Biology subjects

Yang, D.

Publications and source records attributed to Yang, D..

At least 181 records · Page 10Linked to original sources

bioRxiv2022

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor -1 (PTH1R), a member of class B G protein couple receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide, are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.6 and 3.2 [A], respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation and together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

biochemistry↗

bioRxiv2022

Loss of ZNF251 stimulates NHEJ resulting in PARP inhibitor resistance in BRCA1-mutated cancer cells

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a promising new class of agents that have demonstrated efficacy in treating various cancers, particularly those with BRCA1/2 mutations. Cancer-associated BRCA1/2 mutations disrupt DNA double-strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPis) have been used to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, resistance to PARPis is common and can occur through multiple mechanisms, including the restoration of HR and/or stabilization of replication forks. To gain a better understanding of the mechanisms underlying PARPis resistance, we conducted an unbiased CRISPR-pooled genome-wide library screen to identify new genes whose deficiency confers resistance to the PARPi olaparib. Our research revealed that haploinsufficiency of the ZNF251 gene, which encodes zinc finger protein 251, is associated with resistance to PARPis in various breast and ovarian cancer cell lines carrying BRCA1 mutations. Mechanistically, we discovered that ZNF251 haploinsufficiency leads to stimulation of RAD51-mediated HR repair of DSBs in olaparib-treated BRCA1-mutated cancer cells. Moreover, we demonstrated that a RAD51 inhibitor reversed PARPi resistance in ZNF251 haploinsufficient cancer cells harboring BRCA1 mutations. Our findings provide important insights into the mechanisms underlying PARPis resistance by highlighting the role of RAD51 in this phenomenon.

cancer biology↗

bioRxiv2022

Targeting intracellular Neu1 for Coronavirus Infection Treatment

There are no effective therapies for COVID-19 or antivirals against SARS-CoV-2. Furthermore, current vaccines appear less efficacious for new SARS-CoV-2 variants. Thus, there is an urgent need to better understand the virulence mechanisms of SARS-CoV-2 and the host response to develop therapeutic agents. Here, we show host Neu1 regulates coronavirus replication by controlling sialylation on coronavirus nucleocapsid protein. Coronavirus nucleocapsid proteins in COVID-19 patients and in coronavirus HCoV-OC43-infected cells were heavily sialylated; this sialylation controlled the RNA binding activity and replication of coronavirus. Neu1 overexpression increased HCoV-OC43 replication, whereas Neu1 knockdown reduced HCoV-OC43 replication. Moreover, a newly developed Neu1 inhibitor, Neu5Ac2en-OAcOMe, selectively targeted intracellular sialidase, which dramatically reduced HCoV-OC43 and SARS-CoV-2 replication in vitro and rescued mice from HCoV-OC43 infection-induced death. Our findings suggest that Neu1 inhibitors could be used to limit SARS-CoV-2 replication in patients with COVID-19, making Neu1 a potential therapeutic target for COVID-19 and future coronavirus pandemics.

microbiology↗

bioRxiv2022

Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants

The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and illicit psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here we develop methods to isolate SERT from porcine brain (pSERT) using a mild, non-ionic detergent, utilize fluorescence- detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to SERT central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to SERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules. SignificanceThe serotonin transporter (SERT) is the target of antidepressants and illicit psychostimulants. Despite its importance in the nervous, cardiovascular and gastrointestinal systems, there is no direct knowledge of SERTs oligomerization state(s) and interactions with other proteins. Here, we develop methods to isolate porcine SERT (pSERT) from native brain tissue in the presence of a mild, non-ionic detergent, and investigated its properties by biochemical, structural and computational methods. We show how cocaine and methamphetamine exert their pharmacological effect on SERT, binding to a site halfway across the membrane-spanning region of the transporter, stabilizing pSERT in an outward open conformation. pSERT is best described as a monomeric entity, requiring neither oligomerization or additional proteins for its structure or function.

biophysics↗

bioRxiv2022

Activation of ERK by altered RNA splicing in cancer

Many cancers carry change-of-function mutations affecting RNA splicing factors, however, less is known about the functional consequences of upregulated RNA splicing factors in cancer. Here, we demonstrate that SMNDC1, a poorly studied splicing factor, which we found to be upregulated in multiple carcinomas and associated with poor patient prognosis, promotes cell proliferation, clonal expansion, and tumor growth by promoting the retention of G-rich exons, which otherwise would be excluded or retained at a lower rate after RNA splicing in normal cells. Inclusion of exon 4 (E4) of MAPK3 (ERK1), which encodes both kinase phosphorylation sites (Thr202/Tyr204), was among the promoted exons by SMNDC1. Forced exclusion of MAPK3-E4 using anti-sense oligos inhibited the ERK1 phosphorylation, expression of target genes and decreased tumor cell growth. These data support that cancer cells exploit a "splicing switch" to promote ERK kinase activity and offer a druggable alternative to block oncogenic signaling and altered RNA splicing in cancer cells SIGNIFICANCEERK signaling promotes tumor growth and survival. Exon 4 of MAPK3 (ERK1) encodes the activation phosphorylation sites of ERK1 kinase. Aberrant RNA splicing induced by SMNDC1 in cancer cells increases the retention of exon 4 during mRNA splicing, unleashes the kinase activity. SMNDC1 potentializes as a cancer therapeutic target.

cancer biology↗

bioRxiv2022

A unique peptide recognition mechanism by the human relaxin family peptide receptor 4 (RXFP4)

Members of the insulin superfamily regulate a variety of biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs), which are class A G protein-coupled receptors (GPCRs), to exert pleiotropic actions. Here, we report three cryo-electron microscopy structures of RXFP4-Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053, both were discovered through medicinal chemistry efforts. The B chain of INSL5 adopts a single -helix that penetrates into the orthostatic pocket, while the A chain sits above the orthosteric pocket to interact with the extracellular surface of RXFP4, revealing a unique peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. DC591053 selectively mimicked the action of INSL5 at RXFP4 whereas compound 4 activated both RXFP3 and RXFP4. Comparison of peptide binding modes within the insulin superfamily displayed diverse interaction mechanisms distinct to each type of the peptides. Our findings not only provide valuable insights into ligand recognition and subtype selectivity among class A GPCRs, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

molecular biology↗

bioRxiv2022

Study of the anti-inflammatory and anti-apoptosis effect of the traditional Mongolian Hohgardi-9 in acute lung injury

ObjectiveTo explore the main active components of Hohgardi-9 and its mechanism treating in ALI. MethodsThrough searching the TCMSP database, we obtained the main components and action targets of Hohgardi-9, and the targets related to ALI were analyzed as the possible targets of Hohgardi-9. Then, the compound target network was constructed using Cytoscape software and obtained the key compounds of Hohgardi-9 acting on ALI. The blood entering components of Hohgardi-9 were analyzed by metabonomics. Using a string database to investigate the interaction between proteins of possible targets of Hohgardi-9, Gene Ontology (GO) function annotation and Tokyo Encyclopedia of the genome (KEGG) pathway enrichment analysis were carried out at the same time to predict its mechanism. Finally, the ALI rat model verified the pharmacodynamic effects and key targets of Huhgridi-9. ResultsThe network pharmacology and blood component analysis results showed that 27 potentially active components such as quercetin, herbacetin, izoteolin, and columbinetin acetate were the major functional components in Hohgardi-9. Those might act on NF kappa B signalling pathway, toll-like receptor signalling pathway, and TNF signalling pathway through key targets such as RELA (p65), TLR4, etc. In vivo experiments showed that Hohgardi-9 significantly improved lung tissue injury and pulmonary edema in ALI rats. At the same time, the Hohgardi-9 intervention could significantly reduce the mRNA expression levels of TRL4, TNFa, IL-1 {beta}, and ICAM1 in ALI rats. ConclusionHohgardi-9 revealed ALI through the inhibiting inflammatory factor apoptosis-related gene expression.

pharmacology and toxicology↗

bioRxiv2022

DNA packaging via hierarchical chromatin structures revealed by live-cell 3D imaging

Single-molecule localization microscopy is a powerful superresolution imaging technique to study biological questions by visualizing subcellular fine structures with nanometer-scale precision. However, its application in live-cell imaging studies has been impeded by the paucity of self-blinking organic fluorophores that enable high spatiotemporal resolution and labeling/localization density at a moderate laser intensity. Herein, we report a self-blinking Si-rhodamine dye 6-HESiR with a suitably increased "ON" fraction and a fluorogenic self-blinking dsDNA probe 6-HoeHESiR as a powerful tool for 3D superresolution imaging of native chromatin in eukaryotes without the use of photoswitching buffer and high laser intensity. With the probe 6-HoeHESiR, 3D superresolution imaging of in vitro reconstituted nucleosomal arrays and chromatin fibers yielded results consistent with EM analysis. Similar euchromatin and heterochromatin structures were visualized in fixed and live cells with high spatiotemporal resolution and labeling density, providing the first live-cell evidence for a hierarchical model of chromatin organization. 3D imaging results obtained in the presence of selective inhibitors of histone deacetylases also corroborate chromatin fiber decompaction upon hyperacetylation of histones.

cell biology↗

bioRxiv2022

Rapid and Live-cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry

Detection of cellular senescence is important quality analytics for cell therapy products, including mesenchymal stromal cells (MSCs). However, their detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells using heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry ({micro}MRR), which is related to intracellular iron accumulation, correlates strongly with senescent markers in MSC cultures under diverse conditions including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues, and can critically advance our understanding of cellular senescence.

biophysics↗

bioRxiv2022

A Remarkable Adaptive Paradigm Of Heart Performance And Protection Emerges In Response To The Constitutive Challenge Of Marked Cardiac-Specific Overexpression Of Adenylyl Cyclase Type 8

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload ([~]30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide array of biological processes and molecular functions in numerous cellular compartments differed in TGAC8 vs WT; and over 250 canonical signaling pathways characteristic of adaptive survival circuitry of cancers, including PI3K and growth factor signaling, cytokine and T cell receptor signaling, immune responses, ROS scavenging, proliferation, protection from apoptosis, and nutrient sensing, were activated in TGAC8; and compared to WT there was a shift from fatty acid oxidation to increased aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, the adaptive paradigm, that becomes activated in the LV of TGAC8 in response to severe chronic, intense AC/PKA/Ca2+ signaling embodies many hallmarks of cancer.

physiology↗

bioRxiv2022

Neural signals encoded by specific frequencies of closed-loop electrical stimulation structurally and functionally reconstruct spinal sensorimotor circuits after spinal cord injury

Epidural electrical stimulation restores locomotion in animals and humans with spinal cord injury (SCI). However, the coding rules underlying electrical stimulation remain poorly understood, which has greatly limited the application of such electrical neuromodulation techniques in SCI clinical treatment. To elucidate the coding rules of electrical stimulation on spinal sensorimotor circuit reconstruction after complete SCI, we initially developed a spinal-muscle closed-loop stimulation protocol to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. Afterwards, using methods of sensorimotor function evaluation, neural circuit tracing and neural signal recording, we discovered a unique stimulus frequency of 10-20 Hz under closed-loop conditions was required for structural and functional reconstruction of spinal sensorimotor circuits. The single-cell transcriptome analysis of activated motoneurons characterized molecular networks involved in spinal sensorimotor circuit reconstruction. This study provides insights into neural signal decoding during spinal sensorimotor circuit reconstruction, and indicates a technological approach for the clinical treatment of SCI.

neuroscience↗

bioRxiv2022

Three-dose vaccination-induced immune responses protect against SARS-CoV-2 Omicron-BA.2

BackgroundThe ongoing outbreak of SARS-CoV-2 Omicron BA.2 infections in Hong Kong, the model city of universal masking of the world, has resulted in a major public health crisis. Although the third vaccination resulted in strong boosting of neutralization antibody, vaccine efficacy and corelates of immune protection against the major circulating Omicron BA.2 remains to be investigated. MethodsWe investigated the vaccine efficacy against the Omicron BA.2 breakthrough infection among 470 public servants who had received different SARS-CoV-2 vaccine regimens including two-dose BNT162b2 (2xBNT, n=169), three-dose BNT162b2 (3xBNT, n=170), two-dose CoronaVac (2xCorV, n=34), three-dose CoronaVac (3xCorV, n=67) and third-dose BNT162b2 following 2xCorV (2xCorV+1BNT, n=32). Humoral and cellular immune responses after three-dose vaccination were further characterized and correlated with clinical characteristics of BA.2 infection. FindingsDuring the BA.2 outbreak, 27.7% vaccinees were infected. The timely third-dose vaccination provided significant protection with lower incidence rates of breakthrough infections (2xBNT 49.2% vs 3xBNT 13.1%, p <0.0001; 2xCorV 44.1% vs 3xCoV 19.4%, p=0.003). Investigation of immune response on blood samples derived from 92 subjects in three-dose vaccination cohorts collected before the BA.2 outbreak revealed that the third-dose vaccination activated spike (S)-specific memory B cells and Omicron cross-reactive T cell responses, which correlated with reduced frequencies of breakthrough infections and disease severity rather than with types of vaccines. Moreover, the frequency of S-specific activated memory B cells was significantly lower in infected vaccinees than uninfected vaccinees before vaccine-breakthrough infection whereas IFN-{gamma}+ CD4 T cells were negatively associated with age and viral clearance time. Critically, BA.2 breakthrough infection boosted cross-reactive memory B cells with enhanced cross-neutralizing antibodies to Omicron sublineages, including BA.2.12.1 and BA.4/5, in all vaccinees tested. InterpretationOur results imply that the timely third vaccination and immune responses are likely required for vaccine-mediated protection against Omicron BA.2 pandemic. Although BA.2 conferred the highest neutralization resistance compared with variants of concern tested before the emergence of BA.2.12.1 and BA.4/5, the third dose vaccination-activated S-specific memory B cells and Omicron cross-reactive T cell responses contributed to reduced frequencies of breakthrough infection and disease severity. Neutralizing antibody potency enhanced by BA. 2 breakthrough infection with previous 3 doses of vaccines (CoronaVac or BNT162b2) may reduce the risk for infection of ongoing BA.2.12.1 and BA.4/5. FundingHong Kong Research Grants Council Collaborative Research Fund, Health and Medical Research Fund, Wellcome Trust, Shenzhen Science and Technology Program, the Health@InnoHK, Innovation and Technology Commission of Hong Kong, China, National Program on Key Research Project, Emergency Key Program of Guangzhou Laboratory, donations from the Friends of Hope Education Fund and the Hong Kong Theme-Based Research Scheme.

immunology↗

bioRxiv2022

Decision heuristics in contexts exploiting intrinsic skill

Heuristics can inform human decision making in complex environments through a reduction of computational requirements (accuracy-resource trade-off) and a robustness to overparameterisation (less-is-more). However, tasks capturing the efficiency of heuristics typically ignore action proficiency in determining rewards. The requisite movement parameterisation in sensorimotor control questions whether heuristics preserve efficiency when actions are nontrivial. We developed a novel action selection-execution task requiring joint optimisation of action selection and spatio-temporal skillful execution. Optimal choices could be determined by a simple spatial heuristic, or by more complex planning. Computational models of action selection parsimoniously distinguished human participants who adopted the heuristic from those using a more complex planning strategy. Broader comparative analyses then revealed that participants using the heuristic showed combined decisional (selection) and skill (execution) advantages, consistent with a less-is-more framework. In addition, the skill advantage of the heuristic group was predominantly in the core spatial features that also shaped their decision policy, evidence that the dimensions of information guiding action selection might be yoked to salient features in skill learning. Author SummaryWe often must choose between actions and then execute them, e.g., a tennis player chooses between a forehand and backhand and then skilfully executes the shot. To select actions, the player might plan their action with either shot, and select whichever simulated outcome is more advantageous. However, a more efficient approach might instead be to use a "heuristic", i.e., a simpler rule, such as, forehand always on one side of the court, and backhand on the other. In this work, we look at whether styles of planning are related to physical skill performing actions, e.g., would a more skillful tennis player be more likely to use planning or a heuristic? We use a new task that requires people to choose and execute complex actions. Regarding choices, we use computational modeling to identify which people use some degree of planning, and which people use a simpler heuristic. Then, regarding action execution, we reveal that heuristic decision makers are in fact more skilled. However, they are not superiorly skilled in all aspects of performance, showing an advantage solely in the aspect of skill most closely linked to the information (spatial) they use for their heuristic. We therefore reveal the first ever evidence that a relation exists between the complexity of our action-related decisions and how broadly we learn associated motor behaviour.

neuroscience↗

bioRxiv2022

Transcriptional circuitry of NKX2-1 and SOX1 defines an unrecognized lineage subtype of small cell lung cancer

RationaleThe current molecular classification of small cell lung cancer (SCLC) based on expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. ObjectivesTo refine the current SCLC classification with epigenomic profiles and to identify features of the re-defined SCLC subtypes. MethodsWe performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance was evaluated by cell growth, apoptosis and xenograft using CRISPR-Cas9-mediated deletion. The specific cistromic profiles by ChIP-seq and its functional transcriptional partners using co-immunoprecipitation followed by mass spectrometry were determined. Rb1fl/flTrp53fl/fl and Rb1fl/flNkx2-1fl/fl mouse models were engineered to explore the function of Nkx2-1 in tumor initiation and differentiation. H3K27ac profiles were analyzed to reveal 6 human SCLC specimen and 20 mice tumors epigenomic landscapes. Measurements and Main ResultsWe identified an epigenomic subclusters of the major SCLC-A subtype, named SCLC-A and SCLC-A{sigma}. SCLC-A was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-A. We further found maintenance of this neural identity in SCLC-A is mediated by collaborative transcriptional activity with another neuronal transcriptional factor SOX1. ConclusionsWe comprehensively describe an additional epigenomic heterogeneity of the major SCLC-A subtype, and define SCLC-A subtype by the core regulatory circuitry representing NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

molecular biology↗

bioRxiv2022

The structure of insulin granule core determines secretory capacity being reduced in type-2 diabetes

Exocytosis in excitable cells is essential for their physiological functions. Although the exocytotic machinery controlling cellular secretion has been well investigated, the function of the vesicular cargo, i.e. secretory granular content remains obscure. Here we combine dSTORM imaging and single-domain insulin antibody, to dissect the in situ structure of insulin granule cores (IGCs) at nano level. We demonstrate that the size and shape of the IGCs can be regulated by the juxta-granular molecules Nucleobindin-2 and Enolase-1, that further contribute to the stimulated insulin secretion. IGCs located at the plasma membrane are larger than those in the cytosol. The IGCs size is decreased by [~]20% after glucose stimulation due to the release of the peripheral part of IGCs through incomplete granule fusion. Importantly, the reduction of the IGCs size is also observed in non-stimulatory pancreatic {beta}-cells from diabetic db/db mice, Akita (Ins2+/-) mice and human Type-2 diabetic donors, in accordance with impaired secretion. These findings overall highlight the structure of exocytotic insulin cores as a novel modality amenable to targeting in the stimulated exocytosis in {beta}-cells with impaired insulin secretion.

physiology↗

bioRxiv2022

VE607 Stabilizes SARS-CoV-2 Spike In the "RBD-up" Conformation and Inhibits Viral Entry

SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its "up" conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.

microbiology↗

bioRxiv2022

USH2A gene mutations in rabbits lead to progressive retinal degeneration and hearing loss

Mutations in USH2A gene are responsible for the greatest proportion of hearing and vision loss among individuals with Usher Syndrome (USH) and for autosomal recessive non-syndromic retinitis pigmentosa. Mutations on USH2A exon 13 account for more than 35% of the disease causing USH2A variants including the most prevalence point mutation, c.2299delG, a frameshift mutation. The lack of a clinically relevant animal model has been a bottleneck for the development of therapeutics for USH2A related vision loss. Using CRSPR/Cas9 technology, this study establishes a rabbit line carrying an USH2A frameshift mutation on exon12 (equivalent to human USH2A Exon 13) as a novel mammalian animal model of USH2A. The bi-allelic mutant rabbits exhibit hyper reflective signals in FAF indicating RPE damage and OCT changes indicating photoreceptor degeneration as early as 4 months of age. ERG signals of both rod and cone function were reduced in the USH2A mutant rabbits starting from 7 months old and further decreased at 15-22 months old, indicating progressive retinal photoreceptor degeneration, which is further confirmed by retinal histopathology examination. ABR examination showed moderate to server hearing loss in the USH2A mutant rabbits. These results indicated that disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration. To our knowledge, this is the first mammalian animal model of USH2 which closely recapitulates the phenotype of retinitis pigmentosa in human patients. This study supports the use of rabbits as a clinically relevant animal model to understand the pathogenesis and to develop novel therapeutics for Usher Syndrome.

developmental biology↗

bioRxiv2022

Ligand-independent modulation of GIPR signaling by splice variants

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 7 reported (EMBL-EBI, 2022; NCBI, 2022a, 2022b) splice variants (SVs) with previously undefined functions. To explore their roles in endogenous peptide mediated GIPR signaling, we investigated the outcome of co-expressing each of the four SVs in question with GIPR in terms of ligand binding, cAMP accumulation, Gs activation, {beta}-arrestin recruitment and cell surface localization. The effects of these SVs on intracellular cAMP responses modulated by receptor activity-modifying proteins (RAMPs) were also studied. It was found that while SVs of GIPR neither bound to the hormone nor affected its signal transduction per se, they differentially regulated GIPR-mediated cAMP and {beta}-arrestin responses. Specifically, SV1 and SV4 were preferable to Gs signaling, SV3 was biased towards {beta}-arrestin recruitment, whereas SV2 was inactive on both pathways. In the presence of RAMPs, only SV1 and SV4 synergized the repressive action of RAMP3 on GIP-elicited cAMP production. The results suggest a new form of signal bias that is constitutive and ligand-independent, thereby expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-dependent (e.g., SV1 of the growth hormone-releasing hormone receptor).

molecular biology↗