Biology subjects

Zang, J.

Publications and source records attributed to Zang, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural Mechanism of TRPC3 Channel Activation by the Moonwalker Mutation

TRPC3 is a calcium-permeable, non-selective cation channel that is activated by DAG. It is expressed in several tissues, especially in the cerebellum, and has been implicated in various human diseases. Despite recent progress in understanding the structural mechanism of TRPC3, how the channel opens remains elusive. Here, we present structures of hTRPC3 in an agonist-free resting state, determined using a DAG-binding site mutant. We also present the structure of hTRPC3 in a DAG-bound open state, determined using a constitutively active "moonwalker" (T561A) mutant. These structures, together with electrophysiological results, reveal that the T561A mutation activates hTRPC3 by disrupting a polar interaction with N652. A newly formed {pi}-bulge in S6 leads to rotation and outward tilting of the lower half of S6, resulting in dilation of the pore and thus channel opening. Agonist DAG stabilizes hTRPC3 in the open conformation. BTDM exerts its inhibitory effect by pushing S5 and S6 back to the center to close the pore, while preserving the {pi}-bulge. These results shed light on the opening mechanism of hTRPC3.

biophysics↗

bioRxiv2025

Unveiling Eukaryotic Membrane Proteins in High Resolution Using Peptide Solubilization

Integral membrane proteins are vital for numerous biological functions and are typically studied using X-ray crystallography and cryo-electron microscopy (cryo-EM). However, these techniques require the extraction of target membrane proteins from their native membranes using detergents, which might disrupt the lipid environments and alter protein behavior. In this study, we present a novel method for solubilizing membrane proteins using a peptide, thereby eliminating the need for detergents throughout the procedure. We demonstrate that the 4F peptide effectively solubilizes a range of membrane proteins and complexes into peptidiscs, while preserving their functionality and structural integrity. Converting these peptidiscs into nanodiscs further enhances particle homogeneity and facilitates high-resolution structural determination of membrane proteins. Our findings highlight the potential of membrane-solubilizing peptides to advance membrane protein research. HighlightsO_LI4F peptide can effectively solubilize eukaryotic membrane proteins C_LIO_LI4F-peptidisc can be converted to MSP-wrapped nanodisc for high resolution cryo-EM studies C_LIO_LI4F solubilization can preserve endogenous ligands bound to the target membrane protein C_LI

biophysics↗

bioRxiv2024

Time-resolved fluorescent proteins towards fluorescence microscopy in the temporal and spectral domains

Fluorescence microscopy has been widely applied in life sciences. While steady-state fluorescence intensity is mostly used, the time-resolved detection mode relying on fluorescence lifetime remains underexplored. Herein, we present a family of time-resolved fluorescent proteins (TRFP). Using a strategy that regulates lifetime without affecting spectra of FPs, we have developed different TRFPs that cover the visible spectrum and a wide range of lifetime. TRFPs are employed in the temporal-spectral resolved microscopy, allowing the simultaneous imaging of 9 different proteins in live cells and correlation of multiple activities to cell cycles. Finally, TRFPs are integrated into multiplexing super-resolution microscopy to concurrently visualize 4 proteins in live cells, acquiring a remarkable 50 nm spatial resolution using lifetime as signal. TRFPs represent a transformative toolset towards fluorescence microscopy in both the temporal and spectral domains. Beyond the multiplexing imaging, TRFPs could introduce unprecedented opportunities of system complexity and quantitative accuracy into biological research.

biochemistry↗

bioRxiv2024

Mechanisms of urate transport and uricosuric drugs inhibition in human URAT1

High urate levels in circulation lead to the accumulation of urate crystals in joints and ultimately inflammation and gout1. The reabsorption process of urate in the kidney by the urate transporter URAT1 plays a pivotal role in controlling serum urate levels2. Pharmacological inhibition of URAT1 by uricosuric drugs is a valid strategy for gout management3. Despite the clinical significance of URAT1, its structure and mechanism remain elusive. Here, we report the structures of human URAT1 (hURAT1) in complex with substrate urate or inhibitors benzbromarone and verinurad at resolution ranges from 3.0 to 3.3 [A]. Urate-bound hURAT1 adopts the outward-facing conformation. Urate is wrapped in the center of hURAT1 by five phenylalanines and coordinated by two positively charged residues on each side. Uricosuric compounds benzbromarone and verinurad occupy the urate-binding site of hURAT1 in the inward-facing conformation. Structural comparison between different conformations of hURAT1 reveals the rocker-switch-like mechanism for urate transport. Benzbromarone and verinurad exert their inhibitory effect by blocking not only the binding of urate but also the structural isomerization of hURAT1.

biochemistry↗

bioRxiv2024

Magnetoelectric Microrobots for Spinal Cord Injury Regeneration

Regenerative medicine continually seeks effective methods to address spinal cord injuries (SCI), which are known for their limited regenerative potential. Despite advances in neural progenitor cell (NPC) transplants for spinal cord injuries, challenges related to graft survival, reliable in vivo differentiation, and neural integration significantly hinder real functional recovery and limit clinical outcomes. This study introduces NPCbots, biohybrid microrobots engineered by integrating human-induced pluripotent stem cell-derived NPCs with magnetoelectric nanoparticles composed of cobalt ferrite-barium titanate. These enable magnetic navigation and neuronal stimulation, enhancing targeted therapeutic interventions. Our lab-on-a-chip system allows for the mass production of NPCbots, ensuring their differentiation and biocompatibility. Remarkably, in a zebrafish model of SCI, NPCbots stimulated by an alternating magnetic field demonstrated rapid in vivo differentiation and integration into damaged neural pathways, significantly enhancing neural regeneration. Within three days, injured zebrafish treated with NPCbots exhibited almost normal swimming behavior and significantly improved exploratory behavior, showcasing the potential of NPCbots to swiftly repair neural structures and restore the central nervous systems functionality in spinal cord injury models through non-invasive means. Additionally, precise in vitro and in vivo manipulation of NPCbots indicates their broader application in various neurodegenerative disorders, offering a promising route for effective spinal cord and neurological recovery.

neuroscience↗

bioRxiv2024

Defects in exosome biogenesis are associated with sensorimotor defects in zebrafish vps4a mutants

Mutations in human VPS4A are associated with neurodevelopmental defects, including motor delays and defective muscle tone. VPS4A encodes a AAA-ATPase required for membrane scission, but how mutations in VPS4A lead to impaired control of motor function is not known. Here we identified a mutation in zebrafish vps4a, T248I, that affects sensorimotor transformation. In biochemical experiments we show the T248I mutation reduces the ATPase activity of Vps4a and disassembly of its substrate, ESCRT filaments, which mediate membrane scission. Consistent with the established role for Vps4a in the endocytic pathway and exosome biogenesis, vps4aT248I mutants have enlarged endosomal compartments in the CNS and decreased numbers of circulating exosomes. Resembling the central form of hypotonia in human VPS4A patients, motor neurons and muscle cells are unaffected in mutant zebrafish as touch sensitivity is intact. Unlike somatosensory function, optomotor responses, vestibulospinal (VS), and acoustic startle reflexes are severely impaired in vps4aT248I mutants, indicating a greater sensitivity of these circuits to the T248I mutation. ERG recordings indicate that visual ability is largely reduced in the mutants, however, in vivo imaging of tone-evoked responses in the inner ear and ascending auditory pathway show comparable activity. Further investigation of central pathways in vps4aT248I mutants revealed that sensory cues failed to fully activate neurons in the VS and medial longitudinal fasciculus (MLF) nuclei that directly innervate motor neurons. Our results suggest that a defect in sensorimotor transformation underlies the profound yet selective effects on motor reflexes resulting from the loss of membrane scission mediated by Vps4a. Significance StatementHere we present a T248I mutation in vps4a, which causes sensorimotor defects in zebrafish larvae. Spanning biochemical to systems level analyses, our study indicates that a reduction in Vps4a enzymatic activity leads to endosomal defects and reduced exosome biogenesis, resulting in pronounced deficits in vision and sensorimotor transformation of auditory and vestibular cues. We suggest that the mechanisms underlying this type of dysfunction in zebrafish may also contribute to the condition seen in human patients with de novo mutations in the human VPS4A orthologue.

neuroscience↗

bioRxiv2023

Dual Ethanolamine Head Groups in Ionizable Lipids Facilitate Phospholipid-free Stable Nanoparticle Formulation for Augmented and Safer mRNA Delivery

The accelerated advent of mRNA-based therapeutics and vaccines, highlighted by the battle against SARS-CoV-2, underscores the urgency to refine lipid nanoparticles (LNPs) for efficient mRNA delivery. In this work, we introduce a novel series of ionizable lipids characterized by double ethanolamine head groups, significantly amplifying mRNA binding affinity. A succinct three-component formulation is subsequently delineated, obviating the conventional dependency on phospholipids inherent in traditional four-component LNPs. Intriguingly, this formulation enables particle formation under neutral pH conditions, a notable departure from the acidic milieu traditionally required, attributable to the enhanced nonionic interactions predominating in mRNA encapsulation. The resultant particles exhibit exceptional stability, superior mRNA encapsulation efficiency, and maintain robust delivery efficacy. When deployed as a vaccine platform, the formulation elicited pronounced humoral and T-cell immune responses, concurrently exhibiting a favorable toxicity profile with a reduced induction of pro-inflammatory cytokines such as IL-6. Our exploration suggests that by fine-tuning the non-electrostatic interactions between the ionizable lipid and mRNA, the dynamics of particle formation can be considerably divergent from the prevailing paradigms of mRNA-LNP formation, hinting at a broader horizon for lipid optimization within the realm of mRNA delivery systems.

bioengineering↗

bioRxiv2022

Systematic characterization of chromodomain proteins revealsan H3K9me1/2 reader regulating aging in C. elegans

The chromatin organization modifier domain (chromodomain) is an evolutionally conserved motif across eukaryotic species. The chromodomain mainly functions as a histone methyl-lysine reader to modulate gene expression, chromatin spatial conformation and genome stability. Mutations or aberrant expression of chromodomain proteins can result in cancer and other human diseases. Here, we systematically tagged chromodomain proteins with green fluorescent protein (GFP) using CRISPR/Cas9 technology in C. elegans. By combining ChIP-seq analysis and imaging, we delineated a comprehensive expression and functional map of chromodomain proteins. We then conducted a candidate-based RNAi screening and identified factors that regulate the expression and subcellular localization of the chromodomain proteins. Specifically, we revealed a new H3K9me1/2 reader, CEC-5, both by in vitro biochemistry and in vivo ChIP assays. MET-2, an H3K9me1/2 writer, is required for CEC-5 association with heterochromatin. Both MET-2 and CEC-5 are required for the normal lifespan of C. elegans. Furthermore, a forward genetic screening identified a conserved Arginine124 of CEC-5s chromodomain, which was essential for CEC-5s association with chromatin and life span regulation. Thus, our work will serve as a reference to explore chromodomain functions and regulation in C. elegans and allow potential applications in aging-related human diseases.

molecular biology↗

bioRxiv2022

The microtubule-associated protein SlMAP70 interacts with SlIQD21 and regulates fruit shape formation in tomato

The shape of tomato fruits is closely correlated to microtubule organization and the activity of microtubule associated proteins (MAP), but insights into the mechanism from a cell biology perspective are still largely elusive. Analysis of tissue expression profiles of different microtubule regulators revealed that functionally distinct classes of MAPs are highly expressed during fruit development. Among these, several members of the plant-specific MAP70 family are preferably expressed at the initiation stage of fruit development. Transgenic tomato lines overexpressing SlMAP70 produced elongated fruits that show reduced cell circularity and microtubule anisotropy, while SlMAP70 loss-of-function mutant showed an opposite effect with flatter fruits. Microtubule anisotropy of fruit endodermis cells exhibited dramatic rearrangement during tomato fruit development, and SlMAP70-1 is likely implicated in cortical microtubule organization and fruit elongation throughout this stage by interacting with SUN10/SlIQD21a. The expression of SlMAP70 (or co-expression of SlMAP70 and SUN10/SlIQD21a) induces microtubule stabilization and prevents its dynamic rearrangement, both activities are essential for fruit shape establishment after anthesis. Together, our results identify SlMAP70 as a novel regulator of fruit elongation, and demonstrate that manipulating microtubule stability and organization at the early fruit developmental stage has a strong impact on fruit shape.

plant biology↗

bioRxiv2022

An mRNA vaccine candidate for the SARS-CoV-2 Omicron variant

The newly emerged Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains more than 30 mutations on the spike protein, 15 of which are located within the receptor binding domain (RBD). Consequently, Omicron is able to extensively escape existing neutralizing antibodies and may therefore compromise the efficacy of current vaccines based on the original strain, highlighting the importance and urgency of developing effective vaccines against Omicron. Here we report the rapid generation and evaluation of an mRNA vaccine candidate specific to Omicron. This mRNA vaccine encodes the RBD of Omicron (designated RBD-O) and is formulated with lipid nanoparticle. Two doses of the RBD-O mRNA vaccine efficiently induce neutralizing antibodies in mice; however, the antisera are effective only on the Omicron variant but not on the wildtype and Delta strains, indicating a narrow neutralization spectrum. It is noted that the neutralization profile of the RBD-O mRNA vaccine is opposite to that observed for the mRNA vaccine expressing the wildtype RBD (RBD-WT). Our work demonstrates the feasibility and potency of an RBD-based mRNA vaccine specific to Omicron, providing important information for further development of bivalent or multivalent SARS-CoV-2 vaccines with broad-spectrum efficacy.

microbiology↗

bioRxiv2022

Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network.

Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR-Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted towards photoreceptor or Muller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species. Brief Summary Statement for use in emailed and online tables of content alertsThe mutation of vsx genes in zebrafish confirms a conserved role in bipolar cells specification across vertebrates, but do not interfere with the specification of the neural retina domain. Our data reveal the unexpected robustness of the genetic network sustaining the identity of the neural retina.

developmental biology↗

bioRxiv2021

Specialization of the photoreceptor transcriptome by Srrm3-dependent microexons is required for outer segment maintenance and vision

Retinal photoreceptors differ in their transcriptomic profiles from other neuronal subtypes, likely as a reflection of their unique cellular morphology and function in the detection of light thorough the ciliary outer segment. We discovered a new layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and in vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors and its deletion in zebrafish results in widespread downregulation of microexon inclusion, severe photoreceptor alterations and blindness. These results shed light into photoreceptors transcriptomic specialization and functionality, uncovering new cell type-specific roles for Srrm3 and microexons with implication for retinal diseases.

systems biology↗

bioRxiv2021

The Bardet-Biedl protein Bbs1 controls photoreceptor outer segment protein and lipid composition

Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a new bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on isolated OSs show that Bbs1 is required for BBSome-entry into OSs and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a new role for Bbs1/BBSome in OS lipid homeostasis and suggest a new pathomechanism underlying retinal degeneration in BBS.

molecular biology↗

bioRxiv2021

Efficacy of ancestral receptor-binding domain, S1 and trimeric spike protein vaccines against SARS-CoV-2 variants B.1.1.7, B.1.351, and B.1.617.1

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current SARS-CoV-2 vaccines are based on spike (S) protein, S1 subunit, or receptor-binding domain (RBD) of prototype strain. Emergence of several novel SARS-CoV-2 variants has raised concern about potential immune escape. In this study, we performed an immunogenicity comparison of ancestral RBD, S1, and S ectodomain trimer (S-trimer) antigens and tested the efficacy of these prototype vaccines against the circulating variants, especially B.1.617 that has been linked to Indias current COVID-19 surge. We found that RBD and S-trimer proteins could induce significantly higher neutralizing antibody titers than S1 protein. For the three vaccines, the neutralizing titers decreased over time, but still remained high for at least five months after immunization. Importantly, the three prototype vaccines were still effective in neutralizing the variants of concern, although B.1.351 and B.1.617.1 lineages showed varying degrees of reduction in neutralization by the immune sera. The vaccines-induced sera were shown to block receptor binding and inhibit S protein-mediated membrane fusion. In addition, the immune sera did not promote antibody-dependent enhancement (ADE) in vitro. Our work provides valuable information for development of SARS-CoV-2 subunit vaccines and also supports the continued use of ancestral RBD or S-based vaccines to fight the COVID-19 epidemic.

immunology↗

bioRxiv2021

Circadian Regulation of Vertebrate Cone Photoreceptor Function

Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as recoverins, arrestins, opsin kinases, and GTPase-accelerating protein that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. This physiological rhythmicity is mirrored in visual behaviors, such as optokinetic and optomotor responses. Functional rhythmicity persists in continuous darkness, it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.

neuroscience↗

bioRxiv2020

Structural insights into the recognition of histone H3Q5 serotonylation by WDR5

Serotonylation of histone H3Q5 (H3Q5ser) is a recently identified posttranslational modification of histones that apparently acts as a permissive marker for gene activation in synergy with H3K4me3 during neuronal cell differentiation. However, any proteins which specifically recognize H3Q5ser remain unknown. Here, we discovered that WDR5 interacts with the N-terminal tail of histone H3 and functions as a reader for H3Q5ser. Crystal structures of WDR5 in complex with H3Q5ser and H3K4me3Q5ser peptides revealed that the serotonyl group is accommodated in a shallow surface pocket of WDR5. Experiments in neuroblastoma cells demonstrate that WDR5 colocalizes with H3Q5ser in the promoter regions of cancer-promoting genes, where it promotes gene transcription to induce cell proliferation. Thus, beyond revealing a previously unknown mechanism through which WDR5 reads H3Q5ser to activate transcription, our study suggests that this WDR5-H3Q5ser mediated epigenetic regulation apparently promotes tumorigenesis.

biochemistry↗

bioRxiv2020

A comprehensive map of genetic interactions in childhood cancer reveals multiple underlying biological mechanisms

Childhood cancer is a major cause of child death in developed countries. Genetic interactions between mutated genes play an important role in cancer development. They can be detected by searching for pairs of mutated genes that co-occur more (or less) often than expected. Co-occurrence suggests a cooperative role in cancer development, while mutual exclusivity points to synthetic lethality, a phenomenon of interest in cancer treatment research. Little is known about genetic interactions in childhood cancer. We apply a statistical pipeline to detect genetic interactions in a combined dataset comprising over 2,500 tumors from 23 cancer types. The resulting genetic interaction map of childhood cancers comprises 15 co-occurring and 27 mutually exclusive candidates. The biological mechanisms underlying most candidates are either tumor subtype, pathway epistasis or cooperation while synthetic lethality plays a much smaller role. Thus, other explanations beyond synthetic lethality should be considered when interpreting results of genetic interaction tests.

cancer biology↗

bioRxiv2020

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

Mutations in USH2A, encoding usherin, are the most common cause of syndromic and non-syndromic retinitis pigmentosa (RP). The two founder mutations in exon 13 (c.2299delG and c.2276G>T) collectively account for ~34% of USH2A-associated RP cases. Skipping of exon 13 from the USH2A transcript during pre-mRNA splicing presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in larvae of the previously published ush2a exon 13 zebrafish mutant resulted in the production of usherin{Delta}exon13 and completely restored retinal function. RNA antisense oligonucleotides were investigated for their potential to specifically induce human USH2A exon 13 skipping. Lead candidate QR-421a induced dose-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from a patient homozygous for the USH2A c.2299delG mutation. Intravitreal delivery of QR-421a in non-human primates showed that QR-421a penetrates the retinal outer nuclear layer and induces detectable levels of exon 13 skipping until at least 3 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment for RP caused by mutations in exon 13 of the USH2A gene.

genetics↗