Biology subjects

Willard, B.

Publications and source records attributed to Willard, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Multi-Omic Analyses of Dietary Fatty Acid-Microbe-Host Interactions Reveal Metaorganismal Lipid Metabolic Crosstalk Impacting Cardiometabolic Disease

Following a meal, our gut microbiome and human cells collaborate via metaorganismal metabolic circuits to produce diverse nutrient metabolites that systemically circulate to influence health and disease. Although there are now several examples of bacterial fiber-, amino acid-, and micronutrient-derived metabolites impacting cardiometabolic disease, very little is known in regards to how diet-microbe-host interactions impact lipid homeostasis. Here we address this by defining dietary fatty acid substrate availability in germ-free versus conventionally-raised mice coupled to deep multi-omic metabolic phenotyping. Our data demonstrate that the effects of dietary saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) on the host lipidome, transcriptome, proteome and metabolome are uniquely impacted by resident microbiota. Also, the hepatic levels of both pro-inflammatory and pro-resolving lipid mediators are strongly influenced by dietary fatty acid-microbe interactions. This study presents a unique resource to the nutrition and metabolism research community to advance our understanding of metaorganismal lipid metabolism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/705588v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@4fc7e3org.highwire.dtl.DTLVardef@1cc1dc8org.highwire.dtl.DTLVardef@1b7648dorg.highwire.dtl.DTLVardef@12a8088_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

biochemistry↗

bioRxiv2025

BCL-XL Dependence is a Subtype Agnostic Actionable Feature of Difficult-to-Treat Kidney Cancers

The BCL-XL anti-apoptotic protein is a clear cell Renal Cell Carcinoma (ccRCC) dependency; however, the mechanism of this dependence and its relevance in other aggressive kidney cancer contexts, including metastatic and/or rare RCC subtypes [e.g., Fumarate Hydratase (FH)-deficient and sarcomatoid RCCs], is unknown. Computational predictions, using a machine learning model trained on the human RCC TCGA dataset, and cell-based validations, confirmed BCL-XL dependence in all RCC subtypes. Remarkably, cell state changes, anoikis programs, inflammatory state, and metabolic perturbations (e.g., fumarate production in FH-deficient RCCs) independently conferred increased BCL-XL dependence. Correlation studies revealed that increased AMPK isoform 2 (PRKAA2) expression is a kidney-specific biomarker of BCL-XL dependence. Indeed, pharmacological AMPK activation sensitized RCCs to BCL-XL blockade. Finally, using functional studies, we developed a multivariate model that accurately predicted BCL-XL dependence in RCC. Our studies offer biomarkers for patient stratification and credential BCL-XL as a subtype agnostic vulnerability in difficult-to-treat RCCs.

cancer biology↗

bioRxiv2025

Tyrosine phosphorylation and the inhibitory C-terminal SAM domain moderately affect transient interactions in a EphA2 cytoplasmic fragment in solution: A combined experimental and molecular modeling study.

Eph receptors, the largest family of receptor tyrosine kinases (RTKs), orchestrate diverse developmental processes, including axon guidance, tissue patterning, and cell positioning, while also contributing to adult functions such as synaptogenesis and tissue homeostasis. Proteolytic cleavage of Eph receptors by neurodegeneration-associated proteases generates a membrane-detached intracellular region (ICR) fragment whose structural organization and signaling remain poorly understood. Here, we characterize a model for such a fragment from EphA2 and discover that the juxtamembrane region, the kinase domain, and the C-terminally linked sterile alpha motif (SAM) domain comprise a configurational ensemble of domain contacts and orientations which inhibit the intermolecular interactions and tyrosine cross-phosphorylation. Recombinant EphA2 ICR expressed in E. coli retains catalytic activity and undergoes autophosphorylation at multiple tyrosine residues which have previously been identified in eukaryotic systems. Phosphorylation weakens intramolecular interactions, as measured by microscale thermophoresis, indicating a phosphorylation-dependent remodeling of the ICR. While AlphaFold2-Multimer (AF2M) and Alphafold3 (AF3) predictions as well as available crystal structures provide only limited insight into these configurational states, coarse-grained molecular dynamics simulations using the Martini 3 force field reveal highly dynamic and predominantly transient interactions between the kinase and SAM domains that cluster at two discrete regions on the kinase surface. NMR spectroscopy of isotopically labeled EphA2 SAM suggests non-canonical intramolecular contacts that preserve the canonical SAM interaction interface for binding of partner proteins. Addition of the SHIP2 SAM domain promotes formation of the canonical EphA2 and SHIP2 SAM complex while also revealing previously unrecognized interactions of the kinase domain on the SHIP2 SAM domain. Collectively, these findings establish the EphA2 ICR as a dynamic, phosphorylation-responsive signaling module in which transient intramolecular interactions regulate accessibility of several interfaces for binding. Thus, the proteolytically released EphA2 intracellular fragment is likely to have distinct characteristics in cancer and neurodegenerative disease, compared to the membrane bound form in the full receptor.

biophysics↗

bioRxiv2025

dCas9 targeted proteome profiling reveals p300-mediated reciprocal regulation of SMAD and SP1 as a driver of GM2 synthase transcription in renal cell carcinoma

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore is established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasnt clear. This study demonstrated that the histone acetyl transferase (2), p300 acts as a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD 2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

molecular biology↗

bioRxiv2025

MCL1 may not mediate chemoresistance

The anti-apoptotic BCL2 family member MCL1 is overexpressed in many cancers and has been linked to chemoresistance. Unlike other BCL2 family members, MCL1 displays both well-defined mitochondrial anti-apoptotic activities and also emerging nuclear functions. Prior reports suggest that MCL1 enters the nucleus during chemotherapy and promotes chemoresistance by influencing cell cycle progression and DNA repair. These nuclear roles of MCL1, however, remain poorly characterized. Using a newly validated monoclonal antibody across several cell lines and treatments, we find no evidence that MCL1 enhances chemoresistance or preferentially accumulates in the nucleus after drug exposure. Proximity biotinylation identified novel nuclear MCL1 interactors but did not recover previously reported DNA repair or cell cycle partners. Thus, while MCL1 does reach the nucleus and interact with nuclear proteins, our data do not support a role for MCL1 in chemoresistance. Further work is needed to clarify the functional significance of nuclear MCL1. SIGNFIGANCEPrevious studies have implicated MCL1 in promoting chemoresistance via interactions with DNA repair machinery in the nucleus. Using a validated, monoclonal anti-MCL1 antibody, we were unable to replicate these data. We report that MCL1 neither confers chemoresistance, translocates to the nucleus during chemotherapy treatment, nor interacts with DNA repair proteins in live cancer cells.

cell biology↗

bioRxiv2025

Inhibition of KDEL receptors remodels the tumor microenvironment for robust T cell independent tumor regression

Tumor immunotherapy is supported by low-grade inflammatory conditions at the microenvironment, triggered by immunogenic cell death (ICD). However, ICD is dampened when tumors acquire resistance, affecting immune recognition. KDEL receptors (KDELRs), through a retrograde Golgi-to-ER transport, prevent spontaneous secretion of KDEL proteins. We report that inhibition of a single KDELR in a minor fraction of tumor cells, primarily KDELR2, provokes robust infiltration of macrophages and neutrophils into the tumor microenvironment, resulting in a complete regression of both immunogenic and non-immunogenic tumors independently of T cells. Importantly, in the course of regression, anti-tumor T cells are primed, conferring protection against a second challenge. Recapitulated by intratumoral delivery of siDKELR2 utilizing lipid nanoparticles, we implicate KDELR2 as a target to unleash an unusual robust innate immune response, which represents a tractable approach to initiate an adaptive response downstream, bypassing conventional ICD-inducing therapies. We propose KDELR targeting as a strategy to improve immunotherapy across tumor types, including "cold" tumors resistant to T cell-based immunotherapies.

immunology↗

bioRxiv2025

PML1-Mediated Feedforward Loop Through PI3K and MAPK Axes Drives Endocrine Resistance

Treatment of estrogen receptor-positive (ER+) breast cancer is significantly hindered by endocrine resistance. We identified PML1 as a key therapeutic entity that can be targeted to overcome resistance. Endocrine-resistant breast cancer cells share three key characteristics: elevated PML1 protein levels, enhanced activity of PI3K, MAPK, or both signaling pathways, and reduced ER activity. We developed a PML1 gene signature that predicts poor prognosis and correlates strongly with PI3K, MAPK, and endocrine resistance gene signatures, as evident by cellular studies, scRNA-seq analysis, and spatial transcriptomics of endocrine therapy-treated tumors. This signature is present in endocrine-resistant breast cancer cells harboring the Y537S ER mutation. We consistently demonstrate high PML1 protein levels across cells resistant to various treatments, including 4-hydroxytamoxifen, fulvestrant, elacestrant, and CDK4/6 inhibitors. Furthermore, treatments with these therapeutic agents or knockdown of ESR1 mRNA also increase PML1 protein levels. Mechanistically, we show that ER inhibition through fulvestrant treatment activates PI3K and MAPK signaling, which enhance PML1 protein stability and synthesis. PML1 then drives a feedforward loop by stimulating the expression of cytokine and growth factor mRNAs, including CCL5 and HBEGF, further amplifying PI3K and MAPK signaling. Consequently, in endocrine-resistant cells, endocrine therapies, while inactivating ER, paradoxically reinforce this loop through increased PI3K/MAPK activation and PML1 protein accumulation, ultimately compromising therapeutic efficacy. Finally, we demonstrated that arsenic trioxide, an FDA-approved, PML-reducing drug, effectively disrupts this feedforward loop, offering a promising strategy for treating resistant metastatic breast cancer. STATEMENT OF SIGNIFICANCEEndocrine resistance remains a major obstacle in treating estrogen receptor-positive metastatic breast cancer. Our study identifies PML1 as a central mediator of this resistance, revealing how it maintains a self-reinforcing signaling network through PI3K and MAPK pathways by enhancing the production of cytokines and growth factors. The clinical significance of our findings is threefold: we establish PML1 as a biomarker for therapy resistance, demonstrate its mechanistic role in treatment failure, and show that FDA-approved arsenic trioxide can disrupt PML1-driven resistance. These insights provide a direct path to clinical translation, as combining arsenic trioxide with existing therapies could benefit patients with limited treatment options.

cancer biology↗

bioRxiv2025

Gut Microbe-Derived N-Acyl Serinol Lipids Shape Host Postprandial Metabolic Homeostasis

Although strong evidence links the gut microbiome to metabolic disease, the mechanisms linking microbiota to hormonal and metabolic responses to food are not well understood. After a meal, gut bacteria produce a wide array of small molecule, protein, and lipid metabolites originating from bacterial sources. Annotating physiological function to select gut microbe-derived metabolites is critical to understanding diet-microbe-host interactions, and to developing microbiome-inspired therapies to improve human health. Here, we have investigated the role of a poorly annotated class of gut microbiome-derived lipids called N-acyl amides in postprandial metabolic physiology. Here we show both bacterial overproduction and provision of exogenous N-acyl amides reorganize host hormone-driven metabolic transition after a meal. Moreover, N-acyl amides exert broad effects on the meal- and circadian-related reorganization of gene expression, metabolic hormones, and gut microbiome composition. Collectively, these results demonstrate that microbiota-derived N-acyl amides play a physiologic role in postprandial metabolic homeostasis in the host.

physiology↗

bioRxiv2025

Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and skin phenotypes

Weill-Marchesani syndrome (WMS) is characterized by severe short stature, short hands and feet (brachydactyly), joint contractures, tight skin, and heart valve, eye, and skin anomalies. Whereas recessive WMS is caused by mutations in ADAMTS10, ADAMTS17, or LTBP2, dominant WMS is caused by mutations in FBN1 (encoding fibrillin-1). Since bone growth is driven by chondrocyte proliferation and hypertrophy in the growth plates, the genetics of WMS suggests that the affected ECM proteins act within the same pathway to regulate chondrocyte and growth plate function. Here, we investigated the role of the secreted ADAMTS proteases ADAMTS10 and ADAMTS17 in growth plate function and ECM formation. We generated Adamts10;Adamts17 double knockout (DKO) mice, which showed significant postnatal lethality compared to single Adamts10 or Adamts17 KO mice. Importantly, we observed severe bone shortening DKO mice, which correlated with a narrower hypertrophic zone in their growth plates. ADAMTS17 substrates identified by N-terminomics and yeast two-hybrid screening identified the ECM proteins fibronectin and collagen VI (COL6). However, validation experiments did not reveal direct proteolysis of either fibronectin or COL6 by ADAMTS17. We then investigated ECM formation in primary ADAMTS10- and ADAMTS17-deficient skin fibroblasts and observed compromised fibronectin deposition concomitant with aberrant intracellular accumulation of fibrillin-1. These findings support a role for ADAMTS17 in ECM protein secretion and assembly. Collectively, our data suggest that ADAMTS10 and ADAMTS17 regulate bone growth by regulating chondrocyte hypertrophy or hypertrophic chondrocyte turnover. Mechanistically, ADAMTS17 appears to be a critical regulator of ECM protein secretion or pericellular matrix assembly, whereas ADAMTS10 likely modulates ECM formation at later stages, possibly regulating the spatio-temporal deposition of fibrillin isoforms.

cell biology↗

bioRxiv2024

TLR9 signaling requires ligand-induced phosphorylation of two specific tyrosine residues by EGFR and Syk

Toll-like receptors (TLRs) are transmembrane proteins required for recognizing microbial components or cellular danger signals to activate intracellular signaling pathways leading to induction of anti-microbial and inflammatory genes. Inactive TLRs require ligand-induced activation to recruit adaptor proteins, e.g., MyD88, to trigger the synthesis of cytokines and interferons. TLR9 is an endosomal membrane-bound protein, which binds to CpG-containing microbial DNA or endogenous signals from dead cells or tissue damage. We showed that TLR9 activation requires EGFR, a tyrosine (Tyr) kinase, which interacts with and phosphorylates the cytoplasmic domain of TLR9. Blocking EGFR activity pharmacologically, or knocking out EGFR gene in myeloid cells, suppressed lethal TLR9-induced hepatotoxicity. Here, we reveal that TLR9 required two Src family of kinases, Syk and Lyn, which, together with EGFR, led to phosphorylation and activation of TLR9. Lack of either of these kinases inhibited TLR9-MyD88 interaction, thereby inhibiting TLR9-mediated gene induction. Unlike EGFR, which constitutively binds TLR9, activated Syk interacted with TLR9 in a CpG-dependent manner. Activated Syk interacted with TLR9 and was critical for activating TLR9-bound EGFR. Quantitative mass spectrometric analyses revealed TLR9 was phosphorylated, sequentially, on Tyr870 and Tyr980 by Syk and EGFR, respectively. Mutation of either of these tyrosines led to complete loss of TLR9-induced cytokine production. For activation, Syk was phosphorylated by Lyn, which was activated by CpG-mediated scavenger-receptor A, and surprisingly, independent of TLR9. In summary, our results uncovered the molecular details of TLR9 activation by its Tyr-phosphorylation, which is critical for TLR9-mediated intracellular signaling.

immunology↗

bioRxiv2024

Integrated forward and reverse degradomics uncovers the proteolytic landscape of aortic aneurysms and the roles of MMP9 and mast cell chymase

BackgroundDysregulated proteolysis is implicated in thoracic (TAA) and abdominal aortic aneurysm (AAA) pathogenesis, but the proteolytic landscapes (degradomes) of aneurysmal and normal aorta, and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to uncover TAA and AAA degradomes, compare them quantitatively and define the specific role in aortic remodeling of two proteases consistently identified in the aneurysms, mast cell chymase (CMA1) and matrix metalloprotease 9 (MMP9). MethodsThe mass spectrometry-based N-terminomics strategy Terminal Amine Isotopic Labeling of Substrates (TAILS) was applied to Marfan syndrome TAAs (n=5), AAAs (n=16) and corresponding non-diseased aorta (TAs, n=4, and AAs, n=8) as a forward degradomics application, i.e., to define substrate and protease degradomes, and 8-plex iTRAQ-TAILS was used for quantitative comparison. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, i.e., digestion of aortic proteins with these proteases, followed by 6-plex iTRAQ-TAILS. CMA1 and MMP9 proteolysis of biglycan was investigated using Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS). ResultsWe experimentally annotated 16,923 proteolytically derived peptides (substrate degradome) and 90 proteases (protease degradome) in the aorta. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAA and AAA. Reverse degradomics elucidated > 300 CMA1 and MMP9 substrate cleavage sites, of which, many, including orthogonally validated biglycan cleavage, occurred in the disease degradomes. ConclusionsUnbiased, proteome-wide forward degradomics of the aortic wall from TAA, AAA and non-diseased tissue generated the first systems biology view of vascular wall breakdown and public resource for the hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling. The findings provide insights on aortic aneurysm pathways and potential disease biomarkers. Mapping of specific contributions of CMA1 and MMP9 on the aortic forward substrate degradome using reverse degradomics provides a strategy for defining the activities of all proteases involved in aortic disease.

biochemistry↗

bioRxiv2024

In Vivo Proximity Labeling Identifies a New Function for the Lifespan and Autophagy-regulating Kinase Pef1, an Ortholog of Human Cdk5

Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously identified Pef1, the Schizosaccharomyces pombe ortholog of cdk5, as regulator of chronological lifespan. To reveal the processes impacted by Pef1, we developed APEX2-biotin phenol-mediated proximity labeling in S. pombe. Efficient labeling required a short period of cell wall digestion and eliminating glucose and nitrogen sources from the medium. We identified 255 high-confidence Pef1 neighbors in growing cells and a novel Pef1-interacting partner, the DNA damage response protein Rad24. The Pef1-Rad24 interaction was validated by reciprocal proximity labeling and co-immunoprecipitation. Eliminating Pef1 partially rescued the DNA damage sensitivity of cells lacking Rad24. To monitor how Pef1 neighbors change under different conditions, cells induced for autophagy were labeled and 177 high-confidence Pef1 neighbors were identified. Gene ontology (GO) analysis of the Pef1 neighbors identified proteins participating in processes required for autophagosome expansion including regulation of actin dynamics and vesicle-mediated transport. Some of these proteins were identified in both exponentially growing and autophagic cells. Pef1-APEX2 proximity labeling therefore identified a new Pef1 function in modulating the DNA damage response and candidate processes that Pef1 and other cdk5 orthologs may regulate.

genetics↗

bioRxiv2024

Carbapenem-induced β-lactamase-isoform expression trends in Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent bacterial threat to public health, with only a few treatment options and a >50% fatality rate. Although several resistance mechanisms are understood, the appearance of these mutations is generally considered stochastic. Recent reports have, however, begun to challenge this assumption. Here, we demonstrate that independent samples of Ab, exposed to different carbapenems with escalating concentrations, show concentration- and carbapenem-dependent trends in {beta}-lactamase-isoform expression. This result, based on the isoforms identified through label-free-quantification LC-MS/MS measurements of cell-free, gel-separated {beta}-lactamases, suggests that the appearance of antibiotic resistance may be somewhat non-stochastic. Specifically, several minor AmpC/ADC {beta}-lactamase-isoforms were found to exhibit both dose- and carbapenem-dependent expression, suggesting the possibility of non-stochastic mutations. Additionally, these also have high sequence similarity to major expressed isoforms, indicating a potential path over which resistance occurred in independent samples. Antibiotic resistance maybe somewhat antibiotic-directed by a hitherto unknown mechanism and further investigation may lead to new strategies for mitigating antibiotic resistance. TeaserThe emergence of antibiotic-resistant {beta}-lactamase proteins from mutations may exhibit patterns based on specific antibiotics.

microbiology↗

bioRxiv2024

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatory P. aeruginosa culture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II by confocal microscopy. Mechanistically, mass spectrometric analysis of K6a immunoprecipitates identified Sec16A, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.

cell biology↗

bioRxiv2023

Keratin 6a attenuates Toll-like receptor-triggered proinflammatory response in corneal epithelial cells by suppressing ELKS/IKKϵ-dependent activation of NF-κB

The corneal epithelium at the ocular surface is constantly exposed to the environment and represents the first line of defense against infection, mechanical injury or chemical irritation. Through TLR-mediated recognition of pathogen- and damage-associated molecular patterns, it engages in direct antimicrobial responses and alerts the immune system on intruder and tissue damage by secreting pro-inflammatory and chemotactic cytokines that promote immune cell infiltration. How the corneal epithelium downregulates TLR signaling is unclear, yet it highly expresses keratin 6a (K6a), a cytoskeletal protein that has emerged to play essential regulatory roles in corneal innate immune response. Here we report that mice harboring genetic deletion of K6a are more susceptible to developing bacterial keratitis with unresolved corneal opacification and higher bacterial load. Such disease phenotype is caused by the increased pro-inflammatory cytokine and chemokine secretions from the K6a-null corneal epithelium, which further promotes the infiltration of immune cells and their associated pro-inflammatory response. Using human corneal epithelial cells immortalized by telomerase reverse transcriptase (hTCEpi cells), we demonstrated that knocking down K6a enhances NF-{kappa}B/ RelA-dependent cytokine and chemokine expression. Moreover, proteomic screen reveals that K6a interacts with ELKS, a critical NEMO-binding scaffold that links between canonical IKK/{beta} and the principal cytoplasmic inhibitor of RelA, i.e. I{kappa}B., to promote its phosphorylation and degradation. Surprisingly, K6a does not antagonize any of these canonical NF-{kappa}B signaling events. Instead, we found that ELKS in addition to canonical IKKs interacts with the atypical IKK member IKK{varepsilon}. Furthermore, knockdown of K6a in hTCEpi cells promotes ELKS-dependent phosphoactivation of IKK{varepsilon}, which in turn phosphorylates and activates RelA. Our study thus demonstrated an unexpected role of cytosolic K6a as a novel negative regulator of TLR/NF-{kappa}B signaling in preventing excess proinflammatory cytokine and chemokine expressions. It further highlighted the functional importance of ELKS as a common signaling scaffold for both canonical and atypical IKK-dependent activation of NF-{kappa}B in corneal epithelial cells. Using both IKK classes other than only canonical IKKs for TLR/NF-{kappa}B induction as in other cell types including myeloid immune cells suggest that the cornea epithelium is more flexible in modulating its inflammatory response, which could greatly minimize corneal damage while preserving its essential functions for barrier protection and light refraction.

immunology↗

bioRxiv2022

Increased glucose availability sensitizes pancreatic cancer to chemotherapy

Pancreatic cancer (Pancreatic Ductal Adenocarcinoma; PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, leaving chemotherapy as the best available systematic treatment. The discovery of safe and available adjuncts that improve chemotherapeutic efficacy would potentially improve survival outcomes. We show that a hyperglycemic state enhances the efficacy of conventional single- and multi-agent chemotherapies against PDAC. Molecular analyses of tumors exposed to relatively high glucose levels revealed that a key metabolic pathway, glutathione biosynthesis, is diminished and underlies chemo-sensitization by enhancing oxidative injury to cancer cells. Inhibition of this pathway under normal conditions phenocopied a hyperglycemic state by enhancing chemotherapeutic efficacy in mouse PDAC, while rescuing the pathway under high glucose abrogated the anti-tumor effects observed with chemotherapy.

cancer biology↗

bioRxiv2022

Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice

BACKGROUNDThere is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. METHODSWe used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. RESULTSWe show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. CONCLUSIONSThe microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

biochemistry↗

bioRxiv2020

Gut Microbe-Targeted Choline Trimethylamine Lyase Inhibition Improves Obesity Via Rewiring of Host Circadian Rhythms

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (ob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake, but is instead associated with beneficial remodeling of the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism, and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have untapped potential as anti-obesity therapeutics.

physiology↗