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Patel, K. M.

Publications and source records attributed to Patel, K. M..

6 recordsLinked to original sources
bioRxiv2025

Sufficient levels of BECLIN1 are required for intestinal epithelial cell homeostasis and protection against unwanted intestinal inflammation

The prototypical autophagy regulator BECLIN1, orchestrates both autophagic and endocytic trafficking, and its homozygous deletion in the intestinal epithelium leads to intestinal disruption bearing similarities to inflammatory bowel disease (IBD). However, complete loss of BECLIN1 is rare in human disease. To model the effects of a partial reduction in BECLIN1, we examined mice with a monoallelic deletion of Becn1 in the intestinal epithelium, which decreased BECLIN1 protein levels by approximately 50%. Unlike the fatal phenotype following homozygous Becn1 deletion, the heterozygous mice were grossly normal, though they presented with significantly shorter small intestines. Gastrointestinal organoids derived from the mice also displayed disrupted endocytic trafficking and altered cytoskeletal dynamics resulting in mislocalisation of junctional proteins such as E-CADHERIN. In the mice, these changes were associated with impaired goblet cell function and maturation accompanied by abnormal mucin production, composition and secretion, resulting in compromised mucus barrier integrity. The mice also exhibited heightened susceptibility to dextran sulfate sodium (DSS)-induced colitis, emphasising the critical role of BECLIN1 in mediating protection against unregulated inflammation. Investigation of BECLIN1 levels in biopsies from patients with inflammatory bowel disease (IBD) revealed changes in its expression across distinct states of inflammation with reduced levels observed in highly inflamed regions in some patients, consistent with the gastrointestinal defects seen in mice with reduced BECLIN1. Hence, this study provides crucial insights into the multifaceted importance of BECLIN1 sufficiency in the intestinal epithelium to prevent pathophysiology. This highlights the potential of BECLIN1 as a biomarker and therapeutic target for IBD management.

cell biology↗
bioRxiv2025

The kinase domain of RIPK3 tunes its scaffolding functions

The pro-inflammatory programmed cell death pathway, necroptosis, relies on phosphorylation of the terminal effector, MLKL, by RIPK3. RIPK3-deficient mice or those harboring the kinase-inactivating mutation, RIPK3K51A, are ostensibly normal in the absence of challenge, indicating that RIPK3 and its kinase activity are dispensable for development. However, another kinase-inactivating mutation, RIPK3D161N, results in embryonic lethality in mice due to widespread apoptosis. As a result, the RIPK3D161N mutation is thought to confer a toxic gain-of-function. Here, to further explore the impacts of RIPK3 inactivation, we compared the stability and cellular interactions of RIPK3D161N and RIPK3K51A to a third previously-uncharacterized kinase-dead variant, RIPK3D143N. Here, we show that RIPK3K51A was unstable and did not associate with RIPK1, RIPK3D161N was unstable but interacted with RIPK1, whereas RIPK3D143N was stable and bound RIPK1 in a manner comparable to wild-type RIPK3. Thus, all three variants scaffold differently, suggesting that the assembly of cell death machinery by RIPK3 is finely tuned, not just by its kinase activity, but also by the conformation of its kinase domain. Physiologically, Ripk3D143N/D143N mice exhibited a partially penetrant lethality in utero. However, once born, Ripk3D143N/D143N mice were fertile and phenotypically indistinguishable from wild-type mice in the absence of challenge, with RIPK3D143N mutation protecting mice lacking intestinal epithelial Caspase-8 from necroptotic ileitis. Our studies support the idea that RIPK3 is a nexus between apoptotic and necroptotic signaling, and highlight the importance of considering kinase domain conformation in RIPK3 inhibitor development.

biochemistry↗
bioRxiv2024

A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic condition caused by altered cytokine signaling, maladaptive immunity, dysbiosis, and intestinal barrier dysfunction. Patients with IBD receive therapy to correct these imbalances and achieve remission. However, most patients relapse, suggesting that pathological mechanisms persist during remission. Here, we show that excess epithelial cell death is an underlying feature of IBD that arises in patients in remission and on advanced therapy. Mechanistically, nascent inflammation reprograms epithelial cells into a macrophage-like state that promotes RIPK1-independent necroptotic signaling, then triggers iNOS-mediated mitochondrial apoptosis of absorptive epithelial cells and PUMA-mediated intestinal stem cell death. These findings reveal aberrant epithelial cell death signaling as a hallmark of IBD that occurs early in mucosal lesion development and persists despite current therapeutic approaches. One-Sentence SummaryEpithelial cell death is dysregulated in patients with inflammatory bowel disease.

cell biology↗
bioRxiv2022

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. We show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-{beta} induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

immunology↗
bioRxiv2020

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.

cell biology↗