Biology subjects

Singh, E.

Publications and source records attributed to Singh, E..

10 recordsLinked to original sources

bioRxiv2026

Targeting proteasome composition reprograms tumor-intrinsic inflammation through selective degradation of STAT1

Proteasome regulatory particles determine cellular protein degradation capacities. Yet specific ubiquitin-independent degradation targets of individual proteasome regulatory particles, and physiological roles of such pathways, remain largely elusive. Here, through a chemical biology-driven approach, we identify a covalent small molecule that selectively targets a conformationally dynamic pocket in the non-catalytic proteasome regulator PSME4 and modulates tumor cell proteasome targets. Using this chemical probe together with orthogonal genetic perturbations, we show that blocking or reducing PSME4 stabilizes STAT1, restores interferon-associated transcription, enhances antigen presentation, and disrupts IL-6/STAT3 signaling, collectively reprogramming tumor inflammation and suppressing tumor growth in vivo. These findings define a PSME4-STAT1-STAT3 axis that couples proteasomal degradation to antigen presentation and cytokine signaling and provide a framework for pharmacologically targeting non-catalytic proteasome regulators to reprogram tumor immunity. More broadly, our findings reveal that proteasome composition can actively govern the balance between immunostimulatory and immunosuppressive signaling programs.

cancer biology↗

bioRxiv2026

On the Robustness of Biomolecular Systems to Perturbations in Translational Resources

The reliable operation of biomolecular circuits depends on the availability of shared cellular resources such as ribosomes, whose levels can vary substantially across growth conditions and cellular contexts. Although resource competition among co-expressed genes is well recognized, the relationship between resource variation and the robustness of circuit dynamics has not been characterized quantitatively. This paper integrates a resource-aware gene expression model, contraction theory-based analytical bounds, and experimental validation to study the effect of translational resource variation on constitutive gene expression and its mitigation through feedback. We show that the constitutive circuit exhibits sensitivity to resource perturbations, and that redesigning it with negative autoregulatory feedback enhances the contraction rate and reduces the steady-state deviation bound, though at lower expression levels. Experiments in E. coli using both plasmid copy number variation and a ribosome sequestration module are consistent with these predictions, confirming that the feedback circuit maintains relatively stable expression under conditions where the constitutive circuit shows large changes. These findings offer a systematic approach for analyzing and improving the robustness of biomolecular circuits operating under variable resource conditions.

systems biology↗

bioRxiv2026

Regulation of the proteasome 20S core particle by the Parkinsonism-associated Proteins FBXO7 and PI31

FBXO7 and PI31 variants are linked to rare Parkinsonian syndromes, implicating their dysfunction in neurodegeneration. We define how both engage each other and regulate the proteasome 20S core particle (CP). In cells, each can associate independently with the proteasome, with multiple domains in FBXO7 contributing. Utilizing cryo-EM we visualized how FBXO7s C-terminal domain engages multiple subunits within the CP interior, blocking the {beta}5 peptidase activity. In contrast, we visualized PI31 engagement of all three catalytic sites within the 20S CP, revealing the previously unknown structural basis for {beta}1 inhibition. Furthermore, we establish how disease-associated variants impact both FBXO7 and PI31 function, including disruption of proteasome inhibition and SKP1-FBXO7-PI31 complex assembly. These results establish an unexpected function for FBXO7, providing a mechanistic basis for investigation of its role in proteasome regulation in Parkinsons disease.

biochemistry↗

bioRxiv2026

Distinct trajectories of urbanization shape the human gut microbiome across South Asia

Human gut microbiomes respond to lifestyle transitions, yet the extent to which these responses are conserved across spatio-cultural contexts remains undercharacterized. We present the South Asian MicroBiome ARray (SAMBAR), a population-scale 16S gut microbiome study of 575 adults from ten geographically and socio-culturally diverse South Asian communities. Each community was sampled in ancestral villages and urban centers, enabling controlled comparisons of geography and lifestyle. Relative to global cohorts, SAMBAR microbiomes occupy a distinct compositional space with stronger correlation to geography and community membership than lifestyle. Although urbanization is consistently associated with increased abundance of disease-linked taxa, microbiome responses to lifestyle transitions are largely community-driven, including the acquisition of wheat- and dairying-associated microbial modules in some communities that may facilitate non-genetic adaptation to lactase non-persistence. Microbiome responses to urbanization are heterogeneous even at regional scales, reflecting local culture and geography and underscoring the need for community-specific investigations of health impacts.

microbiology↗

bioRxiv2025

Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T cell therapies

Patients with T-cell lymphomas and leukemias have overall poor outcomes due to the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. Development of chimeric antigen receptor (CAR) T-cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective anti-tumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, using anti-CD2 CAR T-cells engineered without CD2 expression (CART2), would support CAR T-cell manufacturability and preclinical efficacy. Optimized CD2-knockout CART2, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T-cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, co-stimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel PD-1:CD2 switch receptor that rescues intracellular CD2 signaling, particularly when PD-L1 is engaged, resulting in improved in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T-cells and provides an effective strategy to overcome CD2-signaling deficiencies.

cancer biology↗

bioRxiv2025

TRAP seq in 3D Angiogenesis Assays Reveals a Distinct Endothelial Translatome Associated with Early and Late Stages of Morphogenesis.

Endothelial cells (ECs) are often a minority cell type in a tissue, limiting the utility of bulk sequencing approaches. Single cell sequencing lacks sensitivity and requires disruptive tissue digestion techniques. TRAP seq (Translating Ribosome Affinity Purification) or "RiboTag" has been used to overcome these limitations. Multicellular co-culture systems allow primary ECs to differentiate and undergo tubular morphogenesis in cell culture, however similar limitations exist with these in vitro assays, as ECs are under-represented by as much as a factor of 10 in many assays. We sought to use TRAP seq to better understand the gene expression landscapes that drive these morphogenic events. We found TRAP seq selectively enriches for endothelial RNA in two distinct co-culture paradigms, in both the planar and fibrin bead co-culture assays. Intriguingly, the use of this technology in vitro, revealed distinct gene expression changes in blood vessel development and in the mitotic cell cycle, with genes unique to early and late phases of morphogenesis. It is widely accepted that expression of the NOTCH signaling pathway is a regulator of angiogenesis in vivo. Correspondingly, we found that a large number of NOTCH related genes relevant to endothelial cell biology, were changing across this morphogenic process, underpinning the importance and utility of this technology in 3D multicellular cultures that model in vivo environments.

cell biology↗

bioRxiv2023

Dual localization of JA receptor, CaCOI2, explains JA perception dynamics in chickpea

Jasmonates (JAs) form a group of oxylipin-derived phytohormones involved in multiple biotic and abiotic stress responses and regulate plant development. JAs are perceived by the receptor proteins, COI (coronatine insensitive). These JA receptors encoding F-box protein, form SCFCOI ubiquitin ligase complex (Skp, Cullin, F-box) and activate JA signaling by facilitating degradation of the transcriptional repressor (JAZ; Jasmonate associated ZIM domain containing) proteins through 26S proteasomal pathway. However, JA signaling is poorly understood in chickpea, a vital legume plant. Here, we first identified two putative chickpea JA receptor named CaCOI1, CaCOI2 and demonstrated CaCOI2 as a functional JA receptor. Further subcellular localization studies revealed that CaCOI2 is localized to extra-nuclear region and move to the nucleus on JA perception to activate JA signaling. Using domain swapping (between CaCOI1 and CaCOI2) experiments, we have demonstrated that the LRR region of JA receptors, which interact with bioactive JA i.e., JA-Ile, also plays a critical role in regulating subcellular localization of CaCOI proteins. We report that an acidic amino acid in F-box region of COI proteins (AtCOI1T29) stabilizes COI-InsP8 (1,5-bisdiphosphoinositol-1D-myo-inositol (2,3,4,6) tetrakisphosphate) interaction. This interaction is found to be critical for COI-JAZ interaction and therefore, their functionality. Our study thus identified a functional JA receptor in chickpea and revealed novel aspects of JA signaling and perception, which might also have implications in other plants.

plant biology↗

bioRxiv2023

Exogenous TGFβ1 and its mimic HpTGM attenuate the heart's inflammatory response to ischaemic injury and improve long term cardiac outcomes

RationaleSuccessful and timely coronary reperfusion following acute ST-elevation myocardial infarction (STEMI) is standard therapy to salvage transiently ischaemic heart muscle. However, the subsequent inflammatory response within the infarct can lead to further loss of viable myocardium. Robust interventions are required in the acute MI setting to minimise cardiac injury and reduce risk of further detrimental progression. ObjectiveTGF{beta}1 is an anti-inflammatory cytokine released endogenously in response to infection or tissue injury. The goal of this study was to investigate its protective effects when given exogenously following myocardial infarction. Methods and ResultsTGF{beta}1 is found at increased levels in the blood of STEMI patients immediately following myocardial infarction. We observe a significant correlation (p=0.003) between higher circulating TGF{beta}1 levels at 24h post MI and a reduction in infarct size over the following 3 months, suggesting that an early increase in circulating TGF{beta}1 is protective in these patients. Using a mouse model of cardiac ischaemia-reperfusion we demonstrate that additional exogenous TGF{beta}1 delivered in the acute setting has multiple beneficial outcomes. At 24 hours post-reperfusion It leads to a significantly smaller infarct size (30% reduction, p=0.025), reduced inflammatory infiltrate (28% reduction, p=0.015), lower intra-cardiac expression of inflammatory cytokines IL1{beta} and CCL2 (>50 % reduction, p=0.038 and 0.0004, respectively) and reduced scar size at 4 weeks (21% reduction, p=0.015). Furthermore exogenous delivery of an equivalent dose of HpTGM, a recently described low-fibrogenic mimic of TGF{beta}1, secreted by a helminth parasite to evade immune rejection, has an almost identical protective effect on injured mouse hearts. Furthermore using a genetic approach we show the benefit is mediated by the vascular endothelium. ConclusionsThis work reveals the potential of exogenous TGF{beta}1 and HpTGM delivered in the acute MI setting to provide protective anti-inflammatory effects and reduce infarct size, leading to a smaller scar and reduced detrimental progression.

pathology↗

bioRxiv2021

Rapid fall in circulating non-classical monocytes in ST elevation myocardial infarction patients correlates with cardiac injury

ObjectiveMyocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischaemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes. Approach and ResultsWe retrospectively analysed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (p<0.05) and correlated inversely with left ventricular function (r=0.41, p=0.04). The early fall in NC monocytes post reperfusion was confirmed in a second prospective study of 13 STEMI patients. Furthermore, in a mouse cardiac ischaemia model, there was significant monocyte adhesion to coronary vessel endothelium at 2 hours post-reperfusion pointing to a specific and rapid vessel margination response to cardiac injury. ConclusionsRapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/428825v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@11f758eorg.highwire.dtl.DTLVardef@5eed65org.highwire.dtl.DTLVardef@33a0d9org.highwire.dtl.DTLVardef@1854643_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI3-5 bullet points that summarize the major findings of the study. C_LI O_LICirculating non classical monocytes show a rapid fall in STEMI patients within 90 minutes of re-opening the culprit coronary artery. C_LIO_LIThe extent of the drop in non classical monocytes correlates with loss of cardiac function and increased infarct size. C_LIO_LIA mouse model of cardiac ischaemia and reperfusion shows rapid margination of monocytes to the coronary vasculature C_LI

immunology↗

bioRxiv2020

Arterial endoglin does not protect against arteriovenous malformations

Endoglin (ENG) forms a receptor complex with ALK1 in endothelial cells (ECs) to promote BMP9/10 signalling. Loss of function mutations in either ENG or ALK1 genes lead to the inherited vascular disorder Hereditary Haemorrhagic Telangiectasia (HHT), characterised by arteriovenous malformations (AVMs). However, the vessel-specific role of ENG and ALK1 proteins in protecting against AVMs is unclear. For example, AVMs have been described to initiate in arterioles, whereas ENG is predominantly expressed in venous ECs. To investigate whether ENG has any arterial involvement in protecting against AVM formation we specifically depleted the Eng gene in venous and capillary endothelium whilst maintaining arterial expression, and investigated how this affected the incidence and location of AVMs in comparison with pan-endothelial Eng knockdown. MethodsUsing the mouse neonatal retinal model of angiogenesis, we first established the earliest time point at which Apj-Cre-ERT2 activity was present in venous and capillary ECs but absent from arterial ECs. We then compared the incidence of AVMs following pan-endothelial or venous/capillary-specific ENG knockout. ResultsActivation of Apj-Cre-ERT2 with tamoxifen from postnatal day (P) 5 ensured preservation of arterial ENG protein expression. Specific loss of ENG expression in ECs of veins and capillaries led to retinal AVMs at a similar frequency to pan-endothelial loss of ENG. AVMs occurred in the proximal as well as the distal part of the retina consistent with a defect in vascular remodelling during maturation of the vasculature. ConclusionExpression of ENG is not required in arterial ECs to protect against AVM formation.

genetics↗