Biology subjects

Zou, T.

Publications and source records attributed to Zou, T..

12 recordsLinked to original sources

bioRxiv2026

In situ loading of biologics on and tissue redirection of circulating extracellular vesicles with an anchoring nanobody

Extracellular vesicles (EVs) are promising nanocarriers of therapeutic macromolecules, but ex vivo production, cargo loading and quality control constrains clinical translation. Here we demonstrate that NEV3, an EV-anchoring nanobody identified by phage display, binds EVs from multiple sources through interactions involving fibronectin and loads diverse proteins onto EVs, resulting in enhanced intracellular delivery and biological activities in vitro and in vivo. Conjugating NEV3 to target proteins hijacks circulating EVs, prolonging circulation and altering tissue distribution in vivo. When fused with an GFP nanobody or protein A, NEV3 enabled simultaneous capture and loading of circulating GFP or immunoglobulin G (IgG) antibodies on endogenous EVs with enhanced intracellular delivery in vitro and redistribution to liver in vivo. Intravenous protein A-NEV3 significantly reduced circulating IgG and anti-dsDNA antibodies in systemic lupus erythematosus mice with improved survival and reduced pathologies. Intravenous anti-transferrin receptor-protein A-NEV3 redirected circulating EVs to muscle and alleviated muscle pathologies in dystrophin-deficient mdx mice. Our study demonstrates that NEV3 enables in situ loading of protein therapeutics and tissue-specific delivery of circulating EVs, providing a modular strategy for loading cargo onto and redirecting circulating EVs in vivo.

bioengineering↗

bioRxiv2025

REEP6 deficiency impairs ER and Golgi morphologies and causes retinal degeneration by attenuating the expression of phototransduction proteins

REEP6 is a member of the REEP family proteins belonging to the YIP superfamily. REEP proteins were membrane proteins involved in regulating membrane curvature. Mutations in REEP6 are associated with retinitis pigmentosa (RP). Mice lacking REEP6 were generated to model human RP. Reep6 mutant mice exhibit progressive retinal degeneration, which is attributed to abnormal trafficking of PDE6 and the absent expression of guanylate cyclases (GCs), due to ER dysfunction. However, the phenotype regarding the failed expression of GCs is questionable. Here, we generated an independent Reep6 knockout mouse model. Our Reep6-deficient mouse line showed progressive retinal degeneration as previously reported. Nevertheless, unlike the previous report, our Reep6 mutant mice had normal trafficking of PDE6. GCs were not absent from the mutant mice, but their expression decreased by approximately one third. Other membrane phototransduction proteins, such as rhodopsin and rhodopsin kinase (GRK1) were also reduced. Attenuated expression of membrane proteins might stem from abnormal ER and Golgi function, as in vitro expression of REEP6 altered the expression of the ER marker and Golgi morphologies. Additionally, RNA-seq revealed that deletion of Reep6 caused reduced expression of multiple phototransduction proteins at the transcription level and activated the inflammation pathway. Thus, retinal degeneration associated with REEP6 mutations is the consequence of reduced expression of phototransduction proteins mediated by ER and Golgi dysfunction and ensuing retinal inflammation.

pathology↗

bioRxiv2024

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in the PKR pathway. We demonstrate that PACT is a direct and specific suppressor of PKR against endogenous dsRNA ligands like inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. PACT-mediated inhibition does not involve competition for dsRNA binding. Instead, PACT impairs PKRs ability to scan along dsRNA--a process necessary for PKR molecules to encounter and autophoshorylate each other for activation. By scanning along dsRNA and directly interacting with PKR, PACT restricts PKRs movement on dsRNA, reducing the likelihood of PKR molecular collisions and subsequent autophosphorylation, effectively inhibiting PKR without sequestering dsRNA. Consequently, PKR inhibition is more robust with longer and less abundant dsRNA, and minimal with abundant or short dsRNA. Thus, PACT functions to adjust the PKR activation threshold for long endogenous dsRNA without altering its inherent activity, revealing new mechanisms for establishing self-tolerance.

biochemistry↗

bioRxiv2024

Human Amniotic Epithelial Cells Promote Chx10-/Pax6+ Muller Glia Subpopulation Reprogramming into Photoreceptor-like Cells

Reprogramming Muller glia to regenerate neurons is a promising strategy for treating retinal degeneration, but whether Muller glia contain subpopulations with different regenerative fates remains unclear. Here, using single-cell RNA-seq analysis and Muller glia lineage-tracing mice with retinal degeneration, we reveal that Muller glia were heterogeneous and identify a specific Muller glial subpopulation (Chx10-/Pax6+) in healthy retinas that is activated and migrate to the outer nuclear layer (ONL) during photoreceptor degeneration. Transplantation of human amniotic epithelial cells (hAECs) facilitates the activation and extensive migration of the Chx10-/Pax6+ Muller glial subpopulation to the ONL, where they are reprogrammed into photoreceptor-like cells. Mechanistically, hAECs degrade the inhibitory extracellular matrix through regulating matrix metalloproteinases, which probably induces remodeling of the microenvironment of Muller glia and contributes to cell reprogramming. Consequently, hAEC transplantation improves visual function in rd10 mice. Our findings uncover a distinctive Muller glial subpopulation with the potential for reprogramming into photoreceptors.

cell biology↗

bioRxiv2024

Ligand supplementation restores the cancer therapy efficacy of an antirheumatic drug auranofin from serum inactivation

Auranofin, an FDA-approved antirheumatic gold drug, has gained ongoing interest in clinical studies for treating advanced or recurrent tumors. However, gold ions dynamic thiol exchange nature strongly attenuates its bioactivity due to the fast formation of covalent albumin-gold adducts. Here we report that newly-added thiols can modulate the dynamic albumin-gold binding and recover the therapeutic efficacy. Initially, we found that auranofin supplemented with its own thiol ligand, TGTA (1-thio-{beta}-D-glucose tetraacetate), significantly restored the anticancer activities in cells and patient-derived xenograft models. Then, screening a collection of ligand fragments followed by machine learning evaluation unveiled diverse synergizing thiols, including pantethine that effectuates auranofin at a low dosage used for rheumatoid arthritis. Interestingly, the thiol exchange inside cells accounts for a cuproptosis-like phenotype induced by auranofin. Together, we believe the ligand-enabled dynamic modulation strategy is of value to researchers and clinicians contemplating metallodrugs and ligand-like molecules in cancer therapy.

cancer biology↗

bioRxiv2023

Defense mechanism of a bacterial retron supramolecular assembly

Retrons are a class of multigene antiphage defense system typically consisting of a retron reverse transcriptase, a non-coding RNA, and a cognate effector. Although the triggers for several retron systems have been discovered recently, the full picture of how retron systems sense invading phages and mediate defense remains to be elucidated. Here, we focus on the retron Ec86 defense system and report its modes of activation and action. We identified a phage-encoded DNA cytosine methyltransferase (Dcm) as the trigger of the Ec86 system and show that Ec86 senses msDNA methylation and becomes activated. We further determined the structure of a tripartite retron Ec86 supramolecular assembly, which is primed for activation by Dcm, and demonstrated that the activated system confers defense through depletion of nucleoside derivatives. These findings emphasize the role of retrons being a second line of defense and highlight an emerging theme of anti-phage defense through supramolecular complex assemblies.

microbiology↗

bioRxiv2023

XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells

Emerging data suggest that induction of viral mimicry responses through activation of double-stranded RNA (dsRNA) sensors in cancer cells is a promising therapeutic strategy. One approach to induce viral mimicry is to target molecular regulators of dsRNA sensing pathways. Here, we show that the exoribonuclease XRN1 is a negative regulator of the dsRNA sensor protein kinase R (PKR) in cancer cells with high interferon-stimulated gene (ISG) expression. XRN1 deletion causes PKR activation and consequent cancer cell lethality. Disruption of interferon signaling with the JAK1/2 inhibitor ruxolitinib can decrease cellular PKR levels and rescue sensitivity to XRN1 deletion. Conversely, interferon-{beta} stimulation can increase PKR levels and induce sensitivity to XRN1 inactivation. Lastly, XRN1 deletion causes accumulation of endogenous complementary sense/anti-sense RNAs, which may represent candidate PKR ligands. Our data demonstrate how XRN1 regulates PKR and nominate XRN1 as a potential therapeutic target in cancer cells with an activated interferon cell state.

cancer biology↗

bioRxiv2023

A cingulate-hippocampal circuit mediates early depressive symptoms in Alzheimer's disease

AbstractDepressive symptoms are prevalent and precedes cognitive decline in Alzheimers disease (AD), which worsen the clinical outcome and severely challenge the life quality of the patients. However, the neural circuitry mediating mood disturbance in AD remains elusive. Here we report that the glutamatergic projection from the anterior cingulate cortex (ACC) to the ventral hippocampal CA1 (vCA1) acts as the neural substrate of depressive symptoms in AD. We systemically mapped axonal projection in early stage of 5xFAD mice (3 months), which accompanies depressive- like behavior in these animals and identified reduced axonal projection from ACC to vCA1. This is in accordance with reduced axonal calcium signals detected with in vivo fiber photometry. Chemogenetic or optogenetic reversal of ACC-vCA1 circuitry activity efficiently ameliorated the depressive-like behaviors as well as cognitive impairment in 5xFAD mice. We further identified the synaptic molecules neuregulin-1 (Nrg1) as one vital signal regulating the synaptic glutamate transmission from the ACC to the vCA1. Collectively, these results indicated ACC-vCA1 as a critical pathway that regulates emotional states in Alzheimer disease. Targeting cingulate cortices with brain stimulation may treat depression in AD.

neuroscience↗

bioRxiv2023

LCP1 Regulates Cell Motility in Chondrosarcoma and Correlates with Metastatic Potential and Poor Patient Outcomes

Chondrosarcoma (CSA) is the second most common primary malignancy of bone, whose aggressive potential and chemo-resistant nature result in extremely poor outcomes in patients with advanced disease. Grading and prognostication of these tumors remain a significant challenge for pathologists, and medical oncologists have no effective therapies to prevent or treat metastatic disease. In the present study, we sought to explore the pathogenesis and aggressive progression of chondrosarcoma by comparing gene expression differences from metastasizing and non-metastasizing CSA tumors. We hypothesized that metastasizing tumors have inherent differences that may be attributed to the dysregulation of specific oncogenic cell signaling pathways. RNA-Seq analysis from patient-derived low-passage cell lines of metastasizing and non-metastasizing tumors pinpointed the gene LCP1 (lymphocyte cytosolic protein 1) as upregulated in CSA cells from metastasizing tumors. Analysis of a large clinical cohort of CSA demonstrated that increased expression of LCP1 correlated with poor patient survival. In vitro analyses confirmed the ability of LCP1 to promote migration and invasion of CSA cells. These data support the key role of LCP1 on metastatic potential and poor prognosis in CSA. In conclusion, we confirm the ability of LCP1 to drive metastatic behavior and correlate with poor outcomes in patients.

cancer biology↗

bioRxiv2022

Revealing the structure of somatic cell membranes by in situ cryo-electron tomography

The cell membrane, which separates the cell from the environment, plays a key role in signal transduction, energy conversion and substance transport. Although previous membrane models have successfully interpreted some functions of the cell membrane, no consensus has been reached for the lack of direct and in situ evidence. Here, we characterized the high-resolution 3D structure of 293T cell membranes in situ for the first time by cryo-electron tomography. Due to the excellent thickness of our cryo-samples, we could clearly observe membrane proteins with an average molecular weight of 100 kD. By analysing tomograms, we found that the total thickness of a 293T plasma membrane is approximately 20 nm and that there is a 4-nm lipid bilayer structure within the membrane. We observed that membrane proteins and protein complexes with a similar height (7-11 nm) are densely embedded in the ectoplasmic side of 293T plasma membranes, whereas membrane proteins aggregate to form islands with heights reaching dozens of nanometres on the cytoplasmic side. Additionally, we measured the average sizes of membrane proteins on the cytoplasmic side of 293T plasma membranes and found them to be approximately 7 nm in length and 4 nm in width. Moreover, if more precise structural information is obtained in future studies, we will identify the molecular interactions and detailed structures of membrane protein clusters that can be easily distinguished on a 293T cell membrane. Our work represents the first in situ structural characterization of a native somatic cell membrane with cryo-electron tomography and advances cell membrane structural studies from the model prediction stage to the real structure observation stage.

cell biology↗

bioRxiv2021

The high-resolution structure of cell membranes revealed by in situ cryo-electron tomography

As the structural unit of life, cell is defined by the membrane system. The cell membrane separates the internal and external environment of the cell, and the endomembrane system defines the organelles to perform different functions1-3. However, lack of tools to in situ observe membrane proteins at a molecular resolution has limited our understanding of membrane organization and membrane protein interactions. Here we characterize the high-resolution 3D structure of human red blood cell (hRBC) membranes and the membrane proteins for the first time in situ by cryo-electron tomography (CryoET)4-7. By analyzing tomograms, we have obtained the first fine three-dimensional (3D) structure of hRBC membranes and found the asymmetrical distribution of membrane proteins on both sides of the membranes. We found that the membrane proteins are mainly located on the cytoplasmic side of hRBC membranes, with protein sizes ranging from 6nm to 8nm, in contrast to the ectoplasmic side with basically no proteins. Quantitative analysis of the density of hRBC membrane proteins shows that the membranes with higher protein occupancy have less phospholipid, making the membranes more rigid. Meanwhile, we obtained the channel protein-like structures by preliminary analysis of the membrane protein. Our results represent the first in situ structure characterization of the cell membranes and membrane proteins through cryoET and opens the door for understanding the biological functions of cell membranes in their physiological environments.

biophysics↗

bioRxiv2020

Prelimbic-amygdala overexcitability mediates trait vulnerability in a novel mouse model of acute social defeat stress

BACKGROUNDDepression is a debilitating neuropsychiatric disorder with 20% lifetime prevalence in the developed world but only approximately half of afflicted individuals respond to currently available therapies. While there is growing understanding of the neurobiological underpinnings of the depressed brain, much less is known about the preexisting circuitry leading to selective vulnerability versus resilience. Elucidating these networks could lead to novel preventative approaches. METHODSWe developed a model of acute social defeat stress (ASDS) that allows classification of male mice into "susceptible" (socially avoidant) versus "resilient" (expressing control-level social approach) one hour after exposure to six minutes of social stress. Using circuit tracing and high-resolution confocal imaging, we explored differences in activation and dendritic spine density and morphology in the prelimbic to basolateral amygdala (PL[->]BLA) circuit in resilient versus susceptible mice. To test the functional relevance of identified structure/function differences to divergent behavioral responses, we used an intersectional chemogenetic approach to inhibit the PL[->]BLA circuit during or prior to ASDS. RESULTSSusceptible mice had greater PL[->]BLA recruitment during ASDS and activated PL[->]BLA neurons from susceptible mice had more and larger mushroom spines compared to resilient mice. Inhibition of the PL[->]BLA circuit led to a population shift towards resilience. CONCLUSIONPreexisting PL[->]BLA structure/function differences mediate divergent behavioral responses to ASDS in male mice. These results support the PL[->]BLA circuit as a biomarker of trait vulnerability and potential target for prevention of stress-induced psychopathology.

neuroscience↗