Biology subjects

Chen, P.-H.

Publications and source records attributed to Chen, P.-H..

8 recordsLinked to original sources

bioRxiv2026

PepRePs: Peptide-Retargeted Phosphatases Designed with Generative Language Models

Phosphorylation is a fast, reversible layer of post-translational control that directly governs protein activity, signaling flux, and cell state. Kinases and phosphatases collectively establish these phosphorylation states, but their dysregulation drives cancer, neurodegenerative diseases, and developmental disorders. Selectively manipulating the phosphorylation of individual proteins in cells remains challenging, particularly for disordered substrates. Here, we introduce Peptide-Retargeted Phosphatases (PepRePs), a genetically encoded system that fuses a peptide binder to a serine/threonine phosphatase subunit to localize phosphatase activity to a target protein. We first establish the PepReP architecture using characterized tau-binding peptides, then apply protein language model-guided peptide design to target Rab8A, PTEN, and STAT3. Across endogenous and exogenous targets, PepRePs reduce phosphorylation, with downstream effects on tau aggregation, Rab8A-associated protein interactions, and cellular proliferation following PTEN and STAT3 targeting. Sequence-based peptide design enables targeting without structural inputs, supporting application to substrates with disordered regions that challenge conventional structure-based binder design. By coupling programmable peptide binders with modular phosphatase subunits, PepRePs enable targeted control of protein phosphorylation to probe cellular signaling and develop strategies for correcting disease-associated protein dysfunction.

synthetic biology↗

bioRxiv2026

Nanobody MET CAR T cells show efficacy in solid tumors

BackgroundMET overexpression is associated with poor prognosis in many solid tumors due to its central role in tumor survival, invasion, metastasis, and chemoresistance. While targeting MET with antibody-drug conjugates has shown promising results, engineered cellular immunotherapeutic approaches have not been extensively explored. Compared to conventional single-chain variable fragments (scFv), naturally occurring single-domain antibodies consisting of variable heavy chains only (VHH or nanobodies) are smaller, retain high specificity, and exhibit remarkable biochemical stability. In this study, we tested the efficacy of MET-targeting VHH-CAR-T (chimeric antigen receptor T cells). MethodsWe generated a panel of VHH-CAR-Ts using mRNA electroporation. VHH-CAR-T cells were evaluated in functional assays including cell binding avidity, cytokine production profiles, hydrogel microwell-based cellular kinetics, and in vitro cytotoxicity. We also assessed the therapeutic efficacy of VHH-CAR-T in an in vivo mouse model of metastatic triple negative breast cancer (TNBC). ResultsAmong the tested VHH, we identified those with intermediate avidity as most effective for in vitro tumor killing. VHH-CAR-Ts with CD28 costimulatory domains demonstrated augmented cytotoxicity with favorable selectivity, requiring a minimum antigen density threshold for activation. Mechanistically, VHH-CAR-Ts demonstrated low tonic signaling, high avidity, potent cytokine production, and rapid tumor killing kinetics. When administered in an mRNA format, VHH-CAR-Ts exhibited potent and prolonged control of tumor growth in an in vivo metastatic model of TNBC. ConclusionTaken together, these results demonstrate that VHH-CAR-Ts exhibit robust MET specificity and potent therapeutic efficacy both in vitro and in vivo. Thus, VHH-CAR-T cell therapy represents a promising immunotherapeutic strategy for targeting MET-overexpressing solid tumors. What is already known on this topicMET signaling is an important contributor to the aggressiveness of many solid tumors, and targeting MET by antibody-drug conjugates has shown efficacy and safety. Targeting MET by CAR-T cells has been under study, though with limited potency. What this study addsThis study is the first to demonstrate effectiveness of anti-MET VHH-CAR-T cells. Compared with other antigen binding domains, VHH-incorporated CAR-T cells show low tonic signaling, a favorable cytokine profile, and potent tumor killing. How this study might affect research, practice or policyWith the multiple advantages of VHHs including small size, stability, and low potential for tonic signaling, VHH-CAR-T cells represent a promising approach for CAR-T design against solid tumors.

immunology↗

bioRxiv2023

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 C atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.

biochemistry↗

bioRxiv2022

TWAS coupled with eQTL analysis reveals the genetic connection between gene expression and flowering time in Arabidopsis

Genome-wide association study (GWAS) has improved our understanding of complex traits, but challenges exist in distinguishing causation versus association caused by linkage disequilibrium. Instead, the transcriptome-wide association study (TWAS) detects direct associations between expression levels and phenotypic variations, providing an opportunity to better prioritize candidate genes. To assess the feasibility of TWAS, we investigated the association among transcriptomes, genomes, and various traits, including flowering time in Arabidopsis. First, the associated genes formerly known to regulate growth allometry or metabolite production were identified by TWAS. Then, for flowering time, six TWAS-newly identified genes were functionally validated. Analysis of expression quantitative trait locus (eQTL) further revealed a trans-regulatory hotspot affecting the expression of several TWAS-identified genes. The hotspot covers the FRIGIDA (FRI) gene body, which possesses multiple haplotypes differentially affecting the expression of downstream genes, such as FLOWERING LOCUS C (FLC) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). We also revealed multiple independent paths towards the loss of FRI function in natural accessions. Altogether, this study demonstrates the potential of combining TWAS with eQTL analysis to identify important regulatory modules of the FRI-FLC-SOC1 for quantitative traits in natural populations. HighlightCombining TWAS with eQTL analyses identifies haplotypes connecting flowering genes with their physiological trait, strengthening the importance of FRI-FLC-SOC1 regulatory module on flowering time among the Arabidopsis natural population.

plant biology↗

bioRxiv2018

Mutant p53 triggers a dynamin-1/APPL1 endosome feedback loop that regulates β1 integrin recycling and migration

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors. We report that gain-of-function (GOF) mutant p53 expression enhances {beta} integrin and EGF receptor recycling and increases cell migration by triggering a positive feedback loop involving the activation of dynamin-1 (Dyn1) and accumulation of a spatially-restricted subpopulation of APPL1-positive perimeter endosomes. DNM1 is upregulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Perimeter APPL1 endosomes are required for rapid recycling of EGFR and {beta}1 integrins and modulate Akt signaling and Dyn1 activation to create the positive feedback loop that culminates in increased focal adhesion turnover and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus, integrating signaling and receptor trafficking, that can be co-opted by cancer cells for mutant p53-driven migration and invasion.

cell biology↗

bioRxiv2018

Unilateral optogenetic inhibition and excitation of basal ganglia output show opposing effects on left/right lick choices and movement initiation in mice

Models of basal ganglia function predict that tonic inhibitory output to motor thalamus suppresses unwanted movements, and that a decrease in such activity leads to action selection. A direct test of these outcomes of thalamic inhibition has not been performed, however. To conduct such a direct test, we utilized rapid optogenetic activation and inactivation of the GABAergic output of the substantia nigra pars reticulata (SNr) to motor thalamus in mice that were trained in a sensory cued left/right licking task. Directional licking tasks have previously been shown to depend on a thalamocortical feedback loop between ventromedial motor thalamus and antero-lateral premotor cortex (Li et al., 2015; Guo et al., 2017). In confirmation of model predictions, we found that 1s of unilateral optogenetic inhibition of GABAergic output from the SNr biased decision making towards the contralateral lick spout with ipsilaterally cued trials while leaving motor performance intact. In contrast, 1s of optogenetic excitation of SNr terminals in motor thalamus resulted in an opposite bias towards the ipsilateral direction confirming a bidirectional effect of tonic nigral output on directional decision making. In a second variant of the task we disallowed anticipatory licking and found that successful suppression of anticipatory licking was also impacted by our optogenetic manipulations in agreement with the suppressive effect of tonic nigral output. Nevertheless, direct unilateral excitation of SNr cell bodies resulted in bilateral movement suppression, suggesting that descending motor pathways from the SNr to superior colliculus also play an important role in the control of licking behavior.\n\nSignificance StatementThis study provides the first evidence that basal ganglia output to motor thalamus can control decision making in left/right licking choices and suppress anticipatory movement initiation. Unilateral optogenetic inhibition or excitation of basal ganglia output via the substantia nigra resulted in opposite changes of directional lick choices and could override the sensory information on lick direction provided by a whisker stimulus. These results suggest that basal ganglia output gates activity in a thalamo-cortical feedback loop previously shown to underlie the control of forced choice directional licking behavior. The results substantiate models stating that tonic inhibition of motor thalamus from the basal ganglia directs action selection and suppresses unwanted movements.

neuroscience↗

bioRxiv2018

Mammalian stringent-like response mediated by the cytosolic NADPH phosphatase MESH1

Nutrient deprivation triggers stringent response in bacteria, allowing rapid reallocation of resources from proliferation toward stress survival. Critical to this process is the accumulation/degradation of (p)ppGpp regulated by the RelA/SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Therefore, the function of MESH1 remains a mystery. Here, we report that human MESH1 is an efficient cytosolic NADPH phosphatase, an unexpected enzymatic activity that is captured by the crystal structure of the MESH1-NADPH complex. MESH1 depletion promotes cell survival under ferroptosis-inducing conditions by sustaining the level of NADPH, an effect that is reversed by the simultaneous depletion of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Importantly, MESH1 depletion also triggers extensive transcriptional changes that are distinct from the canonical integrated stress response but resemble the bacterial stringent response, implicating MESH1 in a previously uncharacterized stress response in mammalian cells.

biochemistry↗

bioRxiv2018

A non-canonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells

Dynamin GTPases are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME); but, they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with dynamin-2 (Dyn2), but inactivated in non-neuronal cells via phosphorylation by GSK3{beta} kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently-tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual and triple-channel total internal reflection fluorescence microscopy we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2, but not Dyn1 exhibits a burst of assembly prior to CCV formation. Activation of Dyn1 by acute inhibition of GSK3{beta} results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation and decreased CCP lifetimes, but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME even when present at low, substoichiometric levels relative to Dyn2, and apparently without assembly into supramolecular collar-like structures. Under physiological conditions Dyn1 is activated downstream of EGF-receptor signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoformspecific regulatory role for Dyn1 in clathrin-mediated endocytosis.

cell biology↗