Biology subjects

Zhu, Z.

Publications and source records attributed to Zhu, Z..

At least 199 records · Page 11Linked to original sources

bioRxiv2022

Propagation dynamics of electrotactic motility in large epithelial cell sheets

Directional migration initiated at the wound edge leads epithelial sheets to migrate in wound healing. How such coherent migration is achieved is not well understood. Here we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate increase in migrations directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at the free edge in space and time. The resultant collective migration is remarkably consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell- cell junction.

cell biology↗

bioRxiv2022

Language models of protein sequences at the scale of evolution enable accurate structure prediction

Artificial intelligence has the potential to open insight into the structure of proteins at the scale of evolution. It has only recently been possible to extend protein structure prediction to two hundred million cataloged proteins. Characterizing the structures of the exponentially growing billions of protein sequences revealed by large scale gene sequencing experiments would necessitate a break-through in the speed of folding. Here we show that direct inference of structure from primary sequence using a large language model enables an order of magnitude speed-up in high resolution structure prediction. Leveraging the insight that language models learn evolutionary patterns across millions of sequences, we train models up to 15B parameters, the largest language model of proteins to date. As the language models are scaled they learn information that enables prediction of the three-dimensional structure of a protein at the resolution of individual atoms. This results in prediction that is up to 60x faster than state-of-the-art while maintaining resolution and accuracy. Building on this, we present the ESM Metage-nomic Atlas. This is the first large-scale structural characterization of metagenomic proteins, with more than 617 million structures. The atlas reveals more than 225 million high confidence predictions, including millions whose structures are novel in comparison with experimentally determined structures, giving an unprecedented view into the vast breadth and diversity of the structures of some of the least understood proteins on earth.

synthetic biology↗

bioRxiv2022

Exotic alleles of EARLY FLOWERING 3 determine plant development and grain yield in barley

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be substantial for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for ten developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared to the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme phenotypic effects could be attributed to one exotic ELF3 allele, differing in only one SNP from the cultivated Barke ELF3 allele. This SNP causes an amino acid change, which predictively has an impact on the protein structure of ELF3, thereby possibly affecting phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also affecting its local cellular interactions causing significant trait differences between HIF sister lines.

genetics↗

bioRxiv2022

IKKα promotes lung adenocarcinoma growth through activation of ERK signaling via DARPP-32-mediated inhibition of PP1 activity

Non-small cell lung cancer (NSCLC) accounts for 80-85% cases of lung cancer cases. Diagnosis at advanced stages is common, after which therapy-refractory disease progression frequently occurs. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies. Overexpression of I{kappa}B kinase (IKK) in NSCLC correlates with poor patient survival. IKK is an NF-{kappa}B-activating kinase that is important in cell survival and differentiation, but its regulation of oncogenic signaling is not well understood. We recently demonstrated that IKK promotes NSCLC cell migration by physically interacting with dopamine- and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP. Here, we show that IKK phosphorylates DARPP-32 at threonine 34, resulting in DARPP-32-mediated inhibition of protein phosphatase 1 (PP1), subsequent PP1-mediated dephosphorylation of ERK, and activation of ERK signaling to promote lung oncogenesis. Correspondingly, DARPP-32 ablation in human lung adenocarcinoma cells reduced their anchorage-independent growth in soft agar. Mice challenged with IKK-ablated HCC827 cells exhibited less lung tumor growth than mice orthotopically administered control HCC827 cells. Our findings suggest that IKK drives NSCLC growth through activation of ERK signaling via DARPP-32-mediated inhibition of PP1 activity.

cancer biology↗

bioRxiv2022

Metformin Alleviates Arthrofibrosis in Rat via Fibroblast Metabolic Reprogramming and Lactate-Mediated Macrophage Polarization

BackgroundEmerging studies have suggested an essential role of fibroblast metabolic reprogramming in the pathogenesis of arthrofibrosis. The metabolic modulator metformin appears to be a therapeutic candidate for fibrotic disorders. However, whether metformin could alleviate arthrofibrosis has not been defined. In this study we have determined if treatment with metformin has the beneficial effect on arthrofibrosis and its underlying mechanism. MethodsArticular capsule samples were collected from patients with/without arthrofibrosis to perform gene and protein expression analysis. Arthrofibrosis animal model was established to examine the anti-fibrotic effect of metformin. Cell culture experiments were conducted to determine the mechanism by which metformin inhibits fibroblast activation. ResultsWe found that glycolysis was upregulated in human fibrotic articular capsules. In an arthrofibrosis animal model, intra-articular injection of metformin mitigated inflammatory reactions, downregulated expression of both fibrotic and glycolytic markers, improved range of motion of the joint, and reduced capsular fibrosis and thickening. At the cellular level, metformin inhibited the activation of fibroblasts and mitigated the abundant influx of glucose into activated fibroblasts. Interestingly, metformin prompted a metabolic shift from oxidative phosphorylation to aerobic glycolysis in activated fibroblasts, resulting in the anti-fibrotic effect of metformin. ConclusionMetformin decreased glycolysis, causing a metabolic shift toward aerobic glycolysis in activated fibroblasts and has beneficial effect on the treatment of arthrofibrosis. The translational potential of this articleThe finding of this study demonstrated the therapeutic effect of metformin on arthrofibrosis and defined novel targets for the treatment of articular fibrotic disorders.

cell biology↗

bioRxiv2022

CircRNA-regulated immune response of Asian honey bee workers to microsporidian infection

Nosema ceranae is a widespread fungal parasite for honey bees, causing bee nosemosis. Based on deep sequencing and bioinformatics, identification of circular RNAs (circRNAs) in Apis cerana cerana workers midguts and circRNA-regulated immune response of host to N. ceranae invasion were conducted in this current work, followed by molecular verification of back-splicing sites and expression trends of circRNAs. Here, 10185 and 7405 circRNAs were identified in the midguts of workers at 7 d (AcT1) and 10 d (AcT2) post inoculation (dpi) with N. ceranae. PCR amplification result verified the back-splicing sites in three specific circRNAs (novel_circ_005123, novel_circ_007177, and novel_circ_015140) expressed in N. ceranae-inoculated midgut. In combination with transcriptome data from corresponding un-inoculated midguts (AcCK1 and AcCK2), 2266 circRNAs were found to be shared by the aforementioned four groups, whereas the numbers of specific ones were 2618, 1917, 5691 and 3723 respectively. Further, 83 (52) differentially expressed circRNAs (DEcircRNAs) were identified in AcCK1 vs AcT1 (AcCK2 vs AcT2) comparison group. Source genes of DEcircRNAs in workers midgut at 7 dpi were involved in two cellular immune-related pathways such as endocytosis and ubiquitin mediated proteolysis. Additionally, competing endogenous RNA network analysis showed that 23 (13) DEcircRNAs in AcCK1 vs AcT1 (AcCK2 vs AcT2) can target 18 (14) miRNAs and further link to 1111 (1093) mRNAs. These target mRNAs were annotated to six cellular immunity pathways including endocytosis, lysosome, phagosome, ubiquitin mediated proteolysis, metabolism of xenobiotics by cytochrome P450, and insect hormone biosynthesis. Moreover, 284 (164) IRES and 54 (26) ORF were identified from DEcircRNAs in AcCK1 vs AcT1 (AcCK2 vs AcT2) comparison group; additionally, ORFs in DEcircRNAs in midgut at 7 dpi with N. ceranae were associated with several crucial pathways including endocytosis and ubiquitin-mediated proteolysis. Finally, RT-qPCR results showed that the expression trends of six DEcircRNAs were consistent with those in transcriptome data. These results demonstrated that N. ceranae altered the expression pattern of circRNAs in A. c. cerana workers midguts, and DEcircRNAs were likely to regulate host cellular and humoral immune response to microsporidian infection. Our findings lay a foundation for clarifying the mechanism underlying host immune response to N. ceranae infection and provide a new insight into interaction between Asian honey bee and microsporidian.

pathology↗

bioRxiv2022

Nonlinear effect of light intensity on normal axial development of rhesus monkeys

PurposeTo investigate the effects of different indoor lighting intensity (500 lx, 750 lx and 1,000 lx) on normal ocular axial length growth by using juvenal rhesus monkeys. MethodsTwenty-four juvenile monkeys were exposed continuously to normal intensity light (NIL, 500 lx, n=16), medium intensity light (MIL, 750 lx, n=8) and high intensity light (HIL, 1 000 lx, n=8), with a same CCT value (about 3000 K) for 246 days. Axial length, anterior chamber depth, corneal curvature radius were measured at about a monthly interval. ResultsAfter 246 days of light exposure, the growth of axial length of the MIL group (750 lx) were 0.151 {+/-} 0.081 mm and 0.139 {+/-} 0.070 mm in the right and left eyes, respectively, and significantly larger in comparison with the NIL group (500lx, OD: 0.068 {+/-} 0.055 mm, OS: 0.074 {+/-} 0.057 mm) and the HIL group (1000lx,OD:0.063 {+/-} 0.093 mm, OS: 0.084 {+/-} 0.052 mm) monkeys. This effect was stable and robust during the whole experimental period. ConclusionThe effects of different intensity lighting on normal ocular axial development was not linear as most people currently think. We must be cautious when it comes to elevate light intensity in classrooms. Whether this conclusion is correct under lights of other CCT value needs further study.

developmental biology↗

bioRxiv2022

Pancreatic tumors activate arginine biosynthesis to adapt to myeloid-driven amino acid stress

Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs to maintain survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors (Sullivan et al., 2019a). Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling study of PDAC metabolism under physiological nutrition. We show that PDAC cells cultured in TIFM, compared to standard laboratory models, adopt a cellular state more similar to PDAC cells in tumors. Further, using the TIFM model we identified arginine biosynthesis as a metabolic adaptation PDAC cells engage to cope with microenvironmental arginine starvation driven by myeloid cells in PDAC tumors. Altogether, these data show that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity and enable the discovery of novel cancer metabolic phenotypes.

cancer biology↗

bioRxiv2022

A Genome-scale Screen Reveals ANTXR1, Heparan Sulfate and Neu5Gc as Host Factors mediating Seneca Valley virus Entry into Porcine Cells

Seneca Valley virus (SVV) disease is a newly emerging infectious disease of pigs caused by SVV, which seriously endangers the pig industry. This study was set out to identify the essential host factors required for SVV entering porcine cells. Using a CRISPR/Cas9 library containing 93,859 sgRNAs that were designed to target approximately 22,707 porcine genes, we generated mutated porcine cell libraries, which were subjected to SVV challenge for enrichment of cells resistant to SVV infection. These resistant cells were subsequently analyzed to identify genes essential for SVV infection. We demonstrated that ANTXR1, a type I transmembrane protein encoded by ANTXR1, heparan sulfate (HS), glycosaminoglycans modified by acetylation and sulfation of HS2ST1, and Neu5Gc, a non-human sialic acid catalyzed by CMAH, were the essential host factors for SVV entry into porcine cells. These results will be helpful to elucidate the pathogenesis of SVV and the development of prevention and control measures.

pathology↗

bioRxiv2022

Structural insights into AT-rich DNA recognition by SALL family proteins

Spalt-like 4 (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells (ESCs) via binding to AT-rich regions of genomic DNA. Here we present crystal structures of the the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different double stranded DNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 coordinatively recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common recognition mode for AT-rich DNA by the SALL family proteins. The DNA binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family in controlling cell fate via preferentially targeted to AT-rich sites within genomic DNAs during cell differentiation.

biochemistry↗

bioRxiv2022

Zinc Finger MYND-Type Containing 8 (ZMYND8) is epigenetically regulated in mutant Isocitrate Dehydrogenase 1 (IDH1) glioma to promote radioresistance

Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. Establishment of glioma-specific methylation patterns by mIDH1 reprogramming drives oncogenic features of cancer metabolism, stemness and therapeutic resistance. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1. Treatment of glioma cells with mIDH1 specific inhibitor AGI-5198, upregulated gene networks involved in replication stress. Specifically, we found that the expression of ZMYND8, a regulator of DNA damage response was decreased in three patient-derived glioma cell cultures (GCC) after treatment with AGI-5198. ZMYND8 functions as a chromatin reader to modulate enhancer activity and recruit DNA repair machinery. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 knockout (KO) exhibit significant phosphorylation of ATM and sustained {gamma}H2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. The accumulation of DNA damage in ZMYND8 KO mIDH1 GCCs promoted the phosphorylation of cell cycle checkpoint proteins Chk1 and Chk2. In addition, the recruitment of ZMYND8 to sites of DNA damage has been shown to be PARP- dependent. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. Translational RelevanceOur understanding of radioresistance mechanisms in patient-derived glioma cell cultures (GCC) that endogenously express mIDH1-R132H are limited. We have uncovered a novel gene target Zinc Finger MYND-Type Containing 8 (ZMYND8) that is downregulated following treatment of human mIDH1 GCCs with mIDH1 specific inhibitors. We demonstrate that suppression of ZMYND8 expression by shRNA knockdown or genetic knockout reduces the cellular viability of mIDH1 GCCs to ionizing radiation (IR). Our findings reveal an epigenetic vulnerability of mIDH1 GCCs to ZMYND8 knockout (KO) which results in impaired resolution of IR-induced DNA damage and induction of cell cycle arrest. Additionally, ZMYND8 KO mIDH1 GCCs display increased radiosensitivity to inhibition of epigenetic regulators BRD4, HDAC, and PARP which could be mediated by enhanced replicative stress.

cancer biology↗

bioRxiv2022

Exploring the alternative conformation of a known protein structure based on contact map prediction

The rapid development of deep learning-based methods has considerably advanced the field of protein structure prediction. The accuracy of predicting the 3D structures of simple proteins is comparable to that of experimentally determined structures, providing broad possibilities for structure-based biological studies. Another critical question is whether and how multistate structures can be predicted from a given protein sequence. In this study, analysis of multiple two-state proteins demonstrated that deep learning-based contact map predictions contain structural information on both states, which suggests that it is probably appropriate to change the target of deep learningbased protein structure prediction from one specific structure to multiple likely structures. Furthermore, by combining deep learning- and physics-based computational methods, we developed a protocol for exploring alternative conformations from a known structure of a given protein, by which we successfully approached the holo-state conformation of a leucine-binding protein from its apo-state structure.

biophysics↗

bioRxiv2022

A Parabrachial to Hypothalamic Neurocircuit Mediates Defensive Behavior

Defensive behaviors are critical for animals survival. Both the paraventricular nucleus of the hypothalamus (PVN) and the parabrachial nucleus (PBN) have been shown to be involved in defensive behaviors. However, whether there are direct connections between them to mediate defensive behaviors remains unclear. Here, by retrograde and anterograde tracing, we uncover that cholecystokinin (CCK)-expressing neurons in the lateral PBN (LPBCCK) directly project to the PVN. By in vivo fiber photometry recording, we found that LPBCCK neurons actively respond to various threat stimuli. Selective photoactivation of LPBCCK neurons promotes aversion and defensive behaviors. Conversely, photoinhibition of LPBCCK neurons attenuates rat or looming stimuli-induced flight responses. Optogenetic activation of LPBCCK axon terminals within the PVN or PVN glutamatergic neurons promote defensive behaviors. Whereas chemogenetic and pharmacological inhibition of local PVN neurons prevent LPBCCK-PVN pathway activation-driven flight responses. These data suggest that LPBCCK neurons recruit downstream PVN neurons to actively engage in flight responses. Our study identifies a previously unrecognized role for the LPBCCK-PVN pathway in controlling defensive behaviors.

neuroscience↗

bioRxiv2022

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation

Root system is critical for plant growth and development. To develop plant growth regulators functioning in root development, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that mimicked the effects of auxin on root development. Genetic, pharmacological and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

plant biology↗

bioRxiv2022

Deeply conserved super-enhancers maintain stem cell pluripotency in placental mammals

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share a deep evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of the transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) which displayed deep conservation in placental mammals and are sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR/Cas9 approach severely impaired the proliferative potential and the ability to form undifferentiated colonies. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals. Significance statementSuper-enhancers (SEs) hold stronger power than regular enhancers to direct gene expression in the regulation of stem cell pluripotency. To dissect how pluripotency-associated SEs have evolved in mammals, we performed a systematic comparison of SEs among pigs, humans, and mice. Our analysis allowed the identification of three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that are highly conserved in Placentalia (accounting for 94% of mammals) as well as many species-specific SEs. All three SEs were sufficient to direct pluripotency-dependent gene expression and disruption of each conserved SE caused the loss of stem cell pluripotency. Our work highlights a small number of highly conserved SEs essential for the maintenance of pluripotency.

cell biology↗

bioRxiv2022

Jointly analyzing the association of human milk nutrients with cognition and temperament traits during the first 6 months of life

Early dietary exposure via human milk (HM) components offers a window of opportunity to support cognitive and temperamental development. While several studies have focused on associations of few pre-selected HM components with cognition and temperament, it is highly plausible that HM components synergistically and jointly support cognitive and behavioral development in early life. We aimed to discern the combined associations of a wide array of HM nutrients with cognition and temperament during the first six months of life and explore if there were persistent effects up to 18 months old, when HM is the primary source of an infants nutrition. The Mullen Scales of Early Learning and Infant Behavior Questionnaires-Revised were used to assess cognition and temperament, respectively, of fifty-four exclusively/predominantly breastfed infants in the first 6 months of life, whose follow-ups were conducted at 6-9, 9-12 and 12-18 months old. HM samples were obtained from the mothers of the participants at less than 6 months of life and analyzed for fatty acids (total monounsaturated fatty acids, polyunsaturated fatty acid, total saturated fatty acid (TSFA), arachidonic acid (ARA), docosahexaenoic acid (DHA), ARA/DHA, omega-6/omega-3 polyunsaturated fatty acids ratio (n-6/n-3)), phospholipids (phosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylinositol (PI), sphingomyelin) and choline (free choline, phosphocholine (PCho), glycerophosphocholine). Feature selection was performed to select nutrients associated with cognition and temperament, respectively. The combined effects of selected nutrients were analyzed using multiple regression. A positive association between the arachidonic acid (ARA) and surgency was observed (p = 0.024). Significant effect of DHA, n-6/n-3, PE and TSFA concentrations on receptive language (R2 = 0.39, p = 0.025), and the elevated ARA, PCho, and PI with increased surgency (R2 = 0.43, p = 0.003) was identified, suggesting that DHA and ARA may have distinct roles for temperament and language functions. Furthermore, the exploratory association analyses suggest that the effects of HM nutrients on R.L. and surgency may persist beyond the first 6 months of life, particularly surgency at 12-18 months (p = 0.002). Our studies highlighted that various HM nutrients work together to support the development of cognition and temperament traits during early infancy.

developmental biology↗

bioRxiv2022

Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

ObjectivesTo investigate the effect and mechanisms of salicin (SA) on osteoarthritis (OA) progression. MethodsPrimary rat chondrocytes were stimulated with TNF- and treated with or without SA. CCK-8 was utilized to determine the cytotoxicity of SA. RT-qPCR, Western Blotting and immunofluorescence staining were used to detect inflammatory factors, cartilage matrix degeneration markers, cell proliferation and apoptosis markers expression at mRNA and protein levels respectively. EdU assay and flow cytometer analysis were utilized for evaluating cell proliferation and apoptosis. RNA-sequencing, molecular docking, drug affinity responsive target stability and WB were applied to clarify mechanisms. Rat OA model was used to evaluate the effect of intra-articular injection of SA on OA progression. ResultsNo obvious cytotoxicity was found with the treatment of 10 M SA. SA rescued TNF- induced degeneration of cartilage matrix, inhibition of chondrocytes proliferation, and promotion of chondrocytes apoptosis. In mechanism, we clarified SA could directly bind on IRE1 and occupy IRE1 phosphorylation site, followed with inhibiting IRE1 phosphorylation and regulating IRE1 mediated endoplasmic reticulum (ER) stress by IRE1-I{kappa}B-p65 signaling. Finally, intra-articular injection of SA loaded PLGA could ameliorate OA progression by inhibiting IRE1 mediated ER stress in OA model. ConclusionsSA alleviates OA by directly binding on ER stress regulator IRE1 and inhibits IRE1 mediated ER stress by IRE1-I{kappa}B-p65 signaling. Topical use of small molecular drug SA holds the potential of modifying OA progression.

cell biology↗

bioRxiv2022

Lifting the ban on nuclear import activates Gdown1-mediated modulation of global transcription and facilitates adaptation to cellular stresses

Dynamic regulation of transcription is crucial for cellular response to various environmental or developmental cues. Gdown1 is a ubiquitously expressed, RNA polymerase II (Pol II) interacting protein, essential for embryonic development. It tightly binds Pol II in vitro and competitively blocks binding of TFIIF and other transcriptional regulatory factors, yet its cellular functions and regulatory circuits remain unclear. Here, we show that Gdown1 strictly localizes in the cytoplasm of mammalian somatic cells and exhibits potent resistance to the imposed driving force for nuclear localization. Combined with genetic and microscope-based approaches, two types of functionally coupled and evolutionally conserved localization regulatory motifs are identified, including the CRM1-dependent nucleus export signal (NES) and a novel Cytoplasm Anchoring Signal (CAS) which mediates nuclear pore retention. Mutagenesis of CAS alleviates the cytoplasmic retention activity thus unlocks its nucleocytoplasmic shuttling properties, and increased nuclear import of Gdown1 causes drastic reduction of Pol II levels and global transcription. Importantly, nuclear translocation of Gdown1 occurs in a stress-responsive manner and ablation of GDOWN1 significantly weakens cellular tolerance. Collectively, our work uncovers the molecular basis of the localization of Gdown1 and highlights that its controlled nuclear translocation serves as a key strategy in modulating global transcription and stress-adaptation.

cell biology↗