Biology subjects

Lian, X.

Publications and source records attributed to Lian, X..

At least 19 recordsLinked to original sources

bioRxiv2026

Targeting Cardiac Fibrosis with Chimeric Antigen Receptor Neutrophils Derived from Human Pluripotent Stem Cells

Heart failure remains a leading cause of global morbidity and mortality, with cardiac fibrosis contributing to disease progression. Following myocardial injury, activated cardiac fibroblasts orchestrate adverse extracellular matrix (ECM) remodeling, which leads to tissue stiffening and compromised contractility. Despite the clinical importance of cardiac fibrosis, current pharmacological interventions modulate disease progression without directly eliminating activated fibrogenic cells. To target these cells directly, we engineered human pluripotent stem cell (hPSC)-derived neutrophils expressing a chimeric antigen receptor (CAR) directed against fibroblast activation protein (FAP). In vitro, FAP CAR neutrophils exhibited increased reactive oxygen species-associated fluorescence during fibroblast co-culture and dose-dependent cytotoxicity in 2D cultures, while reducing fibroblast-associated fluorescence in 3D cardiac organoids without a significant reduction in cardiomyocyte-associated cardiac troponin T staining. FAP CAR neutrophils also showed increased iNOS and decreased Arginase-1 expression compared with unmodified neutrophils, consistent with a shift toward an N1-associated polarization profile. Analysis of public cardiac single-cell and single-nucleus RNA-sequencing datasets identified temporal patterns of immune-associated expression and fibroblast activation that informed treatment timing. In a murine myocardial infarction (MI) model, mice treated systemically with FAP CAR neutrophils showed higher day 7 left ventricular ejection fraction than PBS-treated mice in an exploratory comparison. Together, these findings support further evaluation of hPSC-derived FAP CAR neutrophils as an immunotherapeutic approach to cardiac fibrosis.

bioengineering↗

bioRxiv2026

Plasma Proteomic Profiling Identifies a Subset of HIV Elite Controllers with Inflammatory Profiles Resembling People Without HIV

Background: Elite controllers (ECs) are people with HIV (PWH) who suppress viral replication without antiretroviral therapy (ART). A subset, often termed exceptional elite controllers (EECs), harbor very small viral reservoirs, with few intact proviruses integrated into inactive chromosomal regions. Defining inflammatory signatures in ECs may clarify whether some approach a functionally cured state. Methods: We measured 384 plasma proteins in ART-naive ECs (n=62), ART-suppressed non-controllers (n=37), and people without HIV (PwoH) (n=50) using Olink proteomics, and compared proteomic profiles, clinical parameters, and intact reservoir data using FLIP-Seq. Hierarchical clustering identified ECs whose proteomic profiles most closely resembled PWoH; this subset was further analyzed using machine-learning approaches. Results: ART-suppressed non-controllers had higher inflammatory protein levels than ART-naive ECs, yet ECs still showed elevated inflammation and lower CD4:CD8 ratios compared with PWoH. Clustering analysis revealed a distinct subset of ECs (n=9) whose profiles closely matched those without HIV (ECs with PWoH-like proteomic profiles), a pattern not observed in ART-suppressed non-controllers. Compared to typical ECs, ECs with PWoH-like proteomic profiles had consistently lower IL-1{beta}, SCRN1, and BID, numerically lower intact proviral frequencies, and a high frequency of protective HLA alleles. Machine-learning analyses highlighted features that most strongly differentiated ECs with PWoH-like proteomic profiles from typical ECs, including reduced gut mucosal/intestinal epithelial-associated proteins, higher absolute CD4 counts, and lower levels of pro-inflammatory cytokines and cellular immune activation. Conclusions: These findings identify a subset of ECs with a PWoH-like inflammatory proteomic profile, accompanied by numerically lower intact HIV reservoirs and a high frequency of protective HLA alleles. Defining the mechanisms associated with this low-inflammatory state may provide insights into exceptional HIV control and inform strategies for durable HIV remission.

immunology↗

bioRxiv2026

NK-like and networked CD8+ T cell immunity mediates exceptional HIV control

Durable treatment-free remission remains a defining goal for people living with HIV (PLWH). Studies of spontaneous elite controllers have revealed that functional CD8 T cells targeting structurally networked viral epitopes can mediate durable viral suppression1,2. However, rare reservoir-defined exceptional controllers within the spectrum of elite control3-5, characterized by the absence of intact provirus or proviruses confined to transcriptionally repressed genomic regions6, provide a unique opportunity to define mechanisms of cure-like immunity. Here, we integrate functional epitope mapping, single-cell transcriptomics, and infected cell elimination assays to identify networked HIV epitope targeting and a natural killer (NK)-like killer-cell immunoglobulin-like receptor (KIR) CD8 T cell subset as key features of exceptional control. This NK-like subset was selectively enriched within HIV-specific, but not CMV-specific, CD8 T cells from controllers, and was transcriptionally similar to highly cytotoxic subsets within the broader KIR+ CD8+ T cell compartment. Flow cytometry revealed increased frequencies of KIR CD8 T cells in exceptional controllers relative to antiretroviral therapy (ART)-suppressed individuals, and unexpectedly, enrichment of dual KIR+ NKG2A+ CD8 T cells. Functional depletion of KIR CD8 T cells significantly impaired the elimination of autologous HIV-infected CD4 T cells, despite preserved recognition by proliferative networked HIV-specific CD8 T cells. These findings thereby identify an NK-like KIR CD8 T cell state as a previously unrecognized component of exceptional HIV immunity that complements networked epitope targeting, providing a novel framework for immunotherapeutic HIV cure strategies.

immunology↗

bioRxiv2025

Size-Modulated Mesoderm-Endoderm Divergence and Myocardial Cavitation in Micropatterned Cardioids

The human heart, originating from the splanchnic mesoderm, is the first functional organ to develop, co-evolving with the foregut endoderm through reciprocal signaling. Previously, cardioid models offered new insights on cardiovascular cell lineages and tissue morphogenesis during heart development, while mesoderm-endoderm crosstalk remain incompletely understood. Here, we integrated micropatterned cardioids, CRISPR-engineered reporter hiPSCs, deep-tissue imaging, and single-cell RNA sequencing (scRNA-seq) to explore synergistic mesoderm-endoderm co-development. scRNA-seq with PHATE trajectory mapping reconstructed lineage bifurcations of mesoderm-heart and endoderm-foregut lineages, identifying key cell types in cardiac and hepatic development. Ligand-receptor interaction analysis highlighted mesodermal cells enriched in non-canonical WNT, NRG, and TGF-{beta} signaling, while endodermal cells exhibited VEGF and Hedgehog activity. We found that micropattern sizes influenced cellular composition, cardioid cavitation, contractile functions, and mesoderm-endoderm signaling crosstalk. The cardioids generated from 600 {micro}m diameter circle patterns showed larger cavity formation resembling early heart chamber formation. Our findings establish micropatterned cardioids as a model for mesoderm-endoderm co-development, enhancing our understanding of heart-foregut synergy during early embryogenesis.

bioengineering↗

bioRxiv2025

Collagen Nanofiber Reinforced Alginate Hydrogel Tube Microbioreactors for Cell Culture

The large-scale production of mammalian cells is pivotal for various applications; however, current bioreactor technologies encounter significant technical and economic challenges. Scaling up cell cultures remains problematic due to excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates. Nevertheless, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. To address these limitations, this study reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Utilizing a novel micro-extruder, we efficiently produced cell-loaded ColAlgTubes. Collagen formed a dense nanofiber network interwoven with the alginate mesh, enhancing the hydrogels mechanical properties while providing cell adhesion sites. ColAlgTubes protected cells from hydrodynamic stress and maintained cell mass within a 400 m diameter, ensuring efficient nutrient exchange and waste removal. This optimized microenvironment resulted in high cell viability, rapid proliferation, and exceptional yields of 5x108 cells/mL - 200 times higher than conventional culture methods. With their scalability, cost-effectiveness, and efficiency, ColAlgTubes offers a transformative solution for large-scale cell production with broad applications in biotechnology, regenerative medicine, and therapeutic manufacturing.

bioengineering↗

bioRxiv2025

Gas Vesicle-Expressing Human Pluripotent Stem Cells Enable Multimodal Ultrasound and Optical Coherence Tomographic Imaging

Genetically encoded imaging reporters are critical tools for tracking cell fate and function in regenerative medicine. Gas vesicles (GVs), air-filled protein nanostructures derived from bacteria, offer unique advantages for noninvasive imaging due to their acoustic and optical properties. In this study, we engineered human pluripotent stem cells (hPSCs) to express GVs using a doxycycline-inducible system. Upon doxycycline (Dox) treatment, GVs formed intracellularly and enabled enhanced contrast in both ultrasound and optical coherence tomography (OCT) imaging. Dynamic ultrasound imaging revealed pressure-dependent GV buckling and harmonic signal generation, while OCT imaging confirmed high sensitivity and depth-resolved detection in both in vitro and ex vivo retinal models. Our work establishes a multimodal GV-based reporter platform compatible with human stem cells and clinically relevant imaging modalities. This approach offers a powerful and versatile tool for noninvasively visualizing and tracking therapeutic cells in real time, advancing the development and monitoring of cell-based therapies.

synthetic biology↗

bioRxiv2025

Collagen Hydrogel Tube Microbioreactors for Cell and Tissue Manufacturing

The production of mammalian cells in large quantities is essential for various applications. However, scaling up cell culture using existing bioreactors poses significant technical challenges and high costs. To address this, we previously developed an innovative 3D culture system, known as the AlgTube cell culture system, for high-density cell cultivation. This system involves processing cells into microscale alginate hydrogel tubes, which are suspended in the culture medium within a vessel. These hydrogel tubes shield cells from hydrodynamic stress and maintain the cell mass below 400 {micro}m in diameter, facilitating efficient mass transport and creating a favorable microenvironment for cell growth. Under optimized conditions, AlgTubes supported long-term culture with high cell viability, rapid expansion (1000-fold increase over 9 days per passage), and high yield (5x10 cells/mL), offering significant advantages over conventional methods. Despite these benefits, AlgTubes have critical drawbacks. They are mechanically fragile, with frequent breakage during culture leading to cell leakage and production failures. Additionally, many cell types exhibit poor growth due to the inability to adhere to the alginate surface, making alginate hydrogel microtubes unsuitable for industrial-scale cell production. To overcome these challenges, we developed a novel collagen hydrogel tube-based microbioreactor system in this work. This system provides enhanced robustness and adhesion, enabling scalable, cost-effective, and efficient cell production for a wide range of applications.

bioengineering↗

bioRxiv2024

Discovery of A Chimeric Polyketide Family as Cancer Immunogenic Chemotherapeutic Leads

Discovery of cancer immunogenic chemotherapeutics represents an emerging, highly promising direction for cancer treatment that uses a chemical drug to achieve the efficacy of both chemotherapy and immunotherapy. Herein we report a high-throughput screening platform and the subsequent discovery of a new class of cancer immunogenic chemotherapeutic leads. Our platform integrates informatics-based activity metabolomics for rapid identification of microbial natural products with both novel structures and potent activities. Additionally, we demonstrate the use of microcrystal electron diffraction (MicroED) for direct structure elucidation of the lead compounds from partially purified mixtures. Using this strategy to screen geographically and phylogenetically diverse microbial metabolites against pseudomyxoma peritonei, a rare and severe cancer, we discovered a new class of leads, aspercyclicins. The aspercyclicins feature an unprecedented tightly packed polycyclic polyketide scaffold that comprises continuous fused, bridged, and spiro rings. The biogenesis of aspercyclicins involves two distinct biosynthetic pathways, leading to formation of chimeric compounds that cannot be predicted by bottom-up approaches mining natural products biosynthetic genes. With comparable potency to some clinically used anticancer drugs, aspercyclicins are active against multiple cancer cell types by inducing immunogenic cell death (ICD), including the release of damage-associated molecular patterns and subsequent phagocytosis of cancer cells. The broad-spectrum ICD-inducing activity of aspercyclicins, combined with their low toxicity to normal cells, represents a new class of potential cancer immunogenic chemotherapeutics and particularly the first drug lead for pseudomyxoma peritonei treatment.

biochemistry↗

bioRxiv2024

Human gut commensal Alistipes timonensis modulates the host lipidome and delivers anti-inflammatory outer membrane vesicles to suppress colitis in an Il10-deficient mouse model

Correlative studies have linked human gut microbes to specific health conditions. Alistipes is one such microbial genus negatively linked to inflammatory bowel disease (IBD). However, the protective role of Alistipes in IBD is understudied, and the underlying molecular mechanisms remain unknown. In this study, colonization of Il10-deficient mice with Alistipes timonensis DSM 27924 delays colitis development. Colonization does not significantly alter the gut microbiome composition, but instead shifts the host plasma lipidome, increasing phosphatidic acids while decreasing triglycerides. Outer membrane vesicles (OMVs) derived from Alistipes are detected in the plasma of colonized mice, carrying potentially immunomodulatory metabolites into the host circulatory system. Fractions of A. timonensis OMVs suppress LPS-induced Il6, Il1b, and Tnfa expression in vitro in murine macrophages. We detect putative bioactive lipids in the OMVs, including immunomodulatory sulfonolipids (SoLs) in the active fraction, which are also increased in the blood of colonized mice. Treating Il10-deficient mice with purified SoL B, a representative SoL, suppresses colitis development, suggesting its contribution to the anti-inflammatory phenotype observed with A. timonensis colonization. Thus, A. timonensis OMVs represent a potential mechanism for Alistipes-mediated delay of colitis in Il10-deficient mice via delivery of immunomodulatory lipids and modulation of the host plasma lipidome.

biochemistry↗

bioRxiv2024

Discovery and Biosynthesis of Sulfenicin and Its New-to-Nature Acylsulfenic Acid Functional Group

Lifes organic molecules are built with diverse functional groups that enable biology by fine tuning intimate connections through time and space. As such, the discovery of new-to-nature functional groups can expand our understanding of the natural world and motivate new applications in biotechnology and biomedicine. Herein we report the genome-aided discovery of sulfenicin, a novel polyketide-nonribosomal peptide hybrid natural product from a marine Streptomyces bacterium bearing a unique acylsulfenic acid functionality. Through a series of heterologous biosynthesis, functional genetics, and enzymatic reconstitution experiments, we show that this previously described synthetic functional group is biologically assembled by a set of enzymes from both primary and secondary metabolism, including a novel flavin-dependent S-hydroxylase that hydroxylates a thiocarboxylic acids sulfur atom. While the sulfenicin biosynthetic gene cluster is presently without parallel in public databases, acylsulfenic acid-encoding enzymes are widely distributed in bacterial genomes, implying that this labile functional group may similarly have a broad distribution among specialized metabolites.

biochemistry↗

bioRxiv2024

Telomere-to-telomere African wild rice (Oryza longistaminata) reference genome reveals segmental and structural variation

Rice (Oryza sativa) is one of the most important staple food crops worldwide, and its wild relatives serve as an important gene pool in its breeding. Compared with cultivated rice species, African wild rice (Oryza longistaminata) has several advantageous traits, such as resistance to increased biomass production, clonal propagation via rhizomes, and biotic stresses. However, previous O. longistaminata genome assemblies have been hampered by gaps and incompleteness, restricting detailed investigations into their genomes. To streamline breeding endeavors and facilitate functional genomics studies, we generated a 343-Mb telomere-to-telomere (T2T) genome assembly for this species, covering all telomeres and centromeres across the 12 chromosomes. This newly assembled genome has markedly improved over previous versions. Comparative analysis revealed a high degree of synteny with previously published genomes. A large number of structural variations were identified between the O. longistaminata and O. sativa. A total of 2,466 segmentally duplicated genes were identified and enriched in cellular amino acid metabolic processes. We detected a slight expansion of some subfamilies of resistance genes and transcription factors. This newly assembled T2T genome of O. longistaminata provides a valuable resource for the exploration and exploitation of beneficial alleles present in wild relative species of cultivated rice.

genomics↗

bioRxiv2024

Modulation Of SUDEP By Central Serotonergic Cooperating with Noradrenergic Circuits: A Synergistic-Dependent Manner

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Despite previous accumulating evidence has shown that seizure-induced respiratory arrest (S-IRA) may play the main contributor to SUDEP as an initiating event preeminent cause of mortality, the specific underlying mechanism of action remains unclear. Based on our previous work, serotonin (5-HT) signaling in the dorsal raphe nucleus (DRN) is strongly implicated in S-IRA in animal models, including the DBA/1 mice, on the meanwhile, norepinephrine (NE) neurons of the locus coeruleus (LC) also plays a vital role in regulating respiratory function on its own. Superficially, monoaminergic neuron, as important neurotransmitters in the central nervous system, have similar modes of action in the maintenance of nervous system balance, and each of them has a regulatory effect on SUDEP. However, it remains to be investigated whether monoaminergic neuron family (NE and 5-HT) are related in the mechanism of regulating SUDEP, what is even more curious is whether the two are intrinsically linked. Thus, we hypothesize neural mechanism of central noradrenergic and serotonergic circuits in modulating SUDEP in a synergistic-dependent manner, this endeavor will culminate in a significant breakthrough in elucidating the precise mechanism of action underlying SUDEP. In our study, we will use chemogenetics, optogenetics, calcium signal recording, and bidirectional tracing to explore the internal mechanism of DR-LC regulating the occurrence of SUDEP, and by specifically injecting 5-HT2AR antagonist Ketanserin (KET) and/or NE-1R antagonist Prazosin into the pre-Botzinger complex (PBC), it was finally elucidate that the DR-LC-PBC network can effectively reduce the incidence of SIRA. We firstly proposed a powerful target for exploring the reduction of the incidence of SUDEP, which has great clinical translation potential.

neuroscience↗

bioRxiv2024

Zinc finger transcription factors BnaSTOP2s regulate sulfur metabolism and confer resistance to Sclerotinia sclerotiorum in Brassica napus

Rapeseed (Brassica napus L.) has a high sulfur requirement for optimal growth, development, and pathogen resistance. In this study, we identified zinc finger transcription factors, BnaSTOP2s, that play key roles in sulfur metabolism and Sclerotinia sclerotiorum resistance. First, our results suggested that BnaSTOP2s are involved in sulfur as evidenced from extensive protein interaction screening. Knockout of BnaSTOP2s reduced the response sensitivity in both sulfur-deficient and sulfur-excessive conditions by promoting the elongation of primary roots of seedlings. Furthermore, the content of essential sulfur-containing metabolites, including glucosinolate and glutathione, were substantially down-regulated in roots and leaves of Bnastop2 mutants, which is consistent with the significantly lowered transcriptional levels of key players of GSL synthesis and transportation, BnaMYB28s and BnaGTR2s, respectively. Through comprehensive RNA-seq analysis, we revealed the substantial effect of BnaSTOP2s on sulfur metabolism from source to sink. Additionally, we observed a significant decrease while increase in leaf lesion sizes of the BnaSTOP2-OE and Bnastop2 mutants, respectively, when compared to the wild type during Sclerotinia sclerotiorum infection, suggesting the vital role of BnaSTOP2 in plant defense response. Overall, our findings highlight that BnaSTOP2s seems to be global regulators of sulfur metabolism and confer resistance to Sclerotinia sclerotiorum infection in B. napus.

plant biology↗

bioRxiv2023

pADP-ribosylation Regulates the Cytoplasmic Localization, Cleavage and Pro-apoptotic Function of HuR

HuR (ElavL1) is one of the main posttranscriptional regulators that determines cell fate. While the role of HuR in apoptosis is well-established, the posttranslational modifications that governs this function remain elusive. In this study we show that PARP1/2-mediated poly(ADP)-ribosylation (PARylation) is instrumental in the pro-apoptotic function of HuR. During apoptosis or in cells depleted of PARP1/2 enzymes, a substantial reduction in HuR PARylation is observed. This results in the cytoplasmic accumulation and the cleavage of HuR, both of which are essential events for apoptosis. These effects are mediated by a pADP-ribose (PAR) binding motif within the HuR-HNS region (HuR PAR Binding Site (HuR-PBS)). Under normal conditions, the association of HuR-PBS with PAR is responsible for the nuclear retention of HuR. Mutations within this motif prevents the binding of HuR to its import factor TRN2, leading to its cytoplasmic accumulation and cleavage. Collectively, our findings underscore the role of PARylation in controlling the proapoptotic function of HuR, offering insight into the mechanism by which PARP1/2 enzymes regulate cell fate and adaptation to various assaults.

cell biology↗

bioRxiv2023

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time. As a proof of concept, we performed real-time imaging of Dictyostelium discoideum cells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

bioengineering↗

bioRxiv2023

Persistence of intact HIV-1 proviruses in the brain during suppressive antiretroviral therapy

HIV-1 reservoir cells that circulate in peripheral blood during suppressive antiretroviral therapy (ART) have been well characterized, but little is known about the dissemination of HIV-1-infected cells across multiple anatomical tissues, especially the central nervous system (CNS). Here, we performed single-genome, near full-length HIV-1 next-generation sequencing to evaluate the proviral landscape in distinct anatomical compartments, including multiple CNS tissues, from 3 ART-treated participants at autopsy. While lymph nodes and, to a lesser extent, gastrointestinal and genitourinary tissues represented tissue hotspots for the persistence of intact proviruses, we also observed intact proviruses in CNS tissue sections, particularly in the basal ganglia. Multi-compartment dissemination of clonal intact and defective proviral sequences occurred across multiple anatomical tissues, including the CNS, and evidence for the clonal proliferation of HIV-1-infected cells was found in the basal ganglia, in the frontal lobe, in the thalamus and in periventricular white matter. Deep analysis of HIV-1 reservoirs in distinct tissues will be informative for advancing HIV-1 cure strategies.

microbiology↗

bioRxiv2023

ProtWave-VAE: Integrating autoregressive sampling with latent-based inference for data-driven protein design

Deep generative models (DGMs) have shown great success in the understanding of data-driven design of proteins. Variational autoencoders (VAEs) are a popular DGM approach that can learn the correlated patterns of amino acid mutations within a multiple sequence alignment (MSA) of protein sequences and distill this information into a low-dimensional latent space to expose phylogenetic and functional relationships and guide generative protein design. Autoregressive (AR) models are another popular DGM approach that typically lack a low-dimensional latent embedding but do not require training sequences to be aligned into an MSA and enable the design of variable length proteins. In this work, we propose ProtWave-VAE as a novel and lightweight DGM employing an information maximizing VAE with a dilated convolution encoder and autoregressive WaveNet decoder. This architecture blends the strengths of the VAE and AR paradigms in enabling training over unaligned sequence data and the conditional generative design of variable length sequences from an interpretable low-dimensional learned latent space. We evaluate the models ability to infer patterns and design rules within alignment-free homologous protein family sequences and to design novel synthetic proteins in four diverse protein families. We show that our model can infer meaningful functional and phylogenetic embeddings within latent spaces and make highly accurate predictions within semi-supervised downstream fitness prediction tasks. In an application to the C-terminal SH3 domain in the Sho1 transmembrane osmosensing receptor in bakers yeast, we subject ProtWave-VAE designed sequences to experimental gene synthesis and select-seq assays for osmosensing function to show that the model enables de novo generative design, conditional C-terminus diversification, and engineering of osmosensing function into SH3 paralogs.

synthetic biology↗

bioRxiv2023

Nuclear bodies protect phase separated proteins from degradation in stressed proteome

RNA-binding proteins (RBPs) containing intrinsically disordered domains undergo liquid-liquid phase separation to form nuclear bodies under stress conditions. This process is also connected to the misfolding and aggregation of RBPs, which are associated with a series of neurodegenerative diseases. However, it remains elusive how folding states of RBPs changes upon the formation and maturation of nuclear bodies. Here, we describe SNAP-tag based imaging methods to visualize the folding states of RBPs in live cells via time-resolved quantitative microscopic analyses of their micropolarity and microviscosity. Using these imaging methods in conjunction with immunofluorescence imaging, we demonstrate that RBPs, represented by TDP-43, initially enters the PML nuclear bodies in its native state upon transient proteostasis stress, albeit it begins to misfolded during prolonged stress. Furthermore, we show that heat shock protein 70 co-enters the PML nuclear bodies to prevent the degradation of TDP-43 from the proteotoxic stress, thus revealing a previously unappreciated protective role of the PML nuclear bodies in the prevention of stress-induced degradation of TDP-43. In summary, our imaging methods described in the manuscript, for the first time, reveal the folding states of RBPs, which were previously challenging to study with conventional methods in nuclear bodies of live cells. This study uncovers the mechanistic correlations between the folding states of a protein and functions of nuclear bodies, in particular PML bodies. We envision that the imaging methods can be generally applied to elucidating the structural aspects of other proteins that exhibit granular structures under biological stimulus.

biochemistry↗