Biology subjects

Lee, G. Y.

Publications and source records attributed to Lee, G. Y..

15 recordsLinked to original sources

bioRxiv2026

Salmonella Typhi asparaginase-dependent activation of GCN2 promotes bacterial killing in murine macrophages

Many intracellular pathogens stimulate host cell stress by directly or indirectly causing an imbalance in host nutrients; depletion of amino acid pools in particular can act as a danger signal to infected cells. Using a restrictive host model of Salmonella enterica serovar Typhi (S. Typhi) infection, we identify early induction the integrated stress response (ISR) by viable bacteria, but not heat-killed bacteria. Genetic deletion of the amino acid sensing ISR kinase GCN2 (also known as EIF2AK4) prevented early ISR activation during S. Typhi infection, and murine macrophages lacking GCN2 show impaired bacterial clearance and decreased cytokine output. Supplementation of wildtype C57BL/6 murine macrophages with only the non-essential amino acid asparagine was sufficient to suppress S. Typhi-induced ISR activation and deletion of S. Typhi ansB, encoding an asparaginase, prevented ISR activation during infection. Pharmacological inhibition of mammalian target of rapamycin (mTOR), the other major amino acid sensing pathway in eukaryotic cells, prevented GCN2 activation and ISR induction in murine macrophages, indicating an upstream role for mTOR in signaling to GCN2. These findings suggest a role for the ISR in macrophage innate immune responses to S. Typhi infection and highlight a potential difference in nutrient-dependent signaling between the S. Typhi-susceptible human host and the restrictive murine host centered around asparagine, mTOR, and GCN2.

immunology↗

bioRxiv2025

The Phosphate-Specific Transport System Gene pstA1 Contributes to Rifampin Tolerance in Mycobacterium tuberculosis

Tuberculosis (TB) caused an estimated 10.8 million new cases and 1.25 million deaths in 2023. Antibiotic tolerance, the ability of bacteria to survive exposure to bactericidal antibiotics without genetic resistance mutations, contributes to the need for prolonged treatment. Targeting antibiotic tolerance mechanisms could promote accelerated clearance of Mycobacterium tuberculosis (Mtb), thereby improving medical adherence. Our previous forward genetic screen for rifampin tolerance genes identified pstA1, which is involved in phosphate-specific import. The ABC-type transporter permease PstA1 has previously been implicated in Mtb virulence, as mutants lacking this gene exhibit defective survival during phosphate limitation in vitro, in infected macrophages, and in immunocompetent mice. The minimum inhibitory concentration (MIC) of rifampin was not altered in a pstA1 deletion mutant ({Delta}pstA1), suggesting this difference in susceptibility is not due to antibiotic resistance. Consistent with a role in rifampin tolerance, time-kill assays revealed a shift in the mean duration of killing (2-log reduction) from 1.6 days in wild-type to 1.0 day in {Delta}pstA1, and complementation partially restored this phenotype. We found that pstA1 is specifically required for Mtb survival in the absence of exogenous inorganic phosphate and is important for adaptation to growth in culture without detergent, and within macrophages in an interferon-{gamma}-dependent manner. Differential expression analysis revealed that {Delta}pstA1 exhibited substantial transcriptional reprogramming with 58 differentially expressed genes, including altered expression of metabolic, DNA damage repair, and secretory pathways. PstA1 represents a novel drug target, and inhibitors could serve as adjunctive therapies to shorten treatment times, reducing opportunities for drug resistance emergence. ImportanceTuberculosis remains one of the worlds deadliest infectious diseases, causing over a million deaths annually. Current treatment requires months of antibiotic therapy, and poor adherence to these lengthy regimens contributes to the emergence of drug-resistant strains that are increasingly difficult to treat. A major barrier to shorter treatment is antibiotic tolerance, which allows bacteria to survive drug exposure without genetic resistance mutations. This study identifies the phosphate transport component PstA1 as a critical factor enabling Mycobacterium tuberculosis to tolerate the frontline antibiotic rifampin. Bacteria lacking pstA1 are eliminated more rapidly by rifampin, demonstrating that this transporter actively promotes bacterial survival during treatment. These findings suggest that drugs targeting PstA1 could be combined with standard antibiotics to accelerate bacterial clearance, potentially shortening treatment duration and improving patient adherence. Such adjunctive therapies targeting tolerance mechanisms represent a promising strategy to combat tuberculosis and reduce the global burden of drug-resistant disease.

microbiology↗

bioRxiv2025

Stabilization of the trimeric pre-fusion structures of influenza H1 and H9 hemagglutinins by mutations in the stem helices

Stabilizing the pre-fusion structures of antigenic proteins can enhance the effectiveness of antiviral vaccines. The pre-fusion form of hemagglutinin (HA) from the influenza virus typically adopts a stable trimeric structure. However, the recombinant ectodomain of HA from the A/California/04/2009 (H1N1) influenza virus formed a monomer in solution rather than the expected trimer. To promote trimer formation in the pre-fusion conformation, we redesigned five amino acid residues in the stem region of HA that are involved in trimerization. The engineered HA protein formed a stable trimer at both pH 8.0 and pH 5.5. Additionally, the thermal stability of the modified protein improved, as indicated by an approximately ten-degree increase in its denaturation temperature. Cryo-EM analysis at 2.2 [A] resolution confirmed that the mutant HA protein adopted the pre-fusion structure. Furthermore, the stabilized mutant exhibited enhanced immunogenicity in mice. We applied the same optimization strategy to the HA proteins from A/Malaysia/1706215/2007 (H1N1) and A/swine/Hong Kong/2106/98 (H9N2). These engineered proteins demonstrated increased thermal stability and retained a trimeric pre-fusion structure, as confirmed by cryo-EM analysis. Extending this optimization strategy to the equivalent five residues in hemagglutinins from six additional group 1 influenza viruses successfully stabilized their trimeric structures.

biophysics↗

bioRxiv2025

Cholesterol-Responsive Interaction of NFE2L1-INSIG1 Controls VLDL Secretion and MASH Pathogenesis

Cholesterol overload drives metabolic dysfunction-associated steatohepatitis (MASH). While the liver maintains homeostasis by exporting cholesterol via very low-density lipoprotein (VLDL) secretion, this process is paradoxically suppressed under cholesterol excess by insulin-induced gene 1 (INSIG1), which inhibits sterol regulatory element-binding protein 1 (SREBP1) activity. How VLDL secretion persists during cholesterol overload to prevent lipotoxicity remains unresolved. We identify a cholesterol-responsive interaction between nuclear factor erythroid 2 related factor-1 (NFE2L1) and INSIG1 that sustains cholesterol balance. Hepatic NFE2L1 deficiency elevates INSIG1 levels, suppressing SREBP1 activation and impairing VLDL secretion, leading to cholesterol accumulation and liver injury. Mechanistically, NFE2L1 binds to INSIG1 via its N-terminal homology box 2 (NHB2) domain, with cholesterol enhancing this interaction to drive INSIG1 degradation and SREBP1 activation. In NFE2L1-deficient mice, wild-type NFE2L1 restores SREBP1 activity and VLDL secretion, while the NHB2-deleted mutant ({Delta}NHB2) fails. Lipidomics reveal that NFE2L1 deficiency reduces serum triglyceride composition, restored exclusively by wild-type NFE2L1. In a murine MASH model, NFE2L1 overexpression activates SREBP1/2, enhances cholesterol secretion, and alleviates liver injury, inflammation and fibrosis, without elevating atherogenic lipoproteins due to compensatory LDL receptor upregulation. Our findings resolve the paradox of cholesterol-driven VLDL secretion and establish the NFE2L1-INSIG1 axis as a therapeutic target for metabolic diseases.

physiology↗

bioRxiv2025

Garcinone C suppressed the proliferation of gastric cancer cells through regulating Hedgehog signaling

Garcinone C, a xanthone derived from Garcinia mangostana L, possesses antioxidant and anti-cancer effects. However, its role in gastric cancer remains unexplored. This study aimed to investigate the effects of garcinone C on gastric cancer cell proliferation and its underlying mechanism. We found that garcinone C suppressed gastric cancer cell growth by inducing G0/G1 arrest and apoptosis in a dose-dependent manner. Furthermore, garcinone C downregulated G0/G1 phase markers Cyclin D1 and p21, as well as apoptosis markers Bax, Bcl-2, cleaved-PARP, and c-caspase 3. Following the previous evidence demonstrated that aberrant Hedgehog (Hh) signaling is implicated in gastric cancer development, we confirmed that inhibiting Gli1/2 reduced the growth of AGS and MKN74 cells. Furthermore, garcinone C exerted similar effects to Gant61, inhibiting Hh signaling by reducing Gli1/2 levels and blocking their nuclear translocation. These findings suggest that garcinone C inhibits gastric cancer proliferation via the Hh signaling, indicating its potential as a therapeutic agent for treating gastric cancer.

cancer biology↗

bioRxiv2024

FABP4 as a Therapeutic Host Target Controlling SARS-CoV2 Infection

Host metabolic fitness is a critical determinant of infectious disease outcomes. Obesity, aging, and other related metabolic disorders are recognized as high-risk disease modifiers for respiratory infections, including coronavirus infections, though the underlying mechanisms remain unknown. Our study highlights fatty acid-binding protein 4 (FABP4), a key regulator of metabolic dysfunction and inflammation, as a modulator of SARS-CoV-2 pathogenesis, correlating strongly with disease severity in COVID-19 patients. We demonstrate that loss of FABP4 function, by genetic or pharmacological means, reduces SARS-CoV2 replication and disrupts the formation of viral replication organelles in adipocytes and airway epithelial cells. Importantly, FABP4 inhibitor treatment of infected hamsters diminished lung viral titers, alleviated lung damage and reduced collagen deposition. These findings highlight the therapeutic potential of targeting host metabolism in limiting coronavirus replication and mitigating the pathogenesis of infection.

microbiology↗

bioRxiv2024

Single missense mutations in Vi capsule synthesis genes confer hypervirulence to Salmonella Typhi

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control the length or acetylation of Vi is enough to create different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper-capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo-capsule variants have primarily been identified in Africa, whereas the hyper-capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.

microbiology↗

bioRxiv2023

Co-Targeting IL-6 and EGFR signaling for the treatment of schwannomatosis and associated pain

Patients with Schwannomatosis (SWN) overwhelmingly present with intractable, debilitating chronic pain. There are no effective therapies to treat SWN. The drivers of pain response and tumor progression in SWN are not clear. The pain is not proportionally linked to tumor size and is not always relieved by tumor resection, suggesting that mechanisms other than mechanical nerve compression exist to cause pain. SWN research is limited by the lack of clinically-relevant models. Here, we established novel patient-derived xenograft (PDX) models, dorsal root ganglia (DRG) imaging model, and combined with single-cell resolution intravital imaging and RNASeq, we discovered: i) schwannomas on the peripheral nerve cause macrophage influx into the DRG, via secreting HMGB1 to directly stimulate DRG neurons to express CCL2, the key macrophage chemokine, ii) once recruited, macrophages cause pain response via overproduction of IL-6, iii) IL-6 blockade in a therapeutic setting significantly reduces pain but has modest efficacy on tumor growth, iv) EGF signaling is a potential driver of schwannoma growth and escape mechanism from anti-IL6 treatment, and v) combined IL-6 and EGFR blockade simultaneously controlled pain and tumor growth in SWN models. Our findings prompted the initiation of phase II clinical trial (NCT05684692) for pain relief in patients with SWN.

neuroscience↗

bioRxiv2022

Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with body mass index and obesity-related pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with FABP4 deletion in cells known to express the gene; adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was only reduced by [~]25% in Adipo-KO mice, whereas Endo-KO mice showed [~]75% decreases compared to wildtype controls. In contrast, Adipo-KO mice exhibited [~]62% reduction in FABP4 responses to lipolysis, while there was minimal reduction in Endo-KO mice, indicating that adipocytes are the main FABP4 source in lipolysis. We did not detect any myeloid cell contribution to circulating FABP4. Surprisingly, despite the nearly intact FABP4 responses, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/511807v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1b2d566org.highwire.dtl.DTLVardef@1d82083org.highwire.dtl.DTLVardef@3e9726org.highwire.dtl.DTLVardef@1357648_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

ATGL is differentially required for adipocyte FABP4 secretion in vivo and ex vivo

Fatty acid binding protein 4 (FABP4) is linked with the pathogenesis of metabolic diseases, including diabetes and cardiovascular disease in both mice and humans. It has also been demonstrated that the levels of hormonal FABP4 are strongly associated with obesity, and secretion is stimulated under conditions of fasting and lipolysis both in vivo and in vitro. Here, we utilized adipocyte-specific deficiency of adipose triglyceride lipase (ATGL) in a mouse model (ATGLAdpKO) to evaluate the regulation of FABP4 secretion by lipolytic signals in the absence of actual lipolysis in vivo. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of ATGL, and from adipose tissue explants from ATGLAdpKO mice. Unexpectedly, upon activation beta-adrenergic receptors, ATGLAdpKO mice exhibited significantly higher levels of circulating FABP4 as compared to ATGLfl/fl controls in vivo, with no corresponding increase in non-esterified free fatty acids or glycerol, confirming the lack of lipolysis. We also generated an additional model with adipocyte-specific deletion of FABP4 in the background of ATGLAdpKO mice (ATGL/FABP4AdpKO or DKO) to evaluate the cellular source of circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the elevated FABP4 hormone levels in the ATGLAdpKO mice were indeed from the adipocytes. ATGLAdpKO mice did not exhibit an increase in insulin secretion upon stimulation of lipolysis, but had a normal insulin response to glucose injection along with increased FABP4 secretion, suggesting the elevated FABP4 secretion is not due to lack of insulin. Inhibition of sympathetic signaling during lipolysis using hexamethonium significantly reduced FABP4 secretion in ATGLAdpKO mice compared to controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for stimulated in vivo FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.

physiology↗

bioRxiv2022

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

biophysics↗

bioRxiv2022

Sex and age differences in cognitive bias and neural activation in response to cognitive bias

Cognitive symptoms of depression, including negative cognitive bias, are more severe in women than in men. Current treatments to reduce negative cognitive bias are not effective and sex differences in the neural activity underlying cognitive bias may play a role. Here we examined sex and age differences in cognitive bias and functional connectivity in a novel paradigm. Male and female rats underwent an 18-day cognitive bias procedure, in which they learned to discriminate between two contexts (shock paired context A, no-shock paired context B), during either adolescence (postnatal day (PD 40)), young adulthood (PD 100), or middle-age (PD 210). Cognitive bias was measured as freezing behaviour in response to an ambiguous context (context C), with freezing levels akin to the shock paired context coded as negative bias. All animals learned to discriminate between the two contexts, regardless of sex or age. However, adults (young adults, middle-aged) displayed a greater negative cognitive bias compared to adolescents, and middle-aged males had a greater negative cognitive bias than middle-aged females. Females had greater neural activation of the nucleus accumbens, amygdala, and hippocampal regions to the ambiguous context compared to males, and young rats (adolescent, young adults) had greater neural activation in these regions compared to middle-aged rats. Functional connectivity between regions involved in cognitive bias differed by age and sex, and only adult males had negative correlations between the frontal regions and hippocampal regions. These findings highlight the importance of examining age and sex when investigating the underpinnings of negative cognitive bias and lay the groundwork for determining what age- and sex-specific regions to target in future cognitive bias studies. HighlightsO_LIMiddle-aged males had a greater negative cognitive bias than middle-aged females C_LIO_LIAdult rats displayed a greater negative cognitive bias compared to adolescents C_LIO_LIGreater neural activity in females than males in limbic and reward regions C_LIO_LIGreater role of the frontal cortex activation in the cognitive bias of adults C_LIO_LIFunctional connectivity in response to cognitive bias differed by age and sex C_LI

neuroscience↗

bioRxiv2021

The molecular basis of extensively drug-resistant Salmonella Typhi isolates from pediatric septicemia patients

Sepsis is a syndromic response to infections and is becoming an emerging threat to the public health sector, particularly in developing countries. Salmonella Typhi (S. Typhi), the cause of typhoid fever, is one primary cause of pediatric sepsis in typhoid endemic areas. Extensively drug-resistant (XDR) S. Typhi is more common among pediatric patients, which is responsible for over 90% of the reported XDR typhoid cases, but the majority of antibiotic resistance studies available have been carried out using S. Typhi isolates from adult patients. Here, we characterized XDR S. Typhi isolates from a medium size cohort of pediatric typhoid patients to determine their antibiotic-resistance-related gene signatures associated with common treatment options to typhoid fever patients. This study informs the molecular basis of antibiotic-resistance among recent S. Typhi isolates from pediatric septicemia patients, therefore providing insights into the development of molecular detection methods and control strategies for XDR S. Typhi.

microbiology↗

bioRxiv2021

Novel Paju Apodemus Paramyxovirus 1 and 2, Harbored by Apodemus agrarius in The Republic of Korea

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a potential threat to public health. Currently, 78 species and 17 genera of paramyxoviruses are classified and harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Jeilongvirus has been proposed as a novel paramyxovirus genus containing J-, Beilong, and Tailam viruses, found in wild rodents. Using RT-PCR, 824 Apodemus agrarius individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was detected in 108 (13.1%) rodents captured at 14 trapping sites in Korea. We first present two genetically distinct novel paramyxoviruses (genus Jeilongvirus), Paju Apodemus paramyxoviruses 1 (PAPV-1) and 2 (PAPV-2), from A. agrarius. Six PAPV strains were completely sequenced using next-generation and Sanger sequencing. PAPV-1 genome comprised 19,716 nucleotides, with eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'), whereas PAPV-2 genome contained 17,475 nucleotides, with seven genes (3'-N-P/V/C-M-F-TM-G-L-5'). The disparity between PAPV-1 and -2 revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogenies of PAPV-1 and -2 belong to distinct genetic lineages of Jeilongvirus despite being from the same natural host. PAPV-1 clustered with Beilong and Tailam viruses, while PAPV-2 formed a genetic lineage with Mount Mabu Lophuromys virus-1. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. Therefore, this study provides profound insights into the molecular epidemiology, virus-host interactions, and zoonotic potential of novel rodent-borne paramyxoviruses. ImportanceParamyxoviruses are a critical public health and socio-economic burden to humans. Rodents play a crucial role in transmitting pathogens to humans. In the last decade, novel paramyxoviruses have been discovered in different rodents. Here, we found that Apodemus agrarius harbored two distinct genotypes of the novel paramyxoviruses, Paju Apodemus paramyxovirues 1 (PAPV-1) and 2 (PAPV-2), possessing unique genome structures that are responsible for encoding TM and G proteins of different sizes. In addition, PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III IFNs, ISGs, and proinflammatory cytokines. Thus, this study provides significant insights into molecular prevalence, virus-host interactions of paramyxoviruses. These observations raise the awareness of physicians and scientists about the emergence of new rodent-borne paramyxoviruses.

microbiology↗

bioRxiv2020

Dendrimer-targeted immunosuppression of microglia reactivity super-accelerates photoreceptor regeneration in the zebrafish retina

Muller glia (MG) function as injury-induced retinal stem cells in zebrafish but not mammals. Insights from zebrafish, however, have been used to stimulate limited regenerative responses from mammalian MG. Microglia/macrophages regulate MG stem cell activity in the chick, zebrafish and mouse. We previously showed that dexamethasone can accelerate retinal regeneration in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation may therefore enhance the regenerative potential of human MG. Nanoparticle-based immunomodulation is an emerging field with immense therapeutic potential. Here, we investigated how regeneration-enhancing dexamethasone treatments alter microglia behavior and how dendrimer-based targeting of dexamethasone to reactive microglia impact retinal regeneration kinetics. Intravital time-lapse imaging revealed specific dexamethasone-induced changes in microglia reactivity. Dendrimer-conjugated dexamethasone treatments resulted in: 1) decreased toxicity, 2) selective targeting of reactive microglia and, 3) "super-accelerated" retinal regeneration kinetics. These data support the use of dendrimer-based drug formulations for modulating microglia reactivity in degenerative disease contexts, especially as therapeutic strategies for promoting regenerative responses to neuronal cell loss.

neuroscience↗