Biology subjects

Che, X.

Publications and source records attributed to Che, X..

17 recordsLinked to original sources

bioRxiv2025

Beyond the G-Loop: CRBN Molecular Glues Potently Target VAV1 via a Novel SH3 RT-Loop Degron

This study reports the discovery and characterization of novel CRBN molecular glues that selectively induce the proteasomal degradation of the hematopoietic-specific signaling protein VAV1, a key target in hematological malignancies and autoimmune diseases. Utilizing unbiased global proteomics, we identified phenyl-glutarimide derivatives NGT-201-12, as effective VAV1 degraders, with its C-terminal SH3 domain (SH3-2) being crucial for this interaction. A significant finding is the elucidation of a non-canonical RT-loop degron (RDxS motif, residues 796-799) within VAV1 SH3-2, distinct from previously characterized G-loop degrons. This discovery, supported by advanced computational modeling using the physics- and AI-driven GluePlex workflow and validated by site-directed mutagenesis, highlights versatility of CRBN in recognizing diverse neosubstrate motifs. Furthermore, we demonstrate that applying Free Energy Perturbation (FEP+) calculations to these predicted ternary structures yields cooperativity metrics that correlate with experimental degradation potency, overcoming the limitations of standard molecular docking. This establishes a robust workflow where, once a ternary complex is predicted, even with initial weak binders, FEP+ can be utilized to prospectively rank analogs and optimize molecular glue potency. Additionally, we demonstrate that strategic chemical modifications, particularly conformational restriction via halogen substitution (e.g., NGT-201-18), markedly potentiate VAV1 degradation, a principle supported by density functional theory (DFT) calculations. Comprehensive structure-activity relationship (SAR) studies provided a roadmap for designing next-generation VAV1 degraders. Importantly, dose-response proteomics not only confirmed VAV1 as the primary target but also revealed LIMD1, possessing a canonical G-loop, as an off-target for some analogs, indicating a single molecular glue can engage disparate degron motifs. The identification of the VAV1 RT-loop degron prompted a proteome-wide search, revealing other SH3-containing proteins as potential targets or off-targets. In conclusion, this research unveils a novel non-canonical RT-loop degron in VAV1, demonstrates the utility of conformational restriction in enhancing degrader potency, and underscores the critical role of integrating global proteomics with advanced structural modeling and FEP calculations for understanding degrader potency and selectivity. These findings offer a promising therapeutic strategy for targeting VAV1 and significantly expand the landscape of CRBN neosubstrate recognition and the rational design of molecular glue degraders.

biochemistry↗

bioRxiv2025

FKSFold: Improving AlphaFold3-Type Predictions of Molecular Glue-Induced Ternary Complexes with Feynman-Kac-Steered Diffusion

We introduce an AI model, FKSFold, that uses Feynman-Kac steered diffusion as an inference-time strategy to improve AlphaFold3-type predictions of molecular-glue induced ternary complexes. FKSFold augments reverse diffusion with a Feynman-Kac derived steering term and uses the interface predicted TM-score (ipTM) as the guiding potential, coupled with particle-based sampling and adaptive resampling to bias trajectories toward high-quality interfaces without retraining the base models. Implemented on Chai-1r and Boltz-2 (named FKSFold-Chai and FKSFold-Boltz) - two open-source AF3-type architectures - FKSFold was benchmarked on eight ternary systems. FKSFold-Chai recovered three challenging complexes with sub-3 [A] accuracy: VHL:MG:CDO1 (DockQ 0.922, iRMSD 0.629, fnat 0.963), FKBP12:MG:mTOR-FRB (DockQ 0.590, iRMSD 1.813, fnat 0.519), and FKBP12:MG:BRD9 (DockQ 0.841, iRMSD 0.801, fnat 0.913), while unmodified baselines failed. Other cases highlighted challenges - flexible loop rearrangements (e.g., CRBN:MG:VAV1-SH3c) and large conformational search spaces (e.g., NEK7, QDPR, HDAC1) - and, empirically, using few particles, short resampling intervals, and moderate-to-high lambda (steering strength) gave the best exploration-exploitation balance. Although the strategy is theoretically model-agnostic, on Boltz-2 ipTM head instability currently limits observed gains. The code for the two implementations, FKSFold-Chai and FKSFold-Boltz, is available at https://github.com/YDS-Pharmatech/FKSFold-Chai and https://github.com/YDS-Pharmatech/FKSFold-Boltz.

biophysics↗

bioRxiv2024

YDS-Ternoplex: Surpassing AlphaFold 3-Type Models for Molecular Glue-Mediated Ternary Complex Prediction

Molecular glues represent an innovative class of drugs that enable previously impossible protein-protein interactions, but their rational design remains challenging, a problem that accurate ternary complex modeling can significantly address. Here we present YDS-GlueFold, a novel computational approach that enhances AlphaFold 3-type models by incorporating guided diffusion during inference to accurately predict molecular glue-induced ternary complex structures. We demonstrate YDS-GlueFolds capabilities across eight diverse test cases, including both E3 ligase-based systems (VHL, CRBN complexes with mTOR-FRB, NEK7, and VAV1-SH3c, and KBTBD4 complexes with HDAC1) and non-E3 ligase complexes (FKBP12 complexes with mTOR-FRB, BRD9 and QDPR). The model achieves remarkable accuracy with RMSD values consistently below 2.5 [A] compared to experimental structures. Importantly, 7 out of 8 test cases involve protein-protein pairs that were not present in the AlphaFold 3 training set, providing a rigorous test of the models ability to generalize beyond its training data. Notably, in the FKBP12 case, YDS-GlueFold correctly predicts a novel interface configuration instead of defaulting to known interactions present in training data, further demonstrating true generalization rather than mere memorization. Our results suggest that strategic enhancement of the inference process through guided diffusion can significantly improve ternary complex prediction accuracy, potentially accelerating the development of molecular glue therapeutics for previously undruggable targets.

biophysics↗

bioRxiv2024

FragLlama: Next-fragment prediction for molecular design

The emergence of ChatGPT has drawn significant attention to Large Language Models (LLMs) due to their impressive performance. While LLMs primarily focus on next token/word prediction, we apply this principle to molecular design by reframing the task as predicting the next token/fragment. We present FragLlama, a large language model trained for molecular design, featuring custom tokens that represent molecular fragments and functional groups. The model is for generating molecules given one or two fragments, for application scenarios like general hit-to-lead and lead optimization stage drug design, PROTAC linker design; mapping to commonly used drug design strategies like fragment growing and scaffold hopping. In the pre-training stage, we adapted the Llama 3 architecture to create FragLlama, training it to learn conditional probabilities of these fragment-level tokens. The subsequent alignment stage employed fine-tuning to guide the model towards generating molecules with desired properties. The effectiveness of FragLlama is demonstrated through its applications in designing molecular glue libraries, PROTAC linkers and EGFR binders. FragLlama demonstrates proficiency in reproducing expert-level designs while also exploring novel and promising chemical spaces, highlighting its potential to augment the capabilities of medicinal chemists in drug design.

bioinformatics↗

bioRxiv2024

Physical activity modulates early visual response and improves target detection in humans

Brain state changes affect visual perception by altering spatial resolution. Attention enhances the spatial resolution decorrelating neuronal activity in early nonhuman primate (NHP) visual cortex. Physical activity (PA) amplifies these attentional effects in rodents but impact of PA on visual perception in humans remains uncertain. We investigated the relationship between broadband high-frequency activity (BHA: 80-150 Hz) recorded with magnetoencephalography (MEG) and visual detection performance. We found that PA enhanced visual target detection predicted by a reduction of early BHA responses (<90 msec). This effect may be due to reduced interneuronal correlation to improve spatial resolution. Moreover, PA improved spatial integration time, as indicated by a linear relationship between reaction times and BHA variation with target eccentricity. These findings provide evidence that PA influences neuronal activity critical for early visual perception, optimizing visual processing at the initial stages of the visual hierarchy.

neuroscience↗

bioRxiv2024

Physical Exercise Improves Working Memory through Ripple-Spindle Coupling

Spindle-ripple coupling enhances memory consolidation during sleep. Ripples, representing the compressed reactivation of environmental information, provide a mechanism for retaining memory information in chronological order and are also crucial for working memory (WM) during wakefulness. Brief sessions of physical exercise (PE) are proposed to boost WM. In concurrent EEG/MEG sessions, we investigated the role of PE in WM performance and high-frequency-ripple to spindle coupling. Ripples, identified in MEG sensors covering the medial temporal lobe (MTL) region, predicted individual WM performance. Ripples were locked to robust oscillatory patterns in the EEG defined spindle band. Spindle activity and ripples decrease during initial stimulus presentation and rebound after 1 sec. Behaviorally, PE enhanced WM performance. Neurophysiologically, PE scaled the ripple rate with the number of items to be kept in WM and strengthened the coupling between ripple events and spindle oscillations. These findings reveal that PE enhances WM by coordinating ripple-spindle interaction.

neuroscience↗

bioRxiv2024

Phospholipase C β4 promotes RANKL-dependent osteoclastogenesis by interacting with MKK3 and p38 MAPK

Phospholipase C beta (PLC{beta}) exerts diverse biological processes, including inflammatory responses and neurogenesis; however, its role in bone cell function is largely unknown. Among the PLC{beta} isoforms ({beta}1-{beta}4), we found that PLC{beta}4 was most highly upregulated during osteoclastogenesis. In this study, we used global knockout and osteoclast lineage-specific PLC{beta}4 conditional knockout (LysM-PLC{beta}4-/-) mice and demonstrated that PLC{beta}4 is a crucial regulator of receptor activator of nuclear factor {kappa}B ligand (RANKL)-induced osteoclast differentiation. Deletion of PLC{beta}4, both globally and in the osteoclast lineage, resulted in a significant reduction in osteoclast formation and the downregulation of osteoclast marker genes. Importantly, LysM-PLC{beta}4-/- male mice exhibited greater bone mass and a lower number of osteoclasts in vivo than their wild-type littermates, without altering osteoblast function. Mechanistically, we found that PLC{beta}4 forms a complex with p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (MKK3) in response to RANKL, thereby modulating p38 activation. An immunofluorescence assay further confirmed the colocalization of PLC{beta}4 with p38 after RANKL exposure. Moreover, p38 activation rescued the impaired osteoclast formation and restored the reduced p38 phosphorylation due to PLC{beta}4 deficiency. Thus, our findings reveal that PLC{beta}4 controls osteoclastogenesis via the RANKL-dependent MKK3-p38 MAPK pathway, and PLC{beta}4 may be a potential therapeutic candidate for bone diseases such as osteoporosis.

cell biology↗

bioRxiv2024

Dimeric R25CPTH(1-34) Activates the Parathyroid Hormone-1 Receptor in vitro and Stimulates Bone Formation in Osteoporotic Female Mice

Osteoporosis, characterized by reduced bone density and strength, increases fracture risk, pain, and limits mobility. Established therapies of Parathyroid hormone (PTH) analogs effectively promote bone formation and reduce fractures in severe osteoporosis, but their use is limited by potential adverse effects. In the pursuit of safer osteoporosis treatments, we investigated R25CPTH, a PTH variant wherein the native arginine at position 25 is substituted by cysteine. These studies were prompted by our finding of high bone mineral density in a hypoparathyroidism patient with the R25C homozygous mutation, and we explored its effects on PTH type-1 receptor (PTH1R) signaling in cells and bone metabolism in mice. Our findings indicate that R25CPTH(1-84) forms dimers both intracellularly and extracellularly, and the synthetic dimeric peptide, R25CPTH(1-34), exhibits altered activity in PTH1R-mediated cAMP response. Upon a single injection in mice, dimeric R25CPTH(1-34) induced acute calcemic and phosphaturic responses comparable to PTH(1-34). Furthermore, repeated daily injections increased calvarial bone thickness in intact mice and improved trabecular and cortical bone parameters in ovariectomized (OVX) mice, akin to PTH(1-34). The overall results reveal a capacity of a dimeric PTH peptide ligand to activate the PTH1R in vitro and in vivo as PTH, suggesting a potential path of therapeutic PTH analog development.

genomics↗

bioRxiv2022

Molecular Mechanism for Bacterial Degradation of Plant Hormone Auxin

Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin-degradation (adg) operon was recently identified in the Variovorax genomes, which is responsible for root growth inhibition (RGI) reversion, promoting rhizosphere colonization and root growth. However, the molecular mechanism underlying auxin degradation by Variovorax remains unclear. Here, we systematically screened Variovorax adg operon products and identified two proteins, AdgB and AdgI, that directly associate with auxin indole-3-acetic acid (IAA). Further biochemical and structural studies revealed that AdgB is a highly IAA-specific ABC transporter solute binding protein, likely involved in IAA uptake. AdgI interacts with AdgH to form a functional Rieske non-heme dioxygenase, which works in concert with a FMN-type reductase encoded by gene adgJ to transform IAA into the biologically inactive 2-oxindole-3-acetic acid (oxIAA), representing a new bacterial pathway for IAA inactivation/degradation. Importantly, incorporation of a minimum set of adgH/I/J genes could enable IAA degradation by E. coli, suggesting a promising strategy for repurposing the adg operon for IAA regulation. Together, our study identifies the key components and underlying mechanisms involved in IAA transformation by Variovorax and brings new insights into the bacterial turnover of plant hormones, which would provide the basis for potential applications in rhizosphere optimization and ecological agriculture.

microbiology↗

bioRxiv2022

Peroxiredoxin 5 regulates osteogenic differentiation via interaction with hnRNPK during bone regeneration

Peroxiredoxin 5 (Prdx5) is involved in pathophysiological regulation via the stress-induced cellular response. However, the function of Prdx5 in the bone remains largely unknown. Here, we show that Prdx5 is involved in osteoclast and osteoblast differentiation, resulting in osteoporotic phenotypes in Prdx5 knockout (Prdx5Ko) mice. Through immunoprecipitation and liquid chromatography combined with tandem mass spectrometry analysis, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as a potential binding partner of Prdx5 during osteoblast differentiation in vitro. We found that Prdx5 acts as a negative regulator of hnRNPK-mediated osteocalcin (Ocn) expression. In addition, transcriptomic analysis revealed that in vitro differentiated osteoclasts from the bone marrow-derived macrophages of Prdx5Ko mice showed enhanced expression of several osteoclast-related genes. These findings indicate that Prdx5 might contribute to the maintenance of bone homeostasis by regulating osteoblast differentiation. This study proposes a new function of Prdx5 in bone remodeling that may be used in developing therapeutic strategies for bone diseases.

cell biology↗

bioRxiv2022

Bayesian statistics improves biological interpretability of metabolomics data from human cohorts

BackgroundUnivariate analyses of metabolomics data currently follow a frequentist approach, using p-values to reject a null-hypothesis. However, the usability of p-values is plagued by many misconceptions and inherent pitfalls. We here propose the use of Bayesian statistics to quantify evidence supporting different hypotheses and discriminate between the null hypothesis versus lack of statistical power. MethodsWe use metabolomics data from three independent human cohorts that studied plasma signatures of subjects with myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). Data are publicly available, covering 84-197 subjects in each study with 562-888 identified metabolites of which 777 were common between two studies, and 93 compounds reported in all three studies. By comparing results from classic multiple regression against Bayesian multiple regression we show how Bayesian statistics incorporates results from one study as prior information into the next study, thereby improving the overall assessment of the likelihood of finding specific differences between plasma metabolite levels and disease outcomes in ME/CFS. ResultsWhereas using classic statistics and Benjamini-Hochberg FDR-corrections, study 1 detected 18 metabolic differences, study 2 detected no differences. Using Bayesian statistics on the same data, we found a high likelihood that 97 compounds were altered in concentration in study 2, after using the results of study 1 as prior distributions. These findings included lower levels of peroxisome-produced ether-lipids, higher levels of long chain, unsaturated triacylglycerides, and the presence of exposome compounds that are explained by difference in diet and medication between healthy subjects and ME/CFS patients. Although study 3 reported only 92 reported compounds in common with the other two studies, these major differences were confirmed. We also found that prostaglandin F2alpha, a lipid mediator of physiological relevance, was significantly reduced in ME/CFS patients across all three studies. ConclusionsThe use of Bayesian statistics led to biological conclusions from metabolomic data that were not found through the frequentist analytical approaches more commonly employed. We propose that Bayesian statistics to be highly useful for studies with similar research designs if similar metabolomic assays are used.

bioinformatics↗

bioRxiv2022

The tissue-specific chromatin accessibility landscape of Papaver somniferum

Accessible chromatin regions (ACRs) at promoters, enhancers, and other gene regulatory regions allow transcription factors (TFs) to bind, which regulate gene transcription involved in plant development and metabolism. Papaver somniferum has been widely applied in clinical medicine as one of the most important and oldest medicinal plants due to its unique and effective active ingredients. However, the transcriptional regulatory mechanism of tissue-specific distribution of active ingredients remains unknown. In this study, transcriptome and chromatin accessibility analysis by RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) was performed to investigate these underlying molecular mechanisms. We identified tissue-specific chromatin Tn5 hypersensitive site (THS) and gene expression by examining the variation of THS and transcripts across six tissues (capsule, stem, fine root, tap root, leaf, and petal). Our results provide insight into the epigenetic mechanism of transcriptional plasticity for P. somniferum organ development. Sequence motif analysis within accessible chromatin regions for co-expressed gene modules revealed enriched binding sites of hub transcription factors that regulate tissue-specific functions. Furthermore, we identified regulatory elements for tissue-specific accumulation of morphine and noscapine in P. somniferum. This is the first tissue-specific chromatin accessibility landscape of P. somniferum providing an important resource for functional epigenetic analysis and future molecular breeding in P. somniferum for variety improvement.

plant biology↗

bioRxiv2022

Structural insights into the viral proteins binding by TRIM7 reveal a general C-terminal glutamine recognition mechanism

The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. A recent study found that TRIM7 inhibits human enteroviruses through ubiquitination and proteasomal degradation of viral 2BC protein by targeting the 2C moiety of 2BC protein. Here, we report the crystal structures of TRIM7 in complex with 2C, where the C-terminal region of 2C is inserted into a positively charged groove of the TRIM7 PRY-SPRY domain. Structure-guided biochemical studies revealed the C-terminus glutamine residue of 2C as the primary determinant for TRIM7 binding. Such a glutamine-end motif binding mechanism can be successfully extended to other substrates of TRIM7. More importantly, leveraged by this finding, we were able to identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. We further show that TRIM7 may function as a restriction factor to promote the degradation of the viral proteins of norovirus and SARS-CoV-2, thereby restoring the Type I interferon immune response and inhibiting viral infection. Several crystal structures of TRIM7 in complex with SARS-CoV-2 proteins are also determined, and a conserved C-terminus glutamine-specific interaction is observed. These findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.

biochemistry↗

bioRxiv2021

Progressive Increases in Mesenchymal Cell Diversity Modulate Lung Development and are Attenuated by Hyperoxia

Lung mesenchymal cells play an essential role in development and at birth, as the lung moves from a fluid-filled to an oxygen-rich environment with a stable gas-liquid interface. The molecular details and cellular changes accompanying this highly coordinated process remain incompletely understood. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung in both health and disease to characterize the spectrum of mesenchymal cell states prior to the onset of air-breathing life through late alveolarization to gain insight into their role in orchestrating tissue maturation. We found that cell type diversity in the mesenchymal compartment increases rapidly during normal development but is delayed during neonatal exposure to 80% O2 hyperoxia, a model for bronchopulmonary dysplasia. This study identifies the molecular transitions between populations of mesenchymal cells at discrete developmental time points across fibroblast, smooth muscle, and mural compartments and elucidates the global and cell type-specific effects of neonatal hyperoxia, including the emergence of Acta1+ cells which are absent in normoxic neonatal lungs. These granular insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world.

developmental biology↗

bioRxiv2021

Phenotypic diversity and sensitivity to injury of the pulmonary endothelium during a period of rapid postnatal growth

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2), contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury which impairs angiogenesis, induced both common and unique endothelial gene signatures, dysregulated capillary EC cross-talk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

developmental biology↗

bioRxiv2020

Form and function: diverse homeostatic and immunomodulatory roles of immune cells in the developing lung revealed at single cell resolution

At birth, the lungs experience a sudden transition from a pathogen-free, hypoxic, fluid-filled environment to a pathogen-rich, rhythmically distended air-liquid interface. While many studies focus on adult tissue, the heterogeneity of immune cells in the perinatal lung remains unexplored. Here, we combine single cell transcriptomics with in situ hybridization to present an atlas of the murine lung immune compartment during a critical period of lung development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a complex and dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed a precise orchestration of five distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.

developmental biology↗

bioRxiv2018

Chromatin Blueprint Of Glioblastoma Stem Cells Reveals Common Drug Candidates For Distinct Subtypes

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as Glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

cancer biology↗