Biology subjects

Lee, K.-M.

Publications and source records attributed to Lee, K.-M..

11 recordsLinked to original sources

bioRxiv2025

Structural insights into how metallochaperones UreE and UreG interact to deliver a toxic metal to urease

Maturation of urease, a virulence factor for Helicobacter pylori infection, requires the delivery of nickel ions to the metalloenzyme. To avoid cytotoxicity, nickel ions are transferred within protein complexes of metallochaperones to ensure that the toxic metal do not escape into the cytoplasm. In the urease maturation pathway, metallochaperone UreG receives its nickel ions by forming a 2:2 complex with another metallochaperone UreE. Using C-terminal truncation variants of UreE [UreE(1-158) and UreE(1-148)], we determined the crystal structures of the UreE2G2 complex bound with nickel ions and GMPPNP, a non-hydrolysable GTP analogue. UreE-UreG interactions are asymmetric, where the UreG dimer mainly interacts with the proximal UreE. GTP binding induces conformational changes in the G2 and CPH motifs of distal UreG that enable its anchorage to the UreE2G2 complex. His68 of distal UreG moves towards and chelates a nickel ion at the UreE binding site. Formation of the UreE2G2 complex juxtaposes the nickel binding sites of UreE and UreG. Nickel transfer from UreE to UreG induces conformational changes that weaken UreE-UreG interactions, thereby facilitating UreG dissociation. We further demonstrated that the hydrogenase maturation factor HypA, providing the nickel source, could activate urease in vitro through protein-protein interaction with wild-type UreE but not with its truncation variants. UreG binding induces conformational changes in the C-terminal tail of UreE, promoting dissociation of HypA-UreE complex. Our work presents a paradigm on how GTP and nicking binding allosterically regulate the formation of a metallochaperone complex to facilitate nickel transfer. SignificanceOur work provides insights into how cells solve the problem of trafficking a toxic metal, nickel, to the active site of urease. Colonization of Helicobacter pylori in acidic human stomach requires the biosynthesis of active urease, which involves the delivery of the toxic nickel ions to the active site of the metalloenzyme. To avoid cytotoxicity, nickel ions are transported from one metallochaperone to another via the formation of protein complexes so that the toxic metal ions do not escape into the cytoplasm. Supported by structural and biochemical evidence, we present a paradigm on how GTP and nickel binding allosterically promote the formation of a complex between metallochaperones UreE and UreG to facilitate nickel transfer between the two proteins.

biochemistry↗

bioRxiv2024

Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance

Macroautophagy (hereafter autophagy) is essential for cells to respond to nutrient stress by delivering cytosolic contents to vacuoles for degradation via the formation of a multi-layer vesicle named autophagosome. A set of autophagy-related (ATG) regulators are recruited to the phagophore assembly site for the initiation of phagophore, as well as its expansion and closure and subsequent delivery into the vacuole. However, it remains elusive that how the phagophore assembly is regulated under different stress conditions. Here, we described an unknown Arabidopsis (Arabidopsis thaliana) cytosolic ATG8-interaction protein family (ERC1/2), that binds ATG8 and NBR1 to promote autophagy. ERC1 proteins translocate to the phagophore membrane and develop into classical ring-like autophagosomes upon autophagic induction. However, ERC1 proteins form large droplets together with ATG8e proteins when in the absence of ATG8 lipidation activity. We described the property of these structures as phase-separated membraneless condensates by solving the in vivo organization with spatial and temporal resolution. Moreover, ERC1 condensates elicits a strong recruitment of the autophagic receptor NBR1. Loss of ERC1 suppressed NBR1 turnover and attenuated plant tolerance to heat stress condition. This work provides novel insights into the mechanical principle of phagophore initiation via an unreported ERC1-mediated biomolecular condensation for heat tolerance in Arabidopsis.

plant biology↗

bioRxiv2023

Male cuticular pheromones stimulate removal of the mating plug and promote re-mating through pC1 neurons in Drosophila females

In birds and insects, the female uptakes sperm for a specific duration post-copulation known as the ejaculate holding period (EHP) before expelling unused sperm and the mating plug through sperm ejection. In this study, we found that Drosophila melanogaster females shortens the EHP when incubated with males or mated females shortly after the first mating. This phenomenon, which we termed male-induced EHP shortening (MIES), requires Or47b+ olfactory and ppk23+ gustatory neurons, activated by 2-methyltetracosane and 7-tricosene, respectively. These odorants raise cAMP levels in pC1 neurons, responsible for processing male courtship cues and regulating female mating receptivity. Elevated cAMP levels in pC1 neurons reduce EHP and reinstate their responsiveness to male courtship cues, promoting re-mating with faster sperm ejection. This study established MIES as a genetically tractable model of sexual plasticity with a conserved neural mechanism. Significance StatementSexual plasticity, the adaptation of reproductive behavior to social changes, was explored in the fruit fly, a genetically tractable model insect. Our findings revealed that inseminated females, encountering another courting male post-mating, shorten the ejaculate holding period (EHP). Specific olfactory and gustatory pathways regulating this phenomenon were identified, converging on the pC1 neurons in the brain-a conserved neural circuit that regulates female mating activity. Odors associated with EHP shortening increased the second messenger cAMP. The transient elevation of cAMP heightened the excitability of pC1 neurons, facilitating the prompt removal of the male ejaculate and subsequent re-mating . This study established a behavioral model of sexual plasticity and provided a framework for understanding the neural circuit processes involved.

neuroscience↗

bioRxiv2022

CD244 regulates both innate and adaptive immune axes in melanoma by inhibiting autophagy-mediated M1 macrophage maturation

Accumulating data have highlighted the role of monocytes/macrophages in immune escape by generating immunologically "cold" tumors that do not respond to immunotherapy. CD244 (SLAMF4, 2B4), a member of the signaling lymphocyte activation molecule family, is expressed on myeloid cells, but its precise role has not been elucidated. Using monocyte lineage-specific CD244-deficient (LysM-cre+/-CD244fl/fl;cKO) mice challenged with B16F10 melanoma, we report for the first time that CD244 negatively regulates tumor immunity by inhibiting the differentiation and functional maturation of CD11b+Ly6ChiF4/80lo monocytes into CD11b+Ly6CloF4/80hi macrophages within the tumor microenvironment. CD244-deficient macrophages more effectively activated antigen-specific T cell responses compared to WT macrophages, thus delaying tumor growth in the B16F10 melanoma model. Moreover, combinatorial intervention of anti-PD-L1 antibodies with CD244-KO BMDM markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with WT BMDM. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset (SCP398 from single-cell portal), revealed 221 differentially expressed genes of CD244- monocytes/macrophages were associated with phagocytosis, antigen presentation, and autophagy. Additionally, cell type deconvolution analysis within melanoma patients bulk RNA-seq datasets from TCGA database, revealed presence of CD244- monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Hence, we proposed that CD244 serve as a critical immune checkpoint receptor on macrophages, and CD244-deficient macrophages may represent a novel therapeutic modality to convert immunologically "cold" tumors to "hot" tumors, which can function synergistically with checkpoint blockade therapies.

immunology↗

bioRxiv2020

Chemical Inhibitors of DksA1, a Conserved Bacterial Transcriptional Regulator, Suppressed Quorum Sensing-Mediated Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative bacterium of clinical importance. As an opportunistic pathogen, its virulence is dependent on quorum sensing (QS). Stringent response (SR), a conserved mechanism in the bacterial kingdom, is activated under diverse stress conditions including nutrient starvation. DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in P. aeruginosa SR. Our recent study clearly demonstrated that, apart from SR, DksA1 regulates a wide range of phenotypes including QS-mediated virulence. This suggests that DksA1 is a potential target to be inhibited to control P. aeruginosa infections. Here, we screened a library of 6,970 chemical compounds and identified two compounds (termed Dkstatin-1 and Dkstatin-2) that specifically suppress DksA1 activity. Two Dkstatins substantially suppressed production of elastase and pyocyanin and protected a murine host against lethal infection with PAO1, a prototype strain of P. aeruginosa. Dkstatins also suppressed the production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced in Dkstatin-treated cells was almost identical to that in the {Delta}dksAI mutant of PAO1. Our RNASeq analysis showed that transcript levels of genes involved in QS and virulence were markedly reduced in Dkstatin-treated PAO1 cells, further demonstrating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased antibiotic susceptibilities of PAO1, particularly to the antibiotics that inhibit protein synthesis. Our co-immunoprecipitation assay demonstrated that Dkstatins interfere with the binding of DksA1 to the {beta} subunit of RNA polymerase, which potentially explains the mode of Dkstatin action. Collectively, our results suggest that the inhibition of P. aeruginosa QS, which has often been attempted, can be achieved by DksA1 inhibitors. Dkstatins will prove to be useful when establishing infection control strategies. Author summaryBacterial cells developed numerous systems to handle environmental stresses. Stringent response (SR) is one of those systems, and it is activated under the stress of nutrient starvation. Active SR requires DksA1, a protein conserved across bacterial species. Besides this well-recognized function, DksA1 in P. aeruginosa positively regulates its virulence by activating quorum sensing (QS). This finding led us to hypothesize that inhibiting DksA1 would result in virulence attenuation of the pathogenic P. aeruginosa. Herein, we identified two molecules that effectively suppressed DksA1 function through a large-scale compound library screen. These two compounds suppressed P. aeruginosa virulence and therefore protected a murine host from a deadly infection of P. aeruginosa. Moreover, the compounds also rendered P. aeruginosa more susceptible to antibiotics. Our results demonstrate that dangerous P. aeruginosa infections can be controlled by inhibiting DksA1.

microbiology↗

bioRxiv2020

A Robust Genome-Wide Association Study Uncovers Signature Genetic Alterations among Pseudomonas aeruginosa Cystic Fibrosis Isolates

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections such as chronic airway infection in cystic fibrosis (CF) patients. Although many sequenced genomes are available, a comprehensive comparison between genomes of CF versus non-CF PA isolates remains yet to be conducted. In order to gain a deeper understanding into the PA adaptation in the CF airway, we performed a Genome-Wide Association Study (GWAS) using a total of 1,001 PA genomes. Genetic variations uniquely identified among CF isolates were categorized into (i) alterations in protein-coding regions either large- or small-scale and (ii) polymorphic variations in intergenic regions. We introduced each CF-specific genetic alteration into the genome of PAO1, a prototype PA strain and experimentally validated their outcomes. Loci readily mutated among CF isolates include genes encoding a probable sulphatase and a probable TonB-dependent receptor (PA2332~PA2336), L-cysteine transporter (YecS, PA0313) and a probable transcriptional regulator (PA5438). A promoter region of heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was similarly identified as meaningfully different between the CF and non-CF isolate groups. Our analysis, the first of its kind, highlights how PA evolves its genome to persist and survive within the context of chronic CF infection.

microbiology↗

bioRxiv2020

The SARS-CoV-2 RNA interactome

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its ability to repurpose host RNA-binding proteins (RBPs) to form its own RNA interactome. Here, we developed and applied a robust ribonucleoprotein capture protocol to uncover the SARS-CoV-2 RNA interactome. We report 109 host factors that directly bind to SARS-CoV-2 RNAs including general antiviral factors such as ZC3HAV1, TRIM25, and PARP12. Applying RNP capture on another coronavirus HCoV-OC43 revealed evolutionarily conserved interactions between viral RNAs and host proteins. Network and transcriptome analyses delineated antiviral RBPs stimulated by JAK-STAT signaling and proviral RBPs responsible for hijacking multiple steps of the mRNA life cycle. By knockdown experiments, we further found that these viral-RNA-interacting RBPs act against or in favor of SARS-CoV-2. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

microbiology↗

bioRxiv2020

Nasal commensal, Staphylococcus epidermidis shapes the mucosal environment to prevent influenza virus invasion through Serpine1 induction

Our recent study presented evidence that Staphylococcus epidermidis (S. epidermidis) was the most frequently encountered microbiome component in healthy human nasal mucus and that S. epidermidis could induce interferon (IFN)-dependent innate immunity to control acute viral lung infection. The serine protease inhibitor Serpine1 was identified to inhibit influenza A virus (IAV) spread by inhibiting glycoprotein cleavage, and the current study supports an additional mechanism of Serpine1 induction in the nasal mucosa, which can be regulated through S. epidermidis and IFN signaling. The exposure of in vivo mice to human S. epidermidis increased IFN-{lambda} secretion in nasal mucosa and prevented an increase in the burden of IAV in the lung. S. epidermidis-inoculated mice exhibited the significant induction of Serpine1 in vivo in the nasal mucosa, and by targeting airway protease, S. epidermidis-induced Serpine1 inhibited the intracellular invasion of IAV to the nasal epithelium and led to restriction of IAV spreading to the lung. Furthermore, IFN-{lambda} secretion was involved in the regulation of Serpine1 in S. epidermidis-inoculated nasal epithelial cells and in vivo nasal mucosa, and this was biologically relevant for the role of Serpine1 as an interferon-stimulated gene in the upper airway. Together, our findings reveal that human nasal commensal S. epidermidis manipulates the suppression of serine protease in in vivo nasal mucosa through Serpine1 induction and protects the nasal mucosa from IAV invasion through IFN-{lambda} signaling. IMPORTANCEPreviously, we proved that nasal microbiome could enhance IFN-related innate immune responses to protect the respiratory tract against influenza virus infection. The present study shows a great understanding of the intimate association of S. epidermidis-regulated IFN-lambda induction and serine protease inhibitor in nasal mucosa. Our data demonstrate that S. epidermidis-regulated Serpine1 suppresses the invasion of influenza virus through suppression of airway serine protease at the level of nasal mucosa and impedes IAV spread to the respiratory tract. Thus, human nasal commensal S. epidermidis represents a therapeutic potential for treating respiratory viral infections via the change of cellular environment in respiratory tract. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

Sequence variation among SARS-CoV-2 isolates in Taiwan

Taiwan experienced two waves of imported cases of coronavirus disease 2019 (COVID-19), first from China in January to late February, followed by those from other countries starting in early March. Additionally, several cases could not be traced to any imported cases and were suspected as sporadic local transmission. Twelve full viral genomes were determined in this study by Illumina sequencing either from virus isolates or directly from specimens, among which 5 originated from clustered infections. Phylogenetic tree analysis revealed that these sequences were in different clades, indicating that no major strain has been circulating in Taiwan. A deletion in open reading frame 8 was found in one isolate. Only a 4-nucleotide difference was observed among the 5 genomes from clustered infections.

genetics↗

bioRxiv2020

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

microbiology↗

bioRxiv2018

Predicting Intra- and Intertypic Recombination in Enterovirus 71

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that: (1) EV-A71 strain-type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (2) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase (RdRp); (3) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination suggesting conservation in mechanism(s); and (4) sequencing of intertypic recombinant genomes indicates that template-switching is by a mechanism that requires some sequence homology at the recombination junction and that the triggers for template-switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.

microbiology↗