Biology subjects

Hsu, F.-F.

Publications and source records attributed to Hsu, F.-F..

9 recordsLinked to original sources

bioRxiv2025

Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα-Dependent Fashion

BackgroundCept1 is essential for de novo phopholipogenesis and is impacted by diabetes. We previously demonstrated that conditional knockdown of Cept1 in the endothelium leads to reduced hindlimb angiogenesis and tissue recovery. We hypothesized that Cept1 may also be sufficient in promoting post-ischemic angiogenesis and recovery in the setting of diabetes. MethodsCEPT1 content was evaluated in peripheral arteries of patients with peripheral arterial disease (PAD), and with or without diabetes. An endothelial cell (EC)-specific Cept1 overexpression mouse model was developed (Cept1fl/flCre+) in adult C57BL6 mice. Murine aortae were harvested, for single-cell RNA sequencing (scRNA-seq), and unilateral hindlimb ischemia was used to evaluate angiogenesis in Cept1fl/flCre+ mice. Primary ECs were isolated and HUVECs transduced with Cept1 cDNA were developed, and evaluated using molecular assays, in vitro functional assays, and mass spectrometry. ResultsIn humans, arterial intima CEPT1 was elevated in the setting of PAD and diabetes, along with ACOX1, VEGF2R, p-Akt, and p-eNOS. In mice, scRNA-seq demonstrated that ECs with Cept1 overexpression were enriched with wound healing, angiogenesis, sprouting, and cell migration pathways. Diabetic Cept1fl/flCre+ mice had improved hind-limb perfusion and angiogenesis, and their aortic rings had increased ex vivo capillary sprouting. Cept1 overexpression in ECs significantly increased migration, tubule formation, and proliferation as predicted by scRNA-seq. Cept1 overexpression in ECs led to increased Ppar, Acox1, Vegfa, and Vegf2r. Similarly, treatment with siPpar, and inhibitors for PPAR (GW6471), VEGFR2 (ZM323881), Akt (LY294002), and eNOS (L-NAME) abrogated CEPT1-induced EC migration. ConclusionsCept1 overexpression promotes EC function and post-ischemic recovery. The impact of CEPT1 on ECs is at least in part dependent on p-Akt/p-eNOS angiogenic signaling and PPAR. Since CEPT1 is elevated in diseased human peripheral arterial tissue, these findings suggest that CEPT1 may be playing an important compensatory role in vascular recovery and reperfusion following ischemic injury in the setting diabetes. HighlightsO_LICEPT1 content is higher in the peripheral arteries of individuals with peripheral arterial disease (PAD) and type 2 diabetes. C_LIO_LICept1 over expression induces endothelial cell activation and function and enhances post-ischemia angiogenesis in vivo. C_LIO_LICEPT1 induces endothelial pAkt/p-eNOS signaling and VEGF-A production in a PPAR dependent fashion. C_LIO_LICEPT1 may be an important regenerative signal that is increased in the peripheral arteries in the setting of PAD. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/642511v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@dde6ecorg.highwire.dtl.DTLVardef@63c6c4org.highwire.dtl.DTLVardef@8e753dorg.highwire.dtl.DTLVardef@b314cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

The ACE-inhibitor drug captopril inhibits ACN-1 to control dauer formation and aging

The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in C. elegans, Drosophila, and rodents, but its mechanism is not well defined. Here we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril hypersensitive mutants. We identified a missense mutation that causes a partial loss-of-function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNAi promoted dauer larvae formation, suggesting acn-1 is a daf gene. Captopril-mediated lifespan extension xwas abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 control aging by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control. Summary StatementCaptopril and acn-1 control aging. By demonstrating they regulate dauer formation and interact with daf genes, including a new DAF-2(A261V) mutant corresponding to a human disease variant, we clarified the mechanism.

developmental biology↗

bioRxiv2023

Dual Membrane-Sspanning Anti-Sigma Factors Regulate Vesiculation in Gut Bacteroidota

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by Outer Membrane Vesicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). Our screening led us to the identification of a novel family of Dual Membrane-spanning Anti-sigma factors (Dma), which regulate OMV biogenesis in Bt. We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal {beta}-barrel embedded in the OM; its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

microbiology↗

bioRxiv2023

Ethanolaminephosphate cytidyltransferase is essential for survival, lipid homeostasis and stress tolerance in Leishmania major

Glycerophospholipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are vital components of biological membranes. Trypanosomatid parasites of the genus Leishmania can acquire PE and PC via de novo synthesis and the uptake/remodeling of host lipids. In this study, we investigated the ethanolaminephosphate cytidyltransferase (EPCT) in Leishmania major, which is the causative agent for cutaneous leishmaniasis. EPCT is a key enzyme in the ethanolamine branch of the Kennedy pathway which is responsible for the de novo synthesis of PE. Our results demonstrate that L. major EPCT is a cytosolic protein capable of catalyzing the formation of CDP-ethanolamine from ethanolamine-phosphate and cytidine triphosphate. Genetic manipulation experiments indicate that EPCT is essential in both the promastigote and amastigote stages of L. major as the chromosomal null mutants cannot survive without the episomal expression of EPCT. This differs from our previous findings on the choline branch of the Kennedy pathway (responsible for PC synthesis) which is required only in promastigotes but not amastigotes. While episomal EPCT expression does not affect promastigote proliferation under normal conditions, it leads to reduced production of ethanolamine plasmalogen or plasmenylethanolamine, the dominant PE subtype in Leishmania. In addition, parasites with epsiomal EPCT exhibit heightened sensitivity to acidic pH and starvation stress, and significant reduction in virulence. In summary, our investigation demonstrates that proper regulation of EPCT expression is crucial for PE synthesis, stress response, and survival of Leishmania parasites throughout their life cycle. AUTHOR SUMMARYIn nature, Leishmania parasites alternate between fast replicating, extracellular promastigotes in sand fly gut and slow growing, intracellular amastigotes in macrophages. Previous studies suggest that promastigotes acquire most of their lipids via de novo synthesis whereas amastigotes rely on the uptake and remodeling of host lipids. Here we investigated the function of ethanolaminephosphate cytidyltransferase (EPCT) which catalyzes a key step in the de novo synthesis of phosphatidylethanolamine (PE) in Leishmania major. Results showed that EPCT is indispensable for both promastigotes and amastigotes, indicating that de novo PE synthesis is still needed at certain capacity for the intracellular form of Leishmania parasites. In addition, elevated EPCT expression alters overall PE synthesis and compromises parasites tolerance to adverse conditions and is deleterious to the growth of intracellular amastigotes. These findings provide new insight into how Leishmania acquire essential phospholipids and how disturbance of lipid metabolism can impact parasite fitness.

microbiology↗

bioRxiv2022

Inositol Phosphorlyceramide Synthase null Leishmania major (Δipcs-) are viable and virulent in animal infections where salvage of host sphingomyelin predominates

Many pathogens synthesize inositolphosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate, and the divergence between IPCS and mammalian sphingolipid synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lacking de novo sphingolipid synthesis ({Delta}spt2-) and sphingolipids entirely survive and remain virulent, as infective amastigotes salvage host sphingolipids and continue to produce IPC. To further understand the role of IPC, we generated null IPCS mutants in L. major ({Delta}ipcs-). Unexpectedly and unlike fungi where IPCS is essential, {Delta}ipcs- was remarkably normal in culture and highly virulent in mouse infections. Both IPCS activity and IPC were absent in {Delta}ipcs- promastigotes and amastigotes, arguing against an alternative route of IPC synthesis. Notably, salvaged mammalian sphingomyelin (SM) was highly abundant in purified amastigotes from both WT and {Delta}ipcs-, and salvaged SLs could be further metabolized into IPC. SM was about 7-fold more abundant than IPC in WT amastigotes, establishing that SM is the dominant amastigote SL, thereby rendering IPC partially redundant. These data suggest that SM salvage likely plays key roles in the survival and virulence of both WT and {Delta}ipcs- parasites in the infected host, confirmation of which will require the development of methods or mutants deficient in host SL/SM uptake in the future. Our findings call into question the suitability of IPCS as a target for chemotherapy, instead suggesting that approaches targeting SM/SL uptake or catabolism may warrant further emphasis.

microbiology↗

bioRxiv2022

An Efficient Approach for SARS-CoV-2 Monoclonal Antibody Production via Modified mRNA-LNP Immunization

Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult to express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies. In addition, the mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.

immunology↗

bioRxiv2022

Broadly neutralizing antibodies against Omicron variants of SARS-CoV-2 derived from mRNA-lipid nanoparticle-immunized mice

The COVID-19 pandemic continues to threaten human health worldwide, as new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged. Currently, the predominant circulating strains around the world are Omicron variants, which can evade many therapeutic antibodies. Thus, the development of new broadly neutralizing antibodies remains an urgent need. In this work, we address this need by using the mRNA-lipid nanoparticle immunization method to generate a set of Omicron-targeting monoclonal antibodies. Five of our novel K-RBD-mAbs show strong binding and neutralizing activities toward all SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron). Notably, the epitopes of these five K-RBD-mAbs are overlapping and localized around K417 and F486 of the spike protein receptor binding domain (RBD). Chimeric derivatives of the five antibodies (K-RBD-chAbs) neutralize Omicron sublineages BA.1 and BA.2 with low IC50 values that range from 5.7 to 12.9 ng/mL. Additionally, we performed antibody humanization on a broadly neutralizing chimeric antibody to create K-RBD-hAb-62, which still retains excellent neutralizing activity against Omicron. Our results collectively suggest that these five therapeutic antibodies may effectively combat current and emerging SARS-CoV-2 variants, including Omicron BA.1 and BA.2. Therefore, the antibodies can potentially be used as universal neutralizing antibodies against SARS-CoV-2.

immunology↗

bioRxiv2020

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress in Leishmania major

Lathosterol oxidase (LSO) catalyzes the formation of C5-C6 double bond in the synthesis of various types of sterols in mammals, fungi, plants and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C5-C6 desaturation, we generated a LSO-null mutant line (lso-) in Leishmania major, the causative agent for cutaneous leishmaniasis. Lso- parasites lacked the ergostane-based sterols commonly found in wild type L. major and instead accumulated equivalent sterol species without the C5-C6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso- mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alteration in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C5-C6 double bond is vital for the structure of sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCESterols are essential membrane components in eukaryotes and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. Lathosterol oxidase (aka sterol C5-desaturase) is required for the formation of C5-C6 double bond in the sterol core structure in mammals, fungi, protozoans, plants and algae. Functions of this C5-C6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that the C5-C6 double bond is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.

microbiology↗

bioRxiv2019

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, the L240A mutant known to decrease ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

biophysics↗