studies, including inflammatory cytokine levels, macrophage, CD4+, and CD8+ cells tumor infiltration at 14 days and 28 days after MST inoculation. In vitro differentiation and in vivo dietary glutamine supplementation were used to assess the effects of glutamine on Treg differentiation and tumor expansion. ResultsWe showed that mice deficient in the essential autophagy gene, Atg5, rejected orthotopic allografts of isogenic MST cells. An enhanced antitumor immune response evidenced by reduction of both M1 and M2 macrophages, increased infiltration of CD8+ T cells, elevated IFN-{gamma} production, as well as decreased inhibitory Tregs within TME and spleens of tumor-bearing Atg5flox/flox mice. Mechanistically, ATG5 deficiency increased glutamine level in tumors. We further demonstrated that dietary glutamine supplementation partially increased glutamine levels and restored potent antitumor responses in Atg5+/+ mice. ConclusionsDietary glutamine supplementation exposes a previously undefined difference in plasticity between cancer cells, cytotoxic CD8+ T cells and Tregs.

cancer biology↗

bioRxiv2022

A congenital hydrocephalus causing mutation in Trim71 results in stem cell differentiation defects through inhibiting Lsd1 mRNA translation

Congenital hydrocephalus (CH) is a major cause of childhood morbidity. Mono-allelic mutations in Trim71, a conserved stem-cell-specific RNA-binding protein, cause CH, however, molecular basis for pathogenesis mediated by these mutations remains unknown. Here, using mouse embryonic stem cells as a model, we reveal that the mouse R783H mutation (R796H in human) significantly alters Trim71s mRNA substrate specificity and leads to accelerated stem-cell differentiation and neural lineage commitment. The mutant Trim71, but not the wild-type Trim71, binds Lsd1 (Kdm1a) mRNA and represses its translation. Specific inhibition of this repression or a slight increase of Lsd1 in the mutant cells alleviates the defects in stem cell differentiation and neural lineage commitment. These results determine a functionally relevant target of the CH-causing Trim71 mutant that can potentially be a therapeutic target and provide molecular mechanistic insights into the pathogenesis of this disease.

molecular biology↗

bioRxiv2022

Identification of Piperlongumine (PL) as an new E3 ligase ligand to induce targeted protein degradation

PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-Ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.

cancer biology↗