Biology subjects

Chen, S.-Y.

Publications and source records attributed to Chen, S.-Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Experimental conditions to retrieve intrinsic cooperativity α directly from single binding assay data exemplified by the ternary complex formation of FKBP12, MAPRE1 and macrocyclic molecular glues

The incorporation of disease-relevant targets in a compound-dependent manner into ternary complexes with an assisting chaperone protein has become a common modality. Among the various types of ternary complex-forming compounds, molecular glues are of particular interest because they do not require significant binary (independent) target affinity. Instead, ternary complexes formed by molecular glues retrieve a significant part of their thermodynamic stability through newly induced chaperone-target or glue-target interactions that occur only in the ternary complex. These interactions lead to enhanced ligand binding - termed intrinsic cooperativity - which can be retrieved via the apparent cooperativity either by monitoring ligand binding through the chaperone or through the target protein. In this publication, the advantage of measuring the apparent cooperativity to determine the cooperativity by the weaker binding protein is discussed and illustrated using the example of ternary complexes between FKBP12, MAPRE1 and macrocyclic molecular glues derived from the rapamycin binding motif for FKBP12. Furthermore, the impact of the following three parameters on the apparent cooperativity is illustrated: 1.) the concentration of the monitoring protein, 2.) the excess of the counter protein, 3.) the affinity of the glue to the weaker binding protein in combination with the degree of intrinsic cooperativity . From this, experimental conditions to determine the intrinsic cooperativity with only one binding assay and without the need for a comprehensive mathematical model covering all simultaneous events under non-saturating conditions are highlighted. However, this framework requires a binding assay capable of measuring or at least estimating very weak binary affinities. If this is not possible for experimental reasons, but binding assays for both proteins are available within a normal bandwidth and the affinity to the stronger binding protein is not too high, it is discussed how the binding curve for the weaker binding protein in the presence of an excess of the weaker binding protein can be used to overcome the missing binary Kd for the weakly binding protein.

biochemistry↗

bioRxiv2024

Innovative Carnitine-Fed Rats Model Reveals Resveratrol Butyrate Ester's Multimechanistic Role in Reducing TMAO and Cardiovascular Risk

BACKGROUNDTrimethylamine-N-oxide (TMAO), a metabolite produced from dietary carnitine through gut microbiota, is a recognized risk factor for cardiovascular disease (CVD). High-fat diets and carnitine supplementation exacerbate TMAO levels and related risks, making them valuable in developing experimental models for studying CVD. Resveratrol butyrate ester (RBE) and its monomer ED4 have shown potential in reducing TMAO levels and improving cardiovascular outcomes through metabolic and microbial modulation, but their precise mechanisms remain unclear. METHODS AND RESULTSA novel animal model was established using 36 male Sprague-Dawley rats fed a high-fat diet supplemented with carnitine to elevate serum TMAO levels, simulating dietary-induced CVD risks. Rats were divided into six groups: control diet (CN), high-fat diet (HFD), high-fat diet with carnitine (HFDC), HFDC with dimethylbutanol (HFDCB), CN with ED4 (CNM), and HFDC with ED4 (HFDCM). Physiological parameters, serum lipid profiles, SCFA levels, microbiota composition, and gene expression (OCTN2 and FMO3) were analyzed. ED4 supplementation reduced serum TMAO levels by upregulating OCTN2 expression, promoting urinary TMAO excretion, and restoring SCFA levels. ED4 also modulated gut microbiota, reducing TMA-producing bacteria (e.g., Bacteroides), and improved cardiovascular markers, including reduced blood lipid levels and fat accumulation. While dimethylbutanol inhibited FMO3 expression to reduce TMAO, ED4 acted primarily through carnitine utilization and microbiota modulation. Both treatments enhanced urinary TMAO excretion and altered gut microbiota composition. CONCLUSIONSThis study introduced an innovative animal model combining high-fat and carnitine-enriched diets to study TMAO-related cardiovascular risks. ED4 demonstrated multimechanistic effects in reducing TMAO levels and CVD risk factors by modulating gut microbiota, restoring SCFA levels, and enhancing carnitine metabolism. These findings highlight ED4s therapeutic potential in cardiovascular protection and metabolic regulation. However, further research is needed to elucidate the molecular pathways underlying ED4s effects on TMAO metabolism and its broader translational applications.

molecular biology↗

bioRxiv2024

Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

One of the most common sites of metastasis in ovarian cancer (OVCA) is the peritoneum. Often, this spread is accompanied by the accumulation of a fluid called ascites in the peritoneal cavity. Despite its common occurrence in metastatic OVCA patients, ascites and its influence on the peritoneal spread of OVCA are poorly understood. Interestingly, OVCA cells are vulnerable to ferroptosis, a type of cell death caused by lipid peroxidation. Hence, how these ferroptosis-sensitive OVCA cells persist in their spread to the peritoneum remains unknown. Here, we show that ascites robustly protects OVCA cells and patient-derived organoids against ferroptosis and enhances the peritoneal spread of OVCA cells in mice. Mechanistically, ascites downregulates the mitochondrial enzyme, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), which contributes to an increase in lipid droplets. Additionally, upon ferroptosis induction, ascites represses the upregulation of the transferrin receptor, TFRC, thereby decreasing cellular labile iron levels. Furthermore, we show that lipid-lowering fibrates reverse cellular changes induced by ascites, and they attenuate the peritoneal spread of OVCA cells in mice. Our findings implicate the importance of ascites in ferroptosis protection and the peritoneal spread of OVCA, and they suggest that targeting the ferroptosis protection by ascites may present a novel therapeutic approach to limit OVCA metastasis.

cancer biology↗

bioRxiv2024

Chromosome-level reference genome assembly for the protected resource plant, Zenia insignis

Zenia insignis Chun, from the subfamily Dialioideae of Fabaceae, is a tree species of significant economic and ecological values. It is a threatened species as per the IUCN Red List. The absence of a reference genome has impeded research on Z. insignis despite its importance. In this study, we generated a reference genome for Z. insig n, iws ith a contig N50 of 6.02Mb, a total length of 361.11 Mb, and 97.71% of the sequences assembled into 14 pseudo-chromosomes. The BUSCO assessment score for completeness is 97.30%, and the LAI index assessment score for continuity is 14.57. To our knowledge, this is the first reported nuclear genome of a species belonging to subfamily Dialioideae. The reference genome will provide a valuable resource for the phylogenomic studies of the Fabaceae family and facilitate further research on Z. insignis.

genomics↗

bioRxiv2024

Protein CoAlation on TXNRD2 regulates mitochondrial thioredoxin system to protect against ferroptosis

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

cell biology↗

bioRxiv2024

Cell-specific polymerization-driven biomolecular condensate formation fine-tunes root tissue morphogenesis

Formation of biomolecular condensates can be driven by weak multivalent interactions and emergent polymerization. However, the mechanism of polymerization-mediated condensate formation is less studied. We found lateral root cap cell (LRC)-specific SUPPRESSOR OF RPS4-RLD1 (SRFR1) condensates fine-tune primary root development. Polymerization of the SRFR1 N-terminal domain is required for both LRC condensate formation and optimal root growth. Surprisingly, the first intrinsically disordered region (IDR1) of SRFR1 can be functionally substituted by a specific group of intrinsically disordered proteins known as dehydrins. This finding facilitated the identification of functional segments in the IDR1 of SRFR1, a generalizable strategy to decode unknown IDRs. With this functional information we further improved root growth by modifying the SRFR1 condensation module, providing a strategy to improve plant growth and resilience.

molecular biology↗

bioRxiv2024

Immunomagneto-activated hepatocyte-specific tissue vesicle profiling reveals message diversification from regenerating liver

Background & AimsThe liver possesses a remarkable regenerative capacity involving intricate intercellular communication, the mechanisms of which remain poorly understood. Extracellular vesicles (EVs) emerge as important messengers in diverse pathophysiological conditions. However, tissue-derived, cell-specific functional EV populations in regeneration have not been robustly investigated. MethodsBulk and single-cell RNA sequencing analyses of the regenerating liver after partial hepatectomy (PHx), ultrastructural examinations of in situ liver tissue EVs (LT-EVs), and nanoscale and proteomic profiling of hepatocyte-derived tissue EVs (Hep-EVs) were integrated. Targeted inhibition of Hep-EV release in vivo was performed via AAV-mediated shRNA knockdown of Rab27a, and Hep-EVs were intravenously infused for therapeutically use. Gross, histological, and immunofluorescent examinations of livers with evaluating in vivo and ex vivo hepatocyte proliferation were conducted. ResultsLT-EVs contribute to the liver regenerative process after PHx, and hepatocytes serve as the major origin of tissue EVs in the regenerating liver. Moreover, Hep-EVs play an indispensable role to orchestrate liver regeneration, which is strengthened to release with proliferative messages identified after PHx. Mechanistically, Hep-EVs from the regenerating liver reciprocally promote hepatocyte proliferation, which are hallmarked by and function based on the Cyclin dependent kinase 1 (Cdk1) activity. Importantly, replenishment of Hep-EVs from the regenerating liver holds translational promise and rescues insufficient liver regeneration. ConclusionsOur study establishes a functional and mechanistic framework that release of regenerative Hep-EVs govern rapid hepatocyte proliferation through cell cycle control, shedding light on investigation of physiological and endogenous tissue EV populations in organ regeneration and therapy.

cell biology↗

bioRxiv2024

Thymidine Phosphorylase Promotes the Formation of Abdominal Aortic Aneurysm in Mice Fed a Western Diet

AimsThe precise molecular drivers of abdominal aortic aneurysm (AAA) remain unclear. Thymidine phosphorylase (TYMP) contributes to increased platelet activation, thrombosis, and inflammation, all of which are key factors in AAA development. Additionally, TYMP suppresses the proliferation of vascular smooth muscle cells (VSMCs), which are central to the development and progression of AAA. We hypothesize that TYMP plays a key role in AAA development. Methods and ResultsWe conducted a histological study using human AAA samples and normal abdominal aortas, revealing heightened levels of TYMP in human AAA vessel walls. To validate this observation, we utilized an Ang II perfusion-induced AAA model in wild-type C57BL/6J (WT) and Tymp-/-mice, feeding them a Western diet (TD.88137) starting from 4 weeks of age. We found that Tymp-/-mice were protected from Ang II perfusion-induced AAA formation. Furthermore, by using TYMP-expressing VSMCs as well as primarily cultured VSMCs from WT and Tymp-/- mice, we elucidated the essential role of TYMP in regulating MMP2 expression and activation. TYMP deficiency or inhibition by tipiracil, a selective TYMP inhibitor, led to reduced MMP2 production, release, and activation in VSMCs. Additionally, TYMP was found to promote pro-inflammatory cytokine expression systemically, and its absence attenuates TNF--stimulated activation of MMP2 and AKT. By co-culturing VSMCs and platelets, we observed that TYMP-deficient platelets had a reduced inhibitory effect on VSMC proliferation compared to WT platelets. Moreover, TYMP appeared to enhance the expression of activated TGF{beta}1 in cultured VSMCs in vitro and in human AAA vessel walls in vivo. TYMP also boosted the activation of thrombospondin-1 type 1 repeat domain-enhanced TGF{beta}1 signaling, resulting in increased connective tissue growth factor production. ConclusionOur findings collectively demonstrated that TYMP serves as a novel regulatory force in vascular biology, exerting influence over VSMC functionality and inflammatory responses that promote the development of AAA. Translational PerspectiveThymidine phosphorylase (TYMP) is increased in the vessel walls of patients with abdominal aortic aneurysm (AAA), and TYMP deficiency in mice reduces the incidence of AAA, suggesting that TYMP plays a crucial role in AAA development. This could be attributed to TYMPs role in enhancing systemic inflammation and thrombosis, inhibiting vascular smooth muscle cell function, increasing the activation of matrix metalloproteinase and AKT, as well as enhancing the expression of TGF{beta}1 and connective tissue growth factor. Tipiracil is an FDA-approved drug known to inhibit TYMP-enhanced thrombosis. Targeting TYMP with tipiracil could represent a promising new therapeutic strategy for AAA development.

cell biology↗

bioRxiv2024

NINJ1 regulates ferroptosis via xCT antiporter interaction and CoA modulation

Ninjurin-1 (NINJ1), initially identified as a stress-induced protein in neurons, recently emerged as a key mediator of plasma membrane rupture during apoptosis, necrosis, and pyroptosis. However, its involvement in ferroptosis remains unknown. Here, we demonstrate that NINJ1 also plays a crucial role in ferroptosis, but through a distinct mechanism. NINJ1 knockdown significantly protected cancer cells against ferroptosis induced by xCT inhibitors but no other classes of ferroptosis-inducing compounds (FINs). Glycine, known to inhibit canonical NINJ1-mediated membrane rupture in other cell deaths, had no impact on ferroptosis. A compound screen revealed that NINJ1-mediated ferroptosis protection can be abolished by pantothenate kinase inhibitor (PANKi), buthionine sulfoximine (BSO), and diethylmaleate (DEM). These results suggest that this ferroptosis protection is mediated via Coenzyme A (CoA) and glutathione (GSH), both of which were found to be elevated upon NINJ1 knockdown. Furthermore, we discovered that NINJ1 interacts with the xCT antiporter, which is responsible for cystine uptake for the biosynthesis of CoA and GSH. The removal of NINJ1 increased xCT levels and stability, enhanced cystine uptake, and contributed to elevated CoA and GSH levels, collectively contributing to ferroptosis protection. These findings reveal that NINJ1 regulates ferroptosis via a non-canonical mechanism, distinct from other regulated cell deaths. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/581432v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@9fa073org.highwire.dtl.DTLVardef@1df0137org.highwire.dtl.DTLVardef@1c8e8cborg.highwire.dtl.DTLVardef@12b76b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Epiregulon: Inference of single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response

Transcription factors (TFs) and transcriptional coregulators represent an emerging class of therapeutic targets in oncology. Gene regulatory networks (GRNs) can be used to evaluate pharmacological agents targeting these factors and to identify drivers of disease and drug resistance. However, GRN methods that rely solely on gene expression often fail to account for post-transcriptional modulation of TF function. We present Epiregulon, a method that constructs GRNs from single-cell ATAC-seq and RNA-seq data for accurate prediction of TF activity. This is achieved by considering the co-occurrence of TF expression and chromatin accessibility at TF binding sites in each cell. We leverage ChIP-seq data to extend inference to transcriptional coregulators lacking defined motifs or TF harboring neomorphic mutations. Epiregulon accurately predicted the effects of AR inhibition across various drug modalities including an AR antagonist and an AR degrader, delineated the mechanisms of a SMARCA4 degrader by identifying context-dependent interaction partners and prioritized known and novel drivers of lineage reprogramming and tumorigenesis. By mapping gene regulation across various cellular contexts, Epiregulon can accelerate the discovery of therapeutics targeting transcriptional regulators.

bioinformatics↗

bioRxiv2023

The effects of resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged adults

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged participants (MA, 55{+/-}8 years old, 17 females, 9 males). We obtained vastus lateralis (VL) muscle cross-sectional area (mCSA), VL biopsies, and strength assessments before and after training. Fiber cross-sectional area (fCSA), satellite cells (Pax7+), denervated myofibers (NCAM+), senescent cells (p16+ or p21+), senescence-related proteins, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsied muscle. Leg extensor peak torque increased after training (p<0.001), while VL mCSA trended upward (p=0.082). No significant changes were observed for fCSA, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p=0.037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13/46 proteins related to muscle-nerve communication (all upregulated, p<0.05) and 10/19 proteins related to cellular senescence (9 upregulated, p<0.05). Only 1/17 SASP proteins increased with training (IGFBP-3, p=0.031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increases senescence-related proteins in skeletal muscle, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. Hence, we interpret these collective findings as resistance training being an unlikely inducer of cellular senescence in humans.

physiology↗

bioRxiv2023

APP substrate ectodomain defines A beta length by restraining gamma-secretase processivity and facilitating product release

Sequential proteolysis of the amyloid precursor protein (APP) by {gamma}-secretases (GSECs) generates amyloid-{beta} (A{beta}) and defines the proportion of short-to-long A{beta} peptides, which is tightly connected to Alzheimers disease (AD) pathogenesis. Here, we study the mechanism controlling substrate processing by GSECs and defining product length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of GSEC processive cleavage by destabilizing enzyme-substrate (E-S) interactions. We show that increasing hydrophobicity at APPC99-ECD - due to mutation or ligand binding - attenuates this substrate-driven product release mechanism, and rescues the effects that AD pathogenic variants exert on A{beta} profiles. In addition, our study reveals that APPC99-ECD facilitates the paradoxical production of longer A{beta}s caused by some GSEC inhibitors that act as high-affinity competitors to the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by GSEC and suggest it as a sweet spot for the potential design of APP targeting compounds selectively promoting its processing by GSEC.

biochemistry↗

bioRxiv2023

Hydrogen Sulfide Coordinates Glucose Metabolism Switch through Destabilizing Tetrameric Pyruvate Kinase M2

Cancer cells reprogram their glucose metabolic pathway from oxidative phosphorylation toward aerobic glycolysis. Pyruvate kinase M2 (PKM2), which converts phosphoenolpyruvate (PEP) to pyruvate, is considered the rate-limiting enzyme involved in cancer glucose metabolism. By reducing PKM2 enzyme activity, cancer cells attain a greater fraction of glycolytic metabolites for macromolecule synthesis needed for rapid proliferation. Here we demonstrate that hydrogen sulfide (H2S) destabilizes PKM2 tetramer into dimer/monomer, leading to reduced PKM2 enzyme activity and an increase in the activation of nuclear transcriptional genes mediated by dimeric PKM2. Proteomic profiling of endogenous PKM2 reveals the occurrence of sulfhydration at cysteines, notably at cysteine 326. Blocking PKM2 sulfhydration at cysteine 326 through amino acid mutation stabilizes PKM2 tetramer and crystal structure further indicating that the tetramer organization of PKM2C326S is different from the currently known T or R states, revealing PKM2C326S as a newly identified form. The presence of a PKM2C326S mutant in cancer cells effectively rewires glucose metabolism to mitochondrial respiration, resulting in the significant inhibition of tumor growth. Collectively, PKM2 sulfhydration by H2S serves as a glucose metabolic rewiring mechanism in promoting tumorigenesis, and inhibition of PKM2 sulfhydration may be applied as a new therapeutic approach targeting cancer metabolism. One-Sentence SummaryH2S rewires glucose metabolism by destabilizing PKM2 tetramerization majorly through sulfhydration at cysteine 326 HighlightsO_LIH2S enhances PKM2 dissociation from tetramer to dimer to facilitate dimeric PKM2 nuclear translocation. C_LIO_LIH2S modifies PKM2 sulfhydration, notably at cysteine 326. C_LIO_LIThe crystal structure reveals PKM2C326S as a unique tetramer conformation. C_LIO_LIBlockage of PKM2 sulfhydration at C326 rewires cancer glucose metabolism and significantly inhibits tumor growth. C_LI

cancer biology↗

bioRxiv2023

A novel deep proteomic approach in human skeletal muscle unveils distinct molecular signatures affected by aging and resistance training

We examined the myofibrillar (MyoF) and non-myofibrillar (non-MyoF) proteomic profiles of the vastus lateralis (VL) muscle of younger (Y, 22{+/-}2 years old; n=5) and middle-aged participants (MA, 56{+/-}8 years old; n=6), and MA following eight weeks of knee extensor resistance training (RT, 2d/week). Shotgun/bottom-up proteomics in skeletal muscle typically yields wide protein abundance ranges that mask lowly expressed proteins. Thus, we adopted a novel approach whereby the MyoF and non-MyoF fractions were separately subjected to protein corona nanoparticle complex formation prior to digestion and Liquid Chromatography Mass Spectrometry (LC-MS) analysis. A total of 10,866 proteins (4,421 MyoF and 6,445 non-MyoF) were identified. Across all participants, the number of non-MyoF proteins detected averaged to be 5,645{+/-}266 (range: 4,888-5,987) and the number of MyoF proteins detected averaged to be 2,611{+/-}326 (range: 1,944-3,101). Differences in the non-MyoF (8.4%) and MyoF (2.5%) proteome were evident between age cohorts. Further, most of these age-related non-MyoF proteins (447/543) were more enriched in MA versus Y. Several biological processes in the non-MyoF fraction were predicted to be operative in MA versus Y including (but not limited to) increased cellular stress, mRNA splicing, translation elongation, and ubiquitin-mediated proteolysis. Non-MyoF proteins associated with splicing and proteostasis were further interrogated, and in agreement with bioinformatics, alternative protein variants, spliceosome-associated proteins (snRNPs), and proteolysis-related targets were more abundant in MA versus Y. RT in MA non-significantly increased VL muscle cross-sectional area (+6.5%, p=0.066) and significantly increased knee extensor strength (+8.7%, p=0.048). However, RT modestly altered the MyoF ([~]0.3%, 11 upregulated and two downregulated proteins) and non-MyoF proteomes ([~]1.0%, 56 upregulated and eight downregulated proteins, p<0.01). Further, RT did not affect predicted biological processes in either fraction. Although participant numbers were limited, these preliminary results using a novel deep proteomic approach in skeletal muscle suggest that aging and RT predominantly affects protein abundances in the non-contractile protein pool. However, the marginal proteome adaptations occurring with RT suggest either: a) this may be an aging-associated phenomenon, b) more rigorous RT may stimulate more robust effects, or c) RT, regardless of age, subtly affects skeletal muscle protein abundances in the basal state.

physiology↗

bioRxiv2023

ApoA-I Protects Pancreatic β-cells from Cholesterol-induced Mitochondrial Damage and Restores their Ability to Secrete Insulin

Objective: High cholesterol levels in pancreatic {beta}-cells cause oxidative stress and decrease insulin secretion. {beta}-cells can internalize apolipoprotein (apo) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves {beta}-cell insulin secretion by reducing oxidative stress. Approach: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-{beta}-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mM glucose was quantified by radioimmunoassay. Internalization of fluorescently labelled apoA-I by {beta}-cells was monitored by flow cytometry. The effects of apoA-I internalization on {beta}-cell gene expression was evaluated by RNA sequencing. ApoA-I binding partners on the {beta}-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in {beta}-cells and isolated islets with MitoSOX and confocal microscopy. Results: An F1-ATPase {beta}-subunit on the {beta}-cell surface was identified as the main apoA-I binding partner. {beta}-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol- and F1-ATPase {beta}-subunit-dependent. {beta}-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase {beta}-subunit levels than {beta}-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I co-localized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The ATPase inhibitory factor 1, IF1, attenuated apoA-I internalization and increased oxidative stress in Ins-1E {beta}-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion and mitochondrial function. Conclusions: These results establish that {beta}-cells are functionally heterogeneous and apoA-I restores insulin secretion in {beta}-cells with elevated cholesterol levels by improving mitochondrial redox balance.

cell biology↗

bioRxiv2023

Surfactant protein A promotes atherosclerosis through mediating macrophage foam cell formation

BACKGROUNDAtherosclerosis is a progressive inflammatory disease where macrophage foam cells play a central role in the pathogenesis. Surfactant protein A (SPA) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated. METHODSPrimary resident peritoneal macrophages were extracted from wildtype (WT) and SPA deficient (SPA-/-) mice to determine the functional effects of SPA in macrophage foam cell formation. SPA expression was assessed in healthy vessels and atherosclerotic aortic tissue from the human coronary artery and WT or apolipoprotein e-deficient (ApoE-/-) mice brachiocephalic arteries fed high fat diets (HFD) for 4 weeks. Hypercholesteremic WT and SPA-/- mice fed a HFD for 6 weeks were investigated for atherosclerotic lesions in vivo. RESULTSIn vitro experiments revealed that global SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA-/- dramatically decreased CD36 cellular and mRNA expression. SPA expression was increased in atherosclerotic lesions in humans and ApoE-/- mice. In vivo SPA deficiency attenuated atherosclerosis and reduced the number of lesion-associated macrophage foam cells. CONCLUSIONSOur results elucidate that SPA is a novel factor for atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis through increasing scavenger receptor cluster of differentiation antigen 36 (CD36) expression.

cell biology↗

bioRxiv2022

ERK-dependent DICER1 phosphorylation promotes open chromatin state and lineage plasticity to mediate tumor progression

DICER1 controls micro(mi)RNA-mediated epithelial-to-mesenchymal transition (EMT) to regulate tumorigenesis of lung adenocarcinomas (LUADs). We discovered that DICER1 is phosphorylated by ERK and nuclear translocated and phospho-DICER1 contributes to tumorigenesis. Mechanisms through which phospho-DICER1 regulates tumor progression remain undefined. We show that phospho-nuclear DICER1 associates with invasive human LUADs with oncogenic KRAS mutations and promotes late-stage tumor progression in mice with oncogenic Kras mutations. Surprisingly, phosphomimetic DICER1 regulates LUAD progression independent of miRNAs and EMT. Integrating single-cell RNA sequencing, fluorescent in situ RNA hybridization, immunofluorescence, and ATAC-sequencing, in mice, we discovered that phosphomimetic DICER1 generates an open chromatin state in the lung tumor alveolar cells leading to expression of gastrointestinal genes and altered AT2 cell identity. Strikingly, we also observe the gastric gene signature in human LUADs with phospho-DICER1 and KRAS mutations. We propose that phosphorylated nuclear DICER1 regulates chromatin remodeling leading to tumor cell reprogramming which drives lung cancer progression.

cancer biology↗

bioRxiv2022

Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

Morphological development of the lung requires complex signal crosstalk between the mesenchymal and epithelial progenitors. Elucidating the genetic cascades underlying the signal crosstalk is essential to understanding the morphogenesis of the lung. Here, we have identified Nolz-1/Znf503 as a mesenchymal lineage-specific transcriptional regulator that plays a key role in lung morphogenesis. The null mutation of Nolz-1 resulted in a severe hypoplasia phenotype, including decreased proliferation of mesenchymal cells, aberrant differentiation of epithelial cells, and defective growth of epithelial branches. The deletion of Nolz-1 also downregulated the expressions of Wnt2, Lef1, Fgf10, Gli3 and Bmp4. Mechanistically, we found that Nolz-1 regulated lung morphogenesis primarily through Wnt2 signaling. Loss of function and overexpression studies demonstrated that Nolz-1 transcriptionally activated Wnt2 and downstream {beta}-catenin signaling to control mesenchymal cell proliferation and epithelial branching. The Nolz-1-Wnt2 axis was also supported by evidence that exogenous Wnt2 could causally rescue defective proliferation and epithelial branching in Nolz-1 knockout lungs. Finally, we have identified Fgf9 as an upstream regulator of Nolz-1. Collectively, Fgf9-Nolz-1-Wnt2 signaling represents a novel signaling axis in the control of lung morphogenesis. These findings are also relevant to lung tumorigenesis in which a pathological function of Nolz-1 is involved.

developmental biology↗