Biology subjects

Tiruppathi, C.

Publications and source records attributed to Tiruppathi, C..

8 recordsLinked to original sources

bioRxiv2026

E3 ubiquitin ligase SYVN1 mediates K63-linked ubiquitination of DDX3X to activate Macrophage NLRP3 Inflammasome

DDX3X (DEAD box helicase 3, X linked) is a common and essential component for both stress granules and NLRP3 inflammasome assembly and their activation; however, the upstream cellular stress signals driving DDX3X to activate the contrasting cellular pathway remain unclear. We identified the pivotal role of the E3 ubiquitin ligase SYVN1(Synoviolin) as the upstream regulator of DDX3X and thereby controlling NLRP3 activation and stress granule assembly. We observed SYVN1 silencing in macrophages prevented both NLRP3 driven inflammation and stress granule formation. SYVN1 deficiency prevented LPS induced inflammatory lung injury and increased the survival rate of the mice. SYVN1 sustains DDX3X gene expression and promotes stimulus-dependent ubiquitination of DDX3X. Under inflammatory conditions, SYVN1 mediated 63 linked ubiquitination of DDX3X, a requirement for NLRP3 inflammasome activation. Conversely, stress conditions reduced K63 linked DDX3X ubiquitination in coordination with activity of the deubiquitinase OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1). Thus, the balance between SYVN1 and OTUB1 functioned to optimize DDX3X activity and activation of NLRP3 or stress granule. These findings show the upstream role of SYVN1 OTUB1 axis in integrating cellular stress signals to decide the cell fate and suggest that ubiquitination of DDX3X is a potential target for inflammasome driven inflammation.

immunology↗

bioRxiv2026

Ubiquitin ligase CHFR impairs Tie2 signaling via K48-linked ubiquitylation and degradation of Akt1 in endothelial cells

Vascular endothelial (VE)-cadherin is essential for maintaining endothelial junctional barrier integrity. The Angiopoietin-1 (Ang-1)/Tie2 axis induced Akt1 activation is crucial for maintaining endothelial junctional barrier by inhibiting FoxO1 and suppressing expression of Angiopoietin-2 (Ang-2), a Tie2 antagonist. Systemic inflammatory conditions such as sepsis, Akt1 expression is reduced, whereas FoxO1-dependent Ang-2 expression is increased, resulting in endothelial barrier dysfunction. We previously showed that the TLR4/FoxO1 axis induces the ubiquitin E3 ligase CHFR, which promotes endothelial barrier disruption by targeting VE-cadherin for ubiquitylation and degradation. However, little is known about Akt1 expression during vascular inflammation. Here, we identified FoxO1-dependent CHFR expression as a key mechanism driving K48-linked polyubiquitylation and proteasomal degradation of Akt1 in endothelial cells (EC). LPS-induced K48-linked ubiquitylation of Akt1 was prevented in CHFR-depleted human EC and in endothelial-specific Chfr knockout (Chfr{Delta}EC) mice. Accordingly, CHFR depletion increased Akt1 and VE-cadherin expression in both human lung EC and Chfr{Delta}EC mice. Chfr{Delta}EC mouse lungs also exhibited elevated Ang-1 and Tie2 expression, and Ang-1 stimulation induced sustained Akt1 phosphorylation in CHFR-deficient EC. Moreover, CHFR depletion prevented LPS-induced expression of FoxO1 and Ang-2 in EC. Mechanistically, CHFR interacted with phosphorylated Akt1 and mediated its ubiquitylation at lysine residues K30, K39, K154, and K268. Expression of a ubiquitylation-deficient Akt1 mutant prevented LPS-induced VE-cadherin degradation and vascular injury. Collectively, these findings identify CHFR as a critical regulator of endothelial inflammatory responses by controlling Akt1 stability and VE-cadherin expression during inflammation.

cell biology↗

bioRxiv2026

Innate Immune Function of Neutrophil Cytoplasts Generated Post-Vital NETosis

The migration of polymorphonuclear neutrophils (PMN) into the site of infection such as lungs during pneumonia is a canonical feature of innate immunity. Formation of neutrophil-derived extracellular traps (NETs), web-like strands of varying lengths comprising DNA, histones, elastase, and myeloperoxidase, is an important determinant of PMN-mediated innate immunity. NETs form in microvessels, entrap bacteria and effete matter, and dampen PMN-mediated inflammatory injury at specific sites. However, studies have largely focused on NET release secondary to lytic NETosis and lysis of PMN. Far less is known about vital NETosis occurring in the absence of PMN rupture. As vital NETosis is characterized by generation of anuclear PMN termed cytoplasts (PMNcyto), we addressed the function of PMNcyto as a critical determinant of PMN-mediated innate immunity. Studies were made in mice challenged with live Pseudomonas aeruginosa (PA) i.t. to induce fulminant pneumonia characterized by tissue injury in which we determined the role of generated PMNcyto population. Using Tomato Red (tDTomato) transgenic mice to mark PMN, we observed PA pneumonia induced PMN transmigration leading to PMNcyto generation in the airspace. In contrast, PMNcyto transmigration, was minimal. PMNcyto accumulating in lung tissue actively phagocytosed and killed PA. Instillation of ex vivo generated PMNcyto also prevented PA-induced inflammatory lung injury and reduced mortality as compared to control mice. We demonstrated that the salutary effects of PMNcyto required functional microchondria. Proteomic analysis revealed that PMNcyto retained bactericidal and ROS generating pathways, consistent with an intact plasma membrane. Genetic deletion of peptidyl arginine deaminase 4 (PAD4), which mediates histone citrullination and promotes NETosis, facilitates PMNcyto generation and thereby abrogated pneumonia-induced mortality. Thus, we have identified the crucial host defense function of PMNcyto generated post-vital NETosis, suggesting that PMNcyto hold promise as cell based anti-bacterial therapy in pneumonia.

immunology↗

bioRxiv2025

CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

The expression of endothelial-enriched receptor tyrosine kinase Tie2 and vascular endothelial cadherin VE-cadherin in endothelial cells (EC) is essential for maintaining endothelial barrier integrity and vascular homeostasis. The Ets family of transcription factors plays a critical role in regulating the expression of Tie2 and VE-cadherin in EC. However, the transcriptional regulation of these factors remains poorly understood. In this study, we performed whole genome methylation analysis and found that the gene encoding the Ets family transcription factor Elf2/Nerf2 was hypermethylated in Camkk{beta}-deficient (Camkk{beta}-/-) mice. Notably, these mice exhibited significantly reduced mRNA and protein levels of Elf2, Tie2, and VE-cadherin, along with uncontrolled lung vascular injury following LPS challenge. Additionally, either depletion of CaMKK{beta} in human lung microvascular endothelial cells or EC-specific deletion of Camkk{beta} in mice led to a decrease in the expression of Elf2, Tie2, and VE-cadherin. Furthermore, administration of the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) or EC-specific expression of wild-type CaMKK{beta} but not the kinase-defective mutant restored the expression of Elf2, Tie2, and VE-cadherin in Camkk{beta}-/-mice. We find that under baseline conditions, methyl CpG binding protein MeCP2 repressed Elf2 expression in EC. Importantly, in EC-restricted Elf2 knockout mice (Elf21EC), lung endothelial barrier integrity was compromised due to dramatically reduced levels of Tie2 and VE-cadherin. Together, these findings underscore the crucial role of CaMKK{beta} in regulating endothelial junctional barrier integrity by controlling Elf2 expression.

cell biology↗

bioRxiv2022

Pink1-mediated mitophagy in the endothelium releases proteins encoded by mitochondrial DNA and activates neutrophil responses

Given their ancient evolutionary origins, eukaryotic mitochondria possess multiple vestiges of their prokaryotic ancestors. One such factor is the N-terminal formylation of proteins encoded by mitochondrial DNA. N-formylated proteins are also released by bacteria and trigger activation of immune cells such as neutrophils. Growing evidence indicate that circulating levels of mitochondrial formyl proteins are elevated in the serum of patients with excessive inflammatory responses and trigger neutrophil activation like their bacterial counterparts. However, the cellular source of these proteins, and the mechanism by which they are released into the circulation is not known. In this study, we have identified vascular endothelial cells as a source of mitophagy induced release of formyl proteins in response to inflammatory mediators in vitro. Mechanistically, endothelial mitophagy required activation of the Pink1 pathway. Using liposomal delivery of sgRNA targeting Pink1 in mice expressing endothelial-specific Cas9, we developed a mouse model in which Pink1 is specifically depleted in the endothelium. Deletion of endothelial Pink1 was remarkably protective in endotoxin-induced lung inflammation, resulting in reduced neutrophil infiltration and significantly reduced death in mice. We thus propose that endothelial cells upregulate pro-inflammatory mitophagy in response to inflammation, leading to release of mitochondrial formyl peptides and detrimental neutrophil recruitment into the lung.

cell biology↗

bioRxiv2021

Computer-Vision Stabilized Intravital Imaging Reveals Lung Capillary Neutrophil Dynamics Crucial for Lung Host-Defense Function

Polymorphonuclear neutrophils (PMN) are highly dynamic innate immune cells which are essential for lung host defense. However, in vivo intravital imaging in moving organs such as the lung remains challenging due to motion artifacts. Here we describe a novel intravital imaging method with high-throughput analytical capability based on a computer vision stabilization algorithm, Computer-vision-Assisted STabilized intravital imaging (CASTii). The sub-micron precision of this approach enables analysis of compartmentalized intravital PMN dynamics. We quantified in real-time a novel patrolling function of lung intracapillary circulating PMN. We also describe the dynamics of intracapillary PMN pooling (marginated PMN pool) using direct imaging of PMNs. The pool was formed by repeated catch-and-release kinetics involving PMN deformation inside microvessels during the passage of PMNs in vessels. We observed rapid PMN recruitment into the lung tissue compartments from pooled PMNs in response to alveolar chemoattract exposure. In contrast, endotoxemia-induced intracapillary sequestration of PMN impaired PMN transmigration into the alveolar space and defective phagocytosis of live bacteria. Intravital imaging of PMN dynamics with CASTii provides fundamental insights into host-defense functions of lung capillary PMN.

microbiology↗

bioRxiv2019

TAK1 Regulates Endothelial Integrity Through Stabilization of Junctions via GSK3β and FoxO1

TLR4 signaling in endothelial cells (ECs) induces vascular injury by disrupting the endothelial junctional barrier. However, it is not known whether TLR4 signaling can also promote endothelial barrier repair after vascular injury. Here we addressed the role of TAK1 activation downstream of TLR4 in the mechanism of vascular integrity. In inducible EC-restricted TAK1 knockout (TAK1i{Delta}EC) mice, the endothelial barrier was compromised. Blocking TAK1 activity caused spontaneous loss of the endothelial barrier. Importantly, TAK1 inactivated GSK3{beta} via AKT to prevent {beta}-catenin downregulation. We observed in ECs of GSK3{beta}i{Delta}EC mice an increase in {beta}-catenin transfer to the nucleus to form a complex with transcription factor FoxO1, thus repressing the expression of the tight junction protein claudin-5 and causing vascular leak. Strikingly, in TAK1i{Delta}EC mice, FoxO1 expression was dramatically increased while expression of AKT was suppressed, and in vivo inhibition of FoxO1 prevented sepsis-induced lung vascular leak in GSK3{beta}i{Delta}EC and TAK1i{Delta}EC mice. Further, EC-restricted deletion of FoxO1 in mice suppressed sepsis-induced lung vascular leak and mortality. Our findings point to the potential of targeting the TAK1-AKT-GSK3{beta}-FoxO1 axis as a therapeutic approach to treat uncontrolled lung vascular leak in sepsis.

cell biology↗

bioRxiv2019

EphB1 in Endothelial Cells Regulates Caveolae Formation and Endocytosis

Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important in regulating key functions such as caveolae-mediated endocytosis and generation of nitric oxide. Here we show that deletion of the receptor tyrosine kinase EphB1 (EphB1-/-) in mice markedly reduced the caveolae number in ECs of heart and lung vessels and prevented caveolae-mediated endocytosis. EphB1 expressed in adult ECs was shown to bind the caveolin-1 (Cav-1) scaffold domain (CSD) via the CSD binding motif (CSDBM) on EphB1. We demonstrated that activation of EphB1 by the native ligand Ephrin B1 uncoupled EphB1 from Cav-1, and licensed Src-dependent Y-14 Cav-1 phosphorylation. Deletion of CSDBM on EphB1 prevented EphB1/Cav-1 interaction and the activation of Src and Src mediated Y-14 Cav-1 phosphorylation. These studies identify the central role of endothelium expressed EphB1 in regulating caveolae biogenesis and caveolae-mediated endocytosis.

cell biology↗