Biology subjects

Yang, J.

Publications and source records attributed to Yang, J..

At least 919 records · Page 51Linked to original sources

bioRxiv2020

Integrated gut-liver-on-a-chip platform as an in vitro human model of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) afflicts a large percentage of the population, but no effective treatments have been established so far because of the unsuitability of in vitro assays and experimental models using animals. By co-culturing human gut and liver cell lines interconnected via microfluidics for a closed circulation loop, we created a gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) for the initiation and progression of NAFLD. Microscopic high-content analysis followed by mRNA sequencing showed that co-culturing the gut and liver cells significantly affected each cell type compared to culturing them separately. NAFLD-inducing free fatty acids (FFAs) accumulated in the gut cells and elevated gene expressions associated with retinol metabolism and glucuronidation. The FFA-treated liver cells accumulated intracellular lipid droplets and showed an increase in gene expressions associated with a cellular response to copper ions and endoplasmic reticulum stress. As an in vitro human GLA model, the iGLC platform may serve as an alternative to animal experiments for investigating NAFLD mechanisms.

bioengineering↗

bioRxiv2020

Parabrachial neuron types categorically encode thermoregulation variables during heat defense

Heat defense is crucial for survival and fitness, and its dysregulation may result in deaths due to poor management. Transmission of thermosensory signals into hypothalamic thermoregulation centers represent a key layer of regulation in heat defense. However, the mechanism by which these signals are transmitted into the hypothalamus remains poorly understood. Here, we reveal that glutamatergic prodynorphin and cholecystokinin neuron populations in the lateral parabrachial (LPB) are progressively recruited to defend elevated body temperature. These two nonoverlapping neuron types form circuitries with downstream preoptic hypothalamic neurons to inhibit BAT thermogenesis and activate tail vasodilation, respectively. Both circuitries are selectively activated by warm temperatures and are required for fever limiting. The prodynorphin circuitry is further required for regulation of energy expenditure and weight homeostasis. Thus, these findings establish that the genetic and functional specificity of heat defense neurons occurs as early as in the LPB and uncover categorical neuron types for encoding two heat defense variables, which may provide targets for treating thermoregulation disorders.

neuroscience↗

bioRxiv2020

Powerful Variance-Component TWAS method identifies novel and known risk genes for clinical and pathologic Alzheimer's dementia phenotypes

Transcriptome-wide association studies (TWAS) have been widely used to integrate transcriptomic and genetic data to study complex human diseases. Within a test dataset lacking transcriptomic data, existing TWAS methods first impute gene expression by creating a weighted sum that aggregates SNPs with their corresponding cis-eQTL effects on reference transcriptome. Existing TWAS methods then employ a linear regression model to assess the association between imputed gene expression and test phenotype, thereby assuming the effect of a cis-eQTL SNP on test phenotype is a linear function of the eQTLs estimated effect on reference transcriptome. To increase TWAS robustness to this assumption, we propose a novel Variance-Component TWAS procedure (VC-TWAS) that assumes the effects of cis-eQTL SNPs on phenotype are random (with variance proportional to corresponding reference cis-eQTL effects) rather than fixed. VC-TWAS is applicable to both continuous and dichotomous phenotypes, as well as individual-level and summary-level GWAS data. Using simulated data, we show VC-TWAS is more powerful than traditional TWAS especially when eQTL genetic effects on test phenotype are no longer a linear function of their eQTL genetic effects on reference transcriptome. We further applied VC-TWAS to both individual-level (N=[~]3.4K) and summary-level (N=[~]54K) GWAS data to study Alzheimers dementia (AD). With the individual-level data, we detected 13 significant risk genes including 6 known GWAS risk genes such as TOMM40 that were missed by existing TWAS methods. With the summary-level data, we detected 57 significant risk genes considering only cis-SNPs and 71 significant genes considering both cis- and trans- SNPs; these findings also validated our findings with the individual-level GWAS data. Our VC-TWAS method is implemented in the TIGAR tool for public use. Author SummaryExisting Transcriptome-wide association studies (TWAS) tools make strong assumptions about the relationships among genetic variants, transcriptome, and phenotype that may be violated in practice, thereby substantially reducing the power. Here, we propose a novel variance-component TWAS method (VC-TWAS) that relaxes these assumptions and can be implemented with both individual-level and summary-level GWAS data. Our simulation studies showed that VC-TWAS achieved higher power compared to existing TWAS methods when the underlying assumptions required by existing TWAS tools were violated. We further applied VC-TWAS to both individual-level (N=[~]3.4K) and summary-level (N=[~]54K) GWAS data to study Alzheimers dementia (AD). With individual-level data, we detected 13 significant risk genes including 6 known GWAS risk genes such as TOMM40 that were missed by existing TWAS methods. Interestingly, 5 of these genes were shown to possess significant pleiotropic effects on AD pathology phenotypes, revealing possible biological mechanisms. With summary-level data of a larger sample size, we detected 57 significant risk genes considering only cis-SNPs and 71 significant genes considering both cis- and trans- SNPs, which also validated our findings with the individual-level GWAS data. In conclusion, VC-TWAS provides an important analytic tool for identifying risk genes whose effects on phenotypes might be mediated through transcriptomes.

genetics↗

bioRxiv2020

ISL2 is an epigenetically silenced tumor suppressor and regulator of metabolism in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers. Uncovering mechanisms responsible for the heterogeneous clinical features of this disease is an essential step toward developing improved and more specific therapeutic approaches. Here, we sought to identify transcriptional regulators of aggressive PDAC growth through in vivo CRISPR screening of epigenetic and transcription factors in an orthotopic model. We identified the ISL LIM homeobox 2 (ISL2) gene as a tumor suppressor whose depletion enhances the proliferation of human PDAC cells in vitro and in vivo and cooperates with activated KRAS to initiate PDAC in a murine model. Conversely, the upregulation of ISL2 expression through CRISPR-mediated locus-specific epigenetic editing results in reduced cell proliferation. Importantly, ISL2 is epigenetically silenced through DNA methylation in ~60% of PDAC tumors, which correlates with poor patient outcome. Functional studies showed that ISL2 loss rewires metabolic gene expression, and consequently potentiates oxidative phosphorylation while reducing glycolysis. This metabolic shift creates selective vulnerability to small molecule inhibitors of mitochondrial respiration and fatty acid oxidation. Collectively, these findings reveal ISL2 as a novel tumor suppressor whose inactivation drives metabolic reprogramming in an aggressive PDAC subset and point to potential therapeutic vulnerabilities in these tumors.

molecular biology↗

bioRxiv2020

Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancer to treat. Due to the asymptomatic nature of the disease and ineffective drug treatment modalities, the survival rate of PDAC patients remains one of the lowest. The recurrent genetic alterations in PDAC are yet to be targeted; therefore, identifying effective therapeutic combinations is desperately needed. Here, we performed an in vivo CRISPR screening in a clinically relevant patient-derived xenograft (PDX) model system to identify synergistic drug combinations for PDAC treatment. Our approach revealed protein arginine methyltransferase gene 5 (PRMT5) as a promising druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to gemcitabine. Genetic and pharmacological inhibition results indicate that of PRMT5 depletion results in synergistic cytotoxicity with Gem due to depleted replication protein A (RPA) levels and an impaired non-homology end joining (NHEJ) DNA repair. Thus, the novel combination creates conditional lethality through the accumulation of excessive DNA damage and cell death, both in vitro and in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is an effective approach in identifying synthetic lethal drug combinations for cancer treatment. STATEMENT of SIGNIFICANCEIdentify synergistic drug combinations for PDAC is a significant unmet need. Through CRISPR screening, we discovered and validated that PRMT5 depletion creates synergistic vulnerability of PDAC cells to gemcitabine. Mechanistically, the combination impairs DNA repair, synergistic accumulation of DNA damage and cell death in vitro and in vivo.

cancer biology↗

bioRxiv2020

Oxytocinergic modulation of threat-specific amygdala sensitization in humans is critically mediated by serotonergic mechanisms

BackgroundOverarching conceptualizations propose that the complex social-emotional effects of oxytocin (OXT) in humans are partly mediated by interactions with other neurotransmitter systems. Recent animal models suggest that the anxiolytic effects of OXT are critically mediated by the serotonin (5-HT) system, yet direct evidence in humans is lacking. MethodsTo determine the role of 5-HT in OXT-induced attenuation of amygdala threat reactivity and sensitization/ desensitization, we conducted a parallel-group randomized placebo-controlled double-blind experiment during which n = 121 healthy subjects underwent a transient decrease in 5-HT signaling via acute tryptophan depletion (ATD, TRYP-) or the corresponding placebo-control protocols before the administration of intranasal OXT or placebo intranasal spray, respectively. Mean and repetition-dependent changes in threat-specific amygdala reactivity towards threatening stimuli (angry faces) as assessed by fMRI served as the primary outcome. ResultsNo treatment main or interaction effects on amygdala threat reactivity were observed, yet OXT switched bilateral amygdala threat sensitization to desensitization and this effect was significantly attenuated during decreased central 5-HT signaling via pretreatment with TRYP-. ConclusionsThe present findings provide the first evidence for a role of OXT in threat-specific amygdala desensitization in humans and suggest that these effects are critically mediated by the 5-HT system. OXT may have a therapeutic potential to facilitate amygdala desensitization and adjunct up-regulation of 5-HT neurotransmission may facilitate OXTs anxiolytic potential. The trial was preregistered on clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT03426176, ID NCT03426176)

neuroscience↗

bioRxiv2020

SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

cell biology↗

bioRxiv2020

ERH as a component of the Microprocessor facilitates the maturation of suboptimal microRNAs

The Microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here we identify Enhancer of Rudimentary Homolog (ERH) as a new component of the Microprocessor. ERH binds to a conserved region in the N-terminus of DGCR8. Knockdown of ERH or deletion of the DGCR8 N-terminus results in a decrease of processing of primary miRNAs with suboptimal hairpin structures that reside in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate "cluster assistance" in which the Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA pathway.

molecular biology↗

bioRxiv2020

A systematic dissection of human primary osteoblasts in vivo at single-cell resolution

Osteoblasts are multifunctional bone cells, which play essential roles in bone formation, angiogenesis regulation, as well as maintenance of hematopoiesis. Although both in vivo and in vitro studies on mice have identified several potential osteoblast subtypes based on their different transition stages or biological responses to external stimuli, the categorization of primary osteoblast subtypes in vivo in humans has not yet been achieved. Here, we used single-cell RNA sequencing (scRNA-seq) to perform a systematic cellular taxonomy dissection of freshly isolated human osteoblasts. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct cell clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. This novel subtype was mainly characterized by the nuclear receptor subfamily 4 group A member 1 and 2 (NR4A1 and NR4A2), and its existence was confirmed by immunofluorescence staining. Trajectory inference analysis suggested that the undetermined cluster, together with the preosteoblasts, are involved in the regulation of osteoblastogenesis and also give rise to mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, preosteoblasts and undetermined osteoblasts may also regulate both angiogenesis and hemopoiesis. Finally, we demonstrated that there are systematic differences between the transcriptional profiles of human osteoblasts in vivo and mouse osteoblasts both in vivo and in vitro, highlighting the necessity for studying bone physiological processes in humans rather than solely relying on mouse models. Our findings provide novel insights into the cellular heterogeneity and potential biological functions of human primary osteoblasts at the single-cell level, which is an important and necessary step to further dissect the biological roles of osteoblasts in bone metabolism under various (patho-) physiological conditions.

bioinformatics↗

bioRxiv2020

Once delayed non-invasive remote ischemic preconditioning protects against early stroke by modulating neuroinflammatory responses in rats

Once delayed non-invasive remote ischemic preconditioning (RIPC) has been proven to provide endogenous protection against injury induced by ischemia-reperfusion in the central nervous system. However, for thus ischemic preconditioning method, it is still unclear how long this protection can maintain and what the underlying mechanism is. In this study, we tested the hypothesis that once delayed non-invasive RIPC protects brain injury at short reperfusion time. The rat was stimulated by transient middle cerebral artery occlusion (MCAo) for 90 min, and subsequent reperfusion was performed at 6 h, 72 h and 7 days after MCAo. RIPC was conducted in both hind limbs 24 h before MCAo for 3 cycles (10 min ischemia/ 10 min reperfusion). The infarct size was measured by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining and Cresyl violet (CV) staining. The mRNA and protein levels of inflammatory cytokines in the brain were measured by real-time RT-PCR and ELISA. The results showed that once delayed non-invasive RIPC reduced the infarct size, improved neurological functions and behavioral performance at 6 and 72 h post-stroke. There was no change by reperfusion at 7 d after MCAo. RIPC reduced the levels of TNF, IL-1{beta} and IL-6 in the brain at 72 h post stroke. It also reduced the levels of TNF and IL-1{beta} when reperfusion at 6 h after MCAo. Our results strongly supported that once delayed non-invasive RIPC protects against stroke as a non-invasive neuroprotective strategy, which maintained for both short and middle term ischemic reperfusion time. The protective effect is mediated by the modulation of inflammatory response in the ischemic brain.

neuroscience↗

bioRxiv2020

Developmentally Delayed Epigenetic Reprogramming Underlying the Pathogenesis of Preeclampsia

Preeclampsia, a life-threatening pregnancy complication characterized by hypertension and multiorgan damage, affects 2-5% of pregnancies and causes 76,000 deaths per year. Most preeclampsia associated syndromes immediately dispel after removal of placenta, indicating a casual role of placenta in the pathogenesis. Failed transformation of spiral artery due to insufficient invasion and excessive apoptosis of trophoblast suggested developmental defects in preeclampsia placenta. However, the underlying molecular mechanisms that affected placenta development in preeclampsia remained elusive. Here we show that, in preeclampsia placenta, the epigenetic landscape formed during extraembryonic tissue differentiation was disrupted: dramatic chromatin accessibility shift affected known and novel genes implicated in preeclampsia. DNA methylation defects in preeclampsia affected lineage-defining PcG-controlled loci in trophectoderm. LTR12 retrotransposons associated with VCT/SCT-specific genes were hypermethylated. Meanwhile, hundreds of PcG-regulated EVT-specific gene promoters, which otherwise undergone post-ZGA extraembryonic-tissue-specific de novo methylation, were hypomethylated and hyper-activated. Together, these epigenetic defects resulted in placenta developmental delay in preeclampsia. The defective methylation pattern could be detected in serum cfDNA, and could be used to accurately predict preeclampsia at early pregnancy weeks in independent validation cohorts. Our data suggests that the preeclampsia placenta represents a stalled state of epigenetic reprogramming en route of development from trophectoderm to normal placenta.

genomics↗

bioRxiv2020

A Genetic Model Therapy Proposes a Critical Role for Liver Dysfunction in Mitochondrial Biology and Disease

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Here we present a new animal model that recapitulates key components of Leigh Syndrome, French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the Leucine Rich Pentatricopeptide repeat-containing motif (LRPPRC) gene. LRPPRC has native functions related to mitochondrial mRNA polyadenylation and translation as well as a role in gluconeogenesis. We used the Gene-Breaking Transposon (GBT) cassette to create a revertible, insertional mutant zebrafish line in the LRPPRC gene. lrpprc zebrafish homozygous mutants displayed impaired muscle development, liver function and lowered levels of mtDNA transcripts and are lethal by 12dpf, all outcomes similar to clinical phenotypes observed in patients. Investigations using an in vivo lipidomics approach demonstrated accumulation of non-polar lipids in these animals. Transcript profiling of the mutants revealed dysregulation of clinically important nuclearly encoded and mitochondrial transcripts. Using engineered liver-specific rescue as a genetic model therapy, we demonstrate survival past the initial larval lethality, as well as restored normal gut development, mitochondrial morphology and triglyceride levels functionally demonstrating a critical role for the liver in the pathophysiology of this model of mitochondrial disease. Understanding the molecular mechanism of the liver-mediated genetic rescue underscores the potential to improve the clinical diagnostic and therapeutic developments for patients suffering from these devastating disorders.

genetics↗

bioRxiv2020

Susceptibility of tree shrew to SARS-CoV-2 infection

Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39{degrees} C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches.

microbiology↗

bioRxiv2020

Cytotoxicity evaluation of chloroquine and hydroxychloroquine in multiple cell lines and tissues by dynamic imaging system and PBPK model

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used in treating COVID-19 patients recently. However, both drugs have some contradictions and rare but severe side effects, such as hypoglycemia, retina and cardiac toxicity. To further uncover the toxicity profile of CQ and HCQ in different tissues, we evaluated the cytotoxicity of them in 8 cell lines, and further adopted the physiologically-based pharmacokinetic models (PBPK) to predict the tissue risk respectively. Retina, myocardium, lung, liver, kidney, vascular endothelium and intestinal epithelium originated cells were included in the toxicity evaluation of CQ and HCQ respectively. The proliferation pattern was monitored in 0-72 hours by IncuCyte S3, which could perform long-term continuous image and video of cells upon CQ or HCQ treatment. CC50 and the ratio of tissue trough concentrations to CC50 (RTTCC) were brought into predicted toxicity profiles. The CC50 at 24 h, 48 h, 72 h of CQ and HCQ decreased in the time-dependent manner, which indicates the accumulative cytotoxic effect. HCQ was found to be less toxic in 7 cell types except cardiomyocytes H9C2 cells (CC50-48 h=29.55 M; CC50-72 h=15.26 M). In addition, RTTCC is significant higher in CQ treatment group compared to HCQ group, which indicates that relative safety of HCQ. Both CQ and HCQ have certain cytotoxicity in time dependent manner which indicates the necessity of short period administration clinically. HCQ has the less impact in 7 cell lines proliferation and less toxicity compared to CQ in heart, liver, kidney and lung.

pharmacology and toxicology↗

bioRxiv2020

Interactions between fungal hyaluronic acid and host CD44 promotes internalization by recruiting host autophagy proteins to forming phagosomes

Phagocytosis and autophagy play critical roles in immune defense. Cryptococcus neoformans (Cn), a fungal pathogen that causes fatal infection, subverts the host autophagy initiation complex (AIC) and its upstream regulatory proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKK{beta} and its downstream target AMPK, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/047621v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@72f2b9org.highwire.dtl.DTLVardef@9d5a2corg.highwire.dtl.DTLVardef@8b0218org.highwire.dtl.DTLVardef@3698d0_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefDing et al. reveal that interactions between fungal hyaluronic acid (HA) and host CD44 activate a Ca2+ - CaMKK{beta}-AMPK-ULK1 signaling pathway that recruits autophagy initiation complex components to forming phagosomes to drive fungal internalization. HighlightsO_LIFungal HA interactions with host cells drive a novel non-canonical, ligand-induced, autophagy pathway in phagocytic cells C_LIO_LICryptococcus neoformans recruits host CD44, together with AIC components and regulatory proteins, to forming phagocytic cups to initiate host cell internalization C_LIO_LIFungal HA interactions with CD44 on host cell surfaces elevate intracellular Ca2+ concentrations, leading to activation of CaMKK{beta} C_LIO_LIA Ca2+-CaMKK{beta}-AMPK-ULK1 signaling axis is involved in HA and CD44 induced autophagy protein recruitment during Cn internalization C_LI

microbiology↗

bioRxiv2020

Lysosomal nucleotide metabolism regulates ER proteostasis through mTOR signaling

Organelles in eukaryotic cells are compartmentalized to carry out different functions, and require specific mechanisms governing their coordination. Two of these organelles, the lysosome and the endoplasmic reticulum (ER), play crucial roles in regulating cellular homeostasis and organismal health. The lysosome contains various enzymes devoted to the hydrolysis of specific substrates, and dysfunctions of these hydrolases and their related metabolic processes are implicated in many diseases 1. On the other hand, ER is essential for protein synthesis and utilizes quality control mechanisms to maintain proteostasis. The metabolic status of the lysosome is now known to actively influence nuclear transcription and mitochondrial signaling 2-4. However, whether and how mechanistically lysosomal metabolic activities regulate ER proteostasis remain unclear. Here, we reported that RSH-1, the Caenorhabditis elegans RelA/SpoT Homolog (RSH) protein, carries a lysosomal NADPH phosphatase activity and acts through the mammalian/mechanistic target of rapamycin (mTOR) signaling to regulate ER proteostasis. We discovered that RSH-1 is localized to the lysosome. Its mutation reduces NADPH hydrolysis by the lysosome, leading to a protection against ER stress-induced lethality. We further revealed that this ER stress tolerance requires lysosomal vacuolar-type H+-ATPase (v-ATPase), and Rag GTPases and ribosomal protein S6 kinase (S6K) in the mTOR signaling pathway. Through transcriptome analysis, we discovered that the S6K activation by the RSH-1 mutation increases the basal expression of X-box binding protein 1 (XBP-1) and peptidyl-prolyl cis-trans isomerases, which is necessary for improving ER proteostasis. Moreover, we demonstrated the lysosomal localization of RSH-1 mammalian homolog and its conserved role in regulating mTOR signaling. Together, our findings reveal the key role of a specific lysosomal nucleotide hydrolase in regulating organellar coordination and also signaling mechanisms underlying this active regulation, and suggest that diseases resulted from deficiency in lysosomal hydrolases may be attributed to the distortion of signal transduction and organelle homeostasis.

cell biology↗

bioRxiv2020

Predicting the Viability of Beta-Lactamase: How Folding and Binding Free Energies Correlate with Beta-Lactamase Fitness

One of the long-standing holy grails of molecular evolution has been the ability to predict an organisms fitness directly from its genotype. With such predictive abilities in hand, researchers would be able to more accurately forecast how organisms will evolve and how proteins with novel functions could be engineered, leading to revolutionary advances in medicine and biotechnology. In this work, we assemble the largest reported set of experimental TEM-1 {beta}-lactamase folding free energies and use this data in conjunction with previously acquired fitness data and computational free energy predictions to determine how much of the fitness of {beta}-lactamase can be directly predicted by thermodynamic folding and binding free energies. We focus upon {beta}-lactamase because of its long history as a model enzyme and its central role in antibiotic resistance. Based upon a set of 21 {beta}-lactamase single and double mutants expressly designed to influence protein folding, we first demonstrate that modeling software such as FoldX and PyRosetta designed to compute folding free energies can meaningfully, although not perfectly, predict the experimental folding free energies of single mutants. Interestingly, while these techniques also yield sensible double mutant free energies, we show that they do so for the wrong physical reasons. We then go on to assess how well both experimental and computational folding free energies explain single mutant fitness. We find that folding free energies account for, at most, 24% of the variance in {beta}-lactamase fitness values according to linear models and, somewhat surprisingly, complementing folding free energies with computationally-predicted binding free energies of residues near the active site only increases the folding-only figure by a few percent. This strongly suggests that the majority of {beta}-lactamases fitness is controlled by factors other than free energies. Overall, our results shed a bright light on to what extent the community is justified in using thermodynamic measures to infer protein fitness as well as how applicable modern computational techniques for predicting free energies will be to the large data sets of multiply-mutated proteins forthcoming.

biophysics↗

bioRxiv2020

A spatiotemporal translatome of mouse tissue development

The precise regulation of gene expression in mammalian tissues during development results in their functional specification. Although previous transcriptomic and proteomic analyses have provided great biological insights into tissue-specific gene expression and the physiological relevance of these tissues in development, our understanding of translational regulation in developing tissues is lacking. In this study, we performed a spatiotemporally resolved translatome analysis of six mouse tissues at the embryonic and adult stages to quantify the effects of translational regulation and identify new translational components. We quantified the spatial and temporal divergences of gene expression and detected specific changes in gene expression and pathways underlying these divergences. We further showed that dynamic translational control can be achieved by modulating the translational efficiency, which resulted in the enhancement of tissue specificity during development. We also discovered thousands of actively translated upstream open reading frames (ORFs) that exhibited spatiotemporal patterns and demonstrated their regulatory roles in translational regulation. Furthermore, we identified known and novel micropeptides encoded by small ORFs from long noncoding RNAs that are functionally relevant to tissue development. Our data and analyses facilitate a better understanding of the complexity of translational regulation across tissue and developmental spectra and serve as a useful resource of the mouse translatome.

bioinformatics↗