Biology subjects

Deshmukh, M.

Publications and source records attributed to Deshmukh, M..

10 recordsLinked to original sources

bioRxiv2026

Orthogonal Chemical Proteomic Strategies Reveal the Cholesteryl Ester Interactome in Mammalian Cells

Cholesteryl esters (CEs) are widely regarded as inert storage forms of cholesterol, yet their potential to directly engage cellular proteins remains largely unexplored. Here, we establish an integrated chemical proteomic platform to systematically define CE-protein interactions in mammalian cells. We developed and comparatively deployed three orthogonal photoaffinity labeling strategies: (i) metabolic assembly of a bifunctional CE probe through endogenous acyl-CoA:cholesterol acyltransferase activity, (ii) direct delivery of a structurally defined diazirine-alkyne CE analog, and (iii) fragment-assisted subtraction using sterol and fatty acyl control probes to resolve interactions dependent on the intact esterified scaffold. Each probe system was rigorously validated by lipidomics and UV-dependent crosslinking prior to quantitative proteomic analysis. Integration of these complementary modalities identified 495 CE-associated proteins spanning enzymes, transporters, scaffolding proteins, and canonical sterol-binding families. The limited overlap across strategies reveals that lipid-protein engagement is strongly conditioned by biosynthetic origin, probe topology, and intracellular routing. Functional and database annotations further demonstrate enrichment of druggable and disease-linked proteins, connecting CE interactions to metabolic, neurological, and cardiovascular pathways. Collectively, this work provides the first systems-level map of CE-protein interactions and establishes a generalizable, multimodal framework for chemically resolving lipid-protein interactomes.

biochemistry↗

bioRxiv2026

Targeting DNMT1 augments anti-tumor CD8⁺ T cell function

Chronic stimulation of CD8 T cells within the tumor microenvironment (TME) induces a hypofunctional state characterized by diminished cytotoxicity and functionally impaired anti-tumor function, known as exhaustion. Exhaustion is associated with epigenetic changes that remain relatively stable despite interventions like immune checkpoint inhibition (ICI). Although epigenetic changes are potentially reversible, reports of therapeutic strategies to effectively restore function in exhausted CD8 T cells remain limited. Here, we report DNA methyltransferase 1 (DNMT1) inhibition (DNMT1i) in counteracting CD8+ T cell dysfunction during the anti-tumor response. We show that DNMT1i synergizes with ICI to rescue the tumor cell killing activity of chronically stimulated CD8 T cells in a melanoma model. DNMT1i mitigates transcriptional features of exhaustion while inducing a divergent effector program. DNMT1i attenuates the global increase in chromatin accessibility associated with exhaustion and enables epigenetic remodeling of the exhausted chromatin landscape upon restimulation. Finally, DNMT1i enhances the effector function of melanoma patient-derived tumor infiltrating lymphocytes after prolonged ex vivo expansion. These studies establish DNMT1 targeting as a promising strategy to counteract CD8 T cell exhaustion and potentiate ICI efficacy.

immunology↗

bioRxiv2026

Organelle communication networks rewire to support lipid metabolism during neuronal differentiation

Cell fate transitions require coordinated remodeling of intracellular organelles, but how organelle morphology and interactions rewire during neurogenesis remains unclear. Here we combine multispectral imaging with quantitative organelle signature analysis to simultaneously map eight organelles as human induced pluripotent stem cells differentiate into forebrain-like neurons. We find compartment and time-specific rescaling of organelles and a progressive increase in higher-order membrane contacts, with mitochondria emerging as an early interaction hub. Later, endoplasmic reticulum (ER)-organelle contacts dominate with ER-peroxisome contacts promoting ether lipid biosynthesis, membrane homeostasis and synapse formation. Disrupting this contact impairs plasmalogen production, synaptic organization, and neuronal activity, identifying the ER-peroxisome axis as a key regulator of neuronal maturation.

cell biology↗

bioRxiv2025

Engineering a Juxtamembrane-Targeting CAR T-Cell Against Mesothelin: A Novel Binder Resilient to Shed Antigen in Ovarian and Pancreatic Cancer

Mesothelin is an attractive target for CAR-T therapy on a number of cancer types; however, the efficacy of this therapy is diminished because the bulk of the cell surface-expressed mesothelin is shed through naturally occurring proteolysis leaving behind a short juxtamembrane peptide "stump". The two problems this creates are (1) the bulk of the target protein is no longer on the tumor cell and (2), the free soluble shed mesothelin remains in the tumor microenvironment and becomes present in blood/other body fluids binding to the mesothelin-targeted CAR-T and interfering with their ability to target the mesothelin that remains on the surface of the tumor. These issues have likely contributed at least in part to the lack of desired efficacy of CAR-T cells that target membrane distal regions of mesothelin (i.e., the shed domain) such as those utilizing anti-mesothelin scFvs SS1 and M5. Here we describe CAR T cells that utilize novel antibodies specific for the mesothelin stump domain thus being unaffected by the natural process of mesothelin shedding. Mesothelin "stump" specific CAR T cells (ST4-22) had cytotoxicity and in vivo activity that was comparable to standard anti-mesothelin CAR T cells. Importantly, CAR T cells expressing ST4-22 were effective against tumor cells that were resistant to standard anti-mesothelin CAR T cells.

immunology↗

bioRxiv2022

Aberrant miR-29 is a predictive feature of severe phenotypes in pediatric Crohn's disease

Crohns disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-sequencing of patient-matched non-inflamed colon and ileum biopsies from treatment-naive pediatric patients with CD (n=169) and a control cohort (n=108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we also found that pediatric CD patients with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 up-regulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

genomics↗

bioRxiv2022

miR-29 is an important driver of aging-related phenotypes

Aging is a consequence of complex molecular changes, but the roles of individual microRNAs (miRNAs) in aging remain unclear. One of the few miRNAs that are upregulated during both normal and premature aging is miR-29. We confirmed this finding in our study in both mouse and monkey models. Follow-up analysis of the transcriptomic changes during normal aging revealed that miR-29 is among the top miRNAs predicted to drive the aging-related gene expression changes. We also showed that partial loss of miR-29 extends the lifespan of Zmpste24-/- mice, an established model of progeria, which indicates that miR-29 is functionally important in this accelerated aging model. To examine whether miR-29 upregulation alone is sufficient to promote aging-related phenotypes in vivo, we generated mice in which miR-29 can be conditionally overexpressed (miR-29TG). We found that miR-29 overexpression in mice is sufficient to drive aging-related phenotypes including alopecia, kyphosis, osteoporosis, senescence, and leads to early lethality. Transcriptomic analysis of both young miR-29TG and old WT mice revealed shared downregulation of genes enriched in extracellular matrix and fatty acid metabolism, and shared upregulation of genes in pathways linked to inflammation. Together, these results highlight the functional importance of miR-29 in controlling a gene expression program that drives agingrelated phenotypes.

physiology↗

bioRxiv2021

Constitutive high expression of NOXA sensitizes human embryonic stem cells for rapid cell death

Human embryonic stem (hES) cells are highly sensitive to apoptotic stimuli such as DNA damage, which allows for rapid elimination of mutated cells during development. However, the mechanisms that maintain hES cells in the primed apoptotic state are not completely known. Key activators of apoptosis, the BH3-only proteins, are present at low levels in most cell types. In contrast, hES cells have constitutive high levels of the BH3-only protein, NOXA. We examined the importance of NOXA for enabling apoptosis in hES cells. hES cells deleted for NOXA showed remarkable protection against multiple apoptotic stimuli. NOXA was constitutively localized to the mitochondria, where it interacted with MCL1. Strikingly, inhibition of MCL1 in NOXA knockout cells was sufficient to sensitize these cells to DNA damage, and subsequently, cell death. Our study demonstrates, an essential function of constitutive high levels of NOXA in hES cells is to effectively antagonize MCL1 to permit rapid apoptosis. Significance statementHuman embryonic stem (hES) cells give rise to the entire organism, hence understanding how these cells regulate their survival and death is important. These cells undergo rapid death in response to DNA damage thereby removing mutated cells from the developing embryo. We focused on identifying the mechanism underlying the sensitivity of these cells to DNA damage. We discovered that the protein NOXA is essential for cell death in hES cells. Further, the crucial function of NOXA is to neutralize high levels of antiapototic protein, MCL1, thus enabling hES cells to respond rapidly to DNA damage.

cell biology↗

bioRxiv2020

Modulation of implanted aortic scaffold- involvement of ECM and immune proteins

Biological scaffold or implant is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortage in the clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix by potentially inducing immune responses after implantation. The current study explores an alternative route for the preparation of acellular scaffolds and explores the fate of the prepared scaffolds in a milieu of immune cells following implantation without using immunosuppressant. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs omentum) for transplantation, normal blood vascular scaffolds were implanted which was converted to acellular vascular scaffolds by in vivo natural decellularization at the end of 2 months of observation. We also prepared chemically decellularized acellular scaffolds from normal untreated blood vascular scaffolds using a cocktail of chemicals which was also similarly placed in subcutaneous and omentum sites. Here, we applied in-depth quantitative proteomics along with histology and image analysis to comprehensively describe and compare the proteome of the natural and chemically decellularized scaffold. Our data confirm that site-specific advantages exist in modulating the ECM and regulating the immune responses (macrophage and T cells) following implantation, which possibly led to the production of an acellular scaffold (natural decellularization) under in vivo conditions. The current approach opens up the possibility to create tailor-made acellular scaffolds to build functional blood vessels. In addition, the identification of different tissue sites facilitates differential immune response against the scaffolds. This study provides a rich resource aimed toward an enhanced mechanistic understanding to study immune responses under similar settings in the field of transplantation and regenerative medicine. Impact statementThe development of a scaffold helps in the preparation of a functional organ in the clinics. In the current study, we prepared an acellular vascular scaffold by utilizing site specific tissue changes and vis-a-vis compared with a conventionally chemically prepared biological scaffold at genomic and protein level, which helped us to identify immunological trigger following implantation. The current study which was carried out without any immunosuppressive agents could help to establish (a) alternative strategies for preparing biological scaffolds as well as (b) implantable sites as potential bioreactors to circumvent any adverse immune reactions for acceptance of the scaffold/implant post implantation.

bioengineering↗

bioRxiv2020

Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2

A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with Mpro, 17 were chosen for evaluation in a kinetic assay for Mpro inhibition. Remarkably 14 of the compounds at 100-M concentration were found to reduce the enzymatic activity and 5 provided IC50 values below 40 M: manidipine (4.8 M), boceprevir (5.4 M), lercanidipine (16.2 M), bedaquiline (18.7 M), and efonidipine (38.5 M). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1, and P2 pockets of Mpro. Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.

pharmacology and toxicology↗

bioRxiv2020

Groucho/TLE opposes axial to hypaxial motor neuron development

Spinal cord motor neuron diversity and the ensuing variety of motor circuits allow for the processing of elaborate muscular behaviours such as body posture and breathing. Little is known, however, about the molecular mechanisms behind the specification of axial and hypaxial motor neurons controlling postural and respiratory functions respectively. Here we show that the Groucho/TLE (TLE) transcriptional corepressor is a multi-step regulator of axial and hypaxial motor neuron diversification in the developing spinal cord. TLE first promotes axial motor neuron specification at the expense of hypaxial identity by cooperating with non-canonical WNT5A signalling within the motor neuron progenitor domain. TLE further acts during post-mitotic motor neuron diversification to promote axial motor neuron topology and axonal connectivity whilst suppressing hypaxial traits. These findings provide evidence for essential and sequential roles of TLE in the spatial and temporal coordination of events regulating the development of motor neurons influencing posture and controlling respiration. HIGHLIGHTSO_LIGroucho/TLE mediates non-canonical WNT signalling in developing motor neurons C_LIO_LINon canonical WNT:TLE pathway regulates thoracic motor neuron diversification C_LIO_LITLE promotes axial while inhibiting hypaxial motor neuron development C_LIO_LITLE influences developing motor neuron topology and muscle innervation C_LI IN BRIEFSalin-Cantegrel et al use in ovo engineered approaches to show that a non-canonical WNT:TLE pathway coordinates temporally and spatially separated elements of motor neuron diversification, repressing hypaxial motor neuron development to promote the axial fate. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/986323v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@e2d96dorg.highwire.dtl.DTLVardef@130baf4org.highwire.dtl.DTLVardef@41fa32org.highwire.dtl.DTLVardef@190ebd3_HPS_FORMAT_FIGEXP M_FIG TLE contribution to the development of thoracic somatic motor columns Progenitor cells in the ventral pMN domain are exposed to higher concentrations of non-canonical WNTs and express more TLE. Cooperation of non-canonical WNTs and TLE renders ventral pMN progenitors refractory to a respiratory MN fate, thereby contributing to the separation of MMC and RMC MN lineages. Differentiating MNs that maintain high TLE expression also maintain LHX3 expression, adopt axial motor neuron topology and connect to axial muscles. TLE activity in differentiating MMC MNs prevents the acquisition of respiratory MN topology and innervation traits. C_FIG

neuroscience↗