Biology subjects

Park, Y.-J.

Publications and source records attributed to Park, Y.-J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Whole organism snRNA-seq reveals systemic peripheral changes in Alzheimer's Disease fly models

Peripheral tissues become disrupted in Alzheimers Disease (AD). However, a comprehensive understanding of how the expression of AD-associated toxic proteins, A{beta}42 and Tau, in neurons impacts the periphery is lacking. Using Drosophila, a prime model organism for studying aging and neurodegeneration, we generated the Alzheimers Disease Fly Cell Atlas (AD-FCA): whole-organism single-nucleus transcriptomes of 219 cell types from adult flies neuronally expressing human A{beta}42 or Tau. In-depth analyses and functional data reveal impacts on peripheral sensory neurons by A{beta}42 and on various non-neuronal peripheral tissues by Tau, including the gut, fat body, and reproductive system. This novel AD atlas provides valuable insights into potential biomarkers and the intricate interplay between the nervous system and peripheral tissues in response to AD-associated proteins.

genetics↗

bioRxiv2024

Human coronavirus HKU1 recognition of the TMPRSS2 host receptor

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. Here, we designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2 providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among human type 2 transmembrane serine proteases. We found that human, rat, hamster and camel TMPRSS2 promote HKU1 S-mediated entry into cells and identified key residues governing host receptor usage. Our data show that serum antibodies targeting the HKU1 RBD TMPRSS2 binding-site are key for neutralization and that HKU1 uses conformational masking and glycan shielding to balance immune evasion and receptor engagement.

biochemistry↗

bioRxiv2023

A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines

Continuous evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and monoclonal antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S2 subunit, which folds as a spring-loaded fusion machinery. Here, we describe a protein design strategy enabling prefusion-stabilization of the SARS-CoV-2 S2 subunit and high yield recombinant expression of trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to all sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S2 fusion machineries. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S2 trimer vaccine elicits broadly reactive sarbecovirus antibody responses and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with SARS-CoV-2 XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines motivating future development.

biochemistry↗

bioRxiv2023

Umbrella toxin particles produced by Streptomyces block mycelial growth of competing species

The Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species. Here we show that in addition to small molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-EM structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, leading to our naming them umbrella particles. S. coelicolor encodes three umbrella particles with distinct toxin and lectin composition, and supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, S. griseus, we find inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative mycelial growth. Our data show that Streptomyces umbrella particles mediate competition between vegetative mycelia of related species, a function distinct from small molecule antibiotics, which are produced at the onset of reproductive growth and act broadly. Sequence analyses suggest this role of umbrella particles extends beyond Streptomyces, as we find umbrella loci in nearly one-thousand species across Actinobacteria.

microbiology↗

bioRxiv2023

A method of identifying false positives in the strain-specific variant calling of rice

In this study, we investigated the strain-specific effect in genetic variant calling from next-generation sequencing data. For this purpose, we used two major strains of the rice genome, Indica and Japonica, to build different variant calling models that differ in the composition of samples from the two strains. We found that the more the samples differed in their strains from the reference sequence, the more variants were predicted. In particular, the increase in predicted variants was noticeable when the samples that differed in their strains from the reference were included. We used machine learning approaches to understand this finding and compared the performance of different variant calling models using confusion matrices constructed from the predicted variants. We found that a significant proportion of the incrementally predicted variants are potential false positives, which becomes more pronounced the more phylogenetically different accessions from the reference are included in the samples. For the accuracy of the predicted variants, we proposed a method to identify the false positives that can be excluded from the potential false positives if necessary. The proposed method involves calling true variants from the purebred samples. We demonstrated the validity of the proposed method on the different variant calling models and showed a reduction of false positives in the predicted variants. As an example of practical utility, we applied the method to the dbSNP, a database of known variants, and demonstrated a way to identify false positives in the dbSNP. In these respects, this study provides general recommendations for effective practices in strain-specific variant calling in rice.

bioinformatics↗

bioRxiv2023

Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad ACE2 usage and that RBD mutations further expand receptor promiscuity and enable human ACE2 utilization. We determined a cryoEM structure of the PRD-0038 RBD bound to R. alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryoEM and monoclonal antibody reactivity revealed its distinct antigenicity relative to SARS-CoV-2 and identified PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared to SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.

biochemistry↗

bioRxiv2023

Molecular blueprints of the Langya virus attachment and fusion glycoproteins

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse and hamster target cells using a different, yet unknown, receptor than NiV and HeV and that NiV-and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryo-electron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing previously unknown conformational landscapes and their distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

biochemistry↗

bioRxiv2023

Hierarchical design of pseudosymmetric protein nanoparticles

Discrete protein assemblies ranging from hundreds of kilodaltons to hundreds of megadaltons in size are a ubiquitous feature of biological systems and perform highly specialized functions 1-3. Despite remarkable recent progress in accurately designing new self-assembling proteins, the size and complexity of these assemblies has been limited by a reliance on strict symmetry 4,5. Inspired by the pseudosymmetry observed in bacterial microcompartments and viral capsids, we developed a hierarchical computational method for designing large pseudosymmetric self-assembling protein nanomaterials. We computationally designed pseudosymmetric heterooligomeric components and used them to create discrete, cage-like protein assemblies with icosahedral symmetry containing 240, 540, and 960 subunits. At 49, 71, and 96 nm diameter, these nanoparticles are the largest bounded computationally designed protein assemblies generated to date. More broadly, by moving beyond strict symmetry, our work represents an important step towards the accurate design of arbitrary self-assembling nanoscale protein objects.

bioengineering↗

bioRxiv2023

Antigen spacing on protein nanoparticles influences antibody responses to vaccination

Immunogen design approaches aim to control the specificity and quality of antibody responses to enable the creation of next-generation vaccines with improved potency and breadth. However, our understanding of the relationship between immunogen structure and immunogenicity is limited. Here we use computational protein design to generate a self-assembling nanoparticle vaccine platform based on the head domain of influenza hemagglutinin (HA) that enables precise control of antigen conformation, flexibility, and spacing on the nanoparticle exterior. Domain-based HA head antigens were presented either as monomers or in a native-like closed trimeric conformation that prevents exposure of trimer interface epitopes. These antigens were connected to the underlying nanoparticle by a rigid linker that was modularly extended to precisely control antigen spacing. We found that nanoparticle immunogens with decreased spacing between closed trimeric head antigens elicited antibodies with improved hemagglutination inhibition (HAI) and neutralization potency as well as binding breadth across diverse HAs within a subtype. Our "trihead" nanoparticle immunogen platform thus enables new insights into anti-HA immunity, establishes antigen spacing as an important parameter in structure-based vaccine design, and embodies several design features that could be used to generate next-generation vaccines against influenza and other viruses. HIGHLIGHTSO_LIComputational design of a closed trimeric HA head ("trihead") antigen platform. C_LIO_LIDesign of a rigid, extendable linker between displayed antigen and underlying protein nanoparticle enables precise variation of antigen spacing. C_LIO_LIDecreased antigen spacing of triheads elicits antibodies with the highest HAI, neutralizing activity, and cross-reactivity. C_LIO_LIChanges to antigen spacing alter epitope specificities of vaccine-elicited antibodies. C_LI

immunology↗

bioRxiv2023

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots1. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

immunology↗

bioRxiv2023

MARCH5-dependent NLRP3 ubiquitination is an essential step for NEK7 docking on the mitochondria

The NLRP3 inflammasome is a global immune-sensor that is activated by a repertoire of endogenous and exogenous stimuli. NLRP3 translocates to mitochondria but whether mitochondria involvement in the inflammasome assembly is unclear. Here, we show that the mitochondrial E3 ligase MARCH5 is a key regulator of NLRP3 inflammasome assembly. Myeloid cell-specific March5 conditional knockout (March5 cKO) mice exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice failed to secrete IL-1{beta} and IL-18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27-linked polyubiquitination of K324 and K430 residues of NLRP3. Ubiquitination-defective NLRP3 mutants neither bind to NEK7, nor form NLRP3 oligomers, but remain binding to MAVS. Accordingly, NLRP3 mutants led to abortive ASC speck formation and diminished IL-1{beta} production. We propose that MARCH5-dependent NLRP3 ubiquitination creates a docking site for NEK7 binding, playing as a fundamental step-wise regulator on the mitochondria.

immunology↗

bioRxiv2022

Aging Fly Cell Atlas Identifies Exhaustive Aging Features at Cellular Resolution

Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. Here, we present the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole aging Drosophila. We characterize 163 distinct cell types and perform an in-depth analysis of changes in tissue cell composition, gene expression, and cell identities. We further develop aging clock models to predict the fly age and show that ribosomal gene expression is a conserved predictive factor for age. Combining all aging features, we find unique cell type-specific aging patterns. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.

developmental biology↗

bioRxiv2022

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

immunology↗

bioRxiv2022

Interactions of C. elegans -tubulins with the microtubule inhibitor albendazole

Parasitic nematodes are major human and agricultural pests, and benzimidazoles are amongst the most important broad spectrum anthelmintic drug class used for their control. Benzimidazole resistance is now widespread in many species of parasitic nematodes in livestock globally and an emerging concern for the sustainable control of human soil transmitted helminths. {beta}-tubulin is the major benzimidazole target, although other genes may influence resistance. Among the six C. elegans {beta}-tubulin genes, loss of ben-1 causes resistance without other apparent defects. Here, we explored the genetics of C. elegans {beta}-tubulin genes in relation to the response to the benzimidazole derivative albendazole. The most highly expressed {beta}-tubulin isotypes, encoded by tbb-1 and tbb-2, were known to be redundant with each other for viability, and their products are predicted not to bind benzimidazoles. We found that tbb-2 mutants, and to a lesser extent tbb-1 mutants, were hypersensitive to albendazole. The double mutant tbb-2 ben-1 is uncoordinated and dumpy, resembling the wild type exposed to albendazole, but the tbb-1 ben-1 double mutant did not show the same phenotype. These results suggest that tbb-2 is a modifier of ABZ sensitivity. To better understand how BEN-1 mutates to cause benzimidazole resistance, we isolated mutants resistant to albendazole and found that 15 of 16 mutations occurred in ben-1. Mutations ranged from likely nulls to hypomorphs, and several corresponded to residues that cause resistance in other organisms. Null alleles of ben-1 are albendazole-resistant and BEN-1 shows high sequence identity with tubulins from other organisms, suggesting that many amino acid changes could cause resistance. However, our results suggest that missense mutations conferring resistance are not evenly distributed across all possible conserved sites. Independent of their roles in benzimidazole resistance, tbb-1 and tbb-2 may have specialized functions as null mutants of tbb-1 or tbb-2 were cold or heat sensitive, respectively.

genetics↗

bioRxiv2022

Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-CoV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the cilgavimab (AZD1061) mAb. Here, we show that this residue substitution remodels the ACE2-binding site allosterically, thereby dampening receptor recognition severely and altering the epitopes recognized by these three mAbs. Although vaccine-elicited neutralizing antibody titers are decreased similarly against the E406 mutant and the Delta or Epsilon variants, broadly neutralizing sarbecovirus mAbs, including a clinical mAb, inhibit the E406W spike mutant.

biochemistry↗

bioRxiv2021

Computational design of nanoscale rotational mechanics in de novo protein assemblies

Natural nanomachines like the F1/F0-ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines. One-Sentence SummaryComputationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.

synthetic biology↗

bioRxiv2021

Structure, receptor recognition and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein

The recent isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. Here, we determined cryo-electron microscopy structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and identified that it binds canine, feline and porcine aminopeptidase N (APN encoded by ANPEP) orthologs which serve as entry receptors. Introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single nucleotide polymorphisms could account for the detection of this virus in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 S-mediated entry, indicating elicitation of cross-neutralizing activity among -coronaviruses. These data provide a blueprint of the CCoV-HuPn-2018 infection machinery, unveil the viral entry receptor and pave the way for vaccine and therapeutic development targeting this zoonotic pathogen.

microbiology↗

bioRxiv2021

Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry

Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures effective against SARS-CoV-2 variants and future spillovers of other sarbecoviruses. Here we describe the isolation and characterization of a human monoclonal antibody, designated S2K146, broadly neutralizing viruses belonging to all three sarbecovirus clades known to utilize ACE2 as entry receptor and protecting therapeutically against SARS-CoV-2 beta challenge in hamsters. Structural and functional studies show that most of the S2K146 epitope residues are shared with the ACE2 binding site and that the antibody inhibits receptor attachment competitively. Viral passaging experiments underscore an unusually high barrier for emergence of escape mutants making it an ideal candidate for clinical development. These findings unveil a key site of vulnerability for the development of a next generation of vaccines eliciting broad sarbecovirus immunity.

immunology↗