Biology subjects

Phuyal, S.

Publications and source records attributed to Phuyal, S..

7 recordsLinked to original sources

bioRxiv2026

Tumor-derived stearic acid induces macrophage Egr2 signaling to suppress anti-tumor immunity in breast cancer

BackgroundMacrophages in breast cancer are highly heterogeneous and targeting both tissue-resident and bone marrow-derived macrophages represents a high-potential strategy in breast cancer immunotherapy. MethodsMyeloid-specific Egr2 knockout (KO) mice were used to determine roles of macrophage Egr2 signaling in tumor progression and anti-tumor immune responses. RNA-seq analysis and Flow cytometry were used to examine regulation of Egr2 in tumor-associated macrophages (TAMs) and fatty acid-stimulated macrophages. Tumor cell and macrophage admixture study was used to evaluate effects of fatty acid educated macrophages on anti-tumor responses. ResultsHere we showed that early growth response 2 (Egr2) was highly expressed in mammary tissue macrophages and recruited macrophages upon tumor progression. Depletion of Egr2 in myeloid cells significantly decreased the immunosuppressive function of polarized M2-like macrophages and TAMs. Tumor progression was delayed in myeloid cell Egr2 knockout mice, which was associated with enhanced function of effector CD8+ T cells and NK cells. Mechanistically, we showed that retinol and nicotinamide metabolism in TAMs were impaired after Egr2 depletion. Further, tumor cell-derived stearic acid (SA) was identified as an important fatty acid inducing Egr2 expression. SA stimulation induced macrophage-mediated immunosuppression in an Egr2 dependent manner. Addition of oleic acid (OA) uniquely repressed SA-induced Egr2 expression and immunosuppressive activity in macrophages. ConclusionsOur study thus uncovers a novel SA-Egr2 pathway driving macrophage immunosuppression and novel molecular mechanism of OA mediated anti-tumor effect. WHAT IS ALREADY KNOWN ON THIS TOPICLipid-associated macrophages (LAMs) accumulate lipids and promote immune suppression, tumor growth and metastasis in breast cancers. Fatty acids play a dual, complex role in tumor immunity, acting as both pro-tumorigenic and anti-tumorigenic effects. However, molecular mechanisms underlying fatty acid modulated macrophages and their roles in tumor remain not fully elucidated. WHAT THIS STUDY ADDSWe demonstrate that Egr2 is essential for the immunosuppressive function of IL-4 polarized M2-like macrophages and TAMs. Tumor cell-derived stearic acid (SA) was identified as an important fatty acid inducing Egr2 expression and immunosuppression in macrophages. Adding oleic acid (OA) uniquely repressed SA-induced Egr2 expression and immunosuppression. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYSA-Egr2 axis is a novel pathway driving macrophage immunosuppression. OA treatment enhances macrophage anti-tumor immunity via inhibiting Egr2 signaling and immunosuppression. The data supports the notion that diets enriched in OA may have potential for prevention and suppression of breast cancer.

immunology↗

bioRxiv2025

Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

T cells play a crucial role in the adaptive immune system and depend on tightly regulated intracellular signalling pathways to respond in an appropriate manner. Adapter proteins have flexible and dynamic features, which allow them to regulate T cell signal transduction pathways. As adapter proteins are enzymatically inert and may play multiple roles in parallel, it has been a challenge to fully characterise their functions individually. One such protein in T cells, is T cell specific adapter protein (TSAd), which is upregulated following T cell receptor (TCR) stimulation and is believed to mediate Src family tyrosine kinase signalling. However, the functional role remains elusive, possibly due to limited insight into interactors that potentially bind TSAd. The only structurally well-defined feature within TSAd, is the Src homology 2 (SH2) domain. This conserved domain displays prototypic binding of phosphorylated tyrosines, which suggests that the adapter molecule is implicated in phosphotyrosine signalling pathways. Here, we used an unbiased approach to identify ligands of the TSAd SH2 domain, by using affinity-purification mass spectrometry (AP-MS). Several novel ligands, many of which are known to be implicated in negative regulation of T cell intracellular signalling, were identified. More specifically, we showed that TSAd binds DOK2 and PTPN11 and determined the tyrosines responsible for the TSAd SH2 domain-dependent interaction. Ablation of TSAd and DOK2 by CRISPR/Cas9 in Jurkat T cells resulted in altered tyrosine phosphorylation. Taken together, these findings provide new insight into the possible function of TSAd as a negative signalling node in T cells.

immunology↗

bioRxiv2025

A bioinformatic workflow to facilitate the study of less-understood proteins: the case of SH2D2A

Adaptor proteins are key regulators of immune signalling but are challenging to study due to redundant pathways that obscure clear functional "anchor" phenotypes. Making use of public single-cell RNA sequencing (scRNA-seq) datasets, we theorised that anchors could be identified by working in the reverse direction: starting from the transcriptomic level and working "bottom-up" to identify anchoring phenomena at the cell surface. We have produced a prototype workflow for this method using the case of the lymphocyte-enriched adaptor protein SH2D2A, whose function remains uncertain. Using our bottom-up analysis, we have identified a link between SH2D2A and T cell-T cell (T-T) synapses enriched for ITGB2. The functional implications of this link are currently being explored, but further application of this bioinformatic approach to less-understood proteins may prove useful in understanding the minutiae of immune cell signalling, with implications for immunotherapy.

molecular biology↗

bioRxiv2022

Lysosome repair by ER-mediated cholesterol transfer

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by formation of contacts with the endoplasmic reticulum (ER), which depend on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

cell biology↗

bioRxiv2022

A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression

Few studies so far have investigated the impact of different cell migration traits on tumor progression. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the migration and invasion traits of Giantin depleted cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

cancer biology↗

bioRxiv2022

Mechanical strain stimulates COPII-dependent trafficking via Rac1

Secretory trafficking from the endoplasmic reticulum (ER) is subject to regulation by extrinsic and intrinsic factors. While much of the focus has been on biochemical triggers, little is known whether and how the ER is subject to regulation by mechanical signals. Here, we show that COPII-dependent ER-export is regulated by mechanical strain. Mechanotransduction to the ER was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulate ER-to-Golgi transport. Altogether, we establish an unprecedented link between mechanical strain and export from the ER.

cell biology↗

bioRxiv2020

Computer-assisted beat-pattern analysis and the flagellar waveforms of bovine spermatozoa

Plagued by hurdles in information extraction, handling, and processing, computer-assisted sperm analysis (CASA) systems have typically neglected the complex flagellar waveforms of spermatozoa, despite their defining role in cell motility. Recent developments in imaging techniques and data processing have produced significantly-improved methods of waveform digitisation. Here, we utilise these improvements to demonstrate that near-complete flagellar capture is realisable on the scale of hundreds of cells, and, further, that meaningful statistical comparisons of flagellar waveforms may be readily performed with widely-available tools. Representing the advent of high-fidelity computer-assisted beat-pattern analysis (CABA), we show how such a statistical approach can distinguish between samples using complex flagellar beating patterns rather than crude summary statistics. Dimensionality-reduction techniques applied to entire samples also reveal qualitatively-distinct components of the beat, and a novel data-driven methodology for the generation of representative synthetic waveform data is proposed.

microbiology↗