Biology subjects

Sharma, S. M.

Publications and source records attributed to Sharma, S. M..

8 recordsLinked to original sources

bioRxiv2025

Biliverdin Reductase A is a major determinant of neuroprotective Nrf2 signaling

Biliverdin reductase A (BVRA), the terminal enzyme in heme catabolism, generates the neuroprotective and lipophilic antioxidant bilirubin. Here, we identify a novel non-enzymatic role for BVRA in redox regulation. We show that BVRA directly interacts with nuclear factor erythroid-derived factor-like 2 (Nrf2), the master regulator of redox homeostasis, to modulate target signaling pathways. ChIP-seq and RNA-seq analyses reveal that this interaction coordinates the expression of neuroprotective genes that are typically dysregulated in Alzheimers disease and other neurodegenerative conditions. Thus, this previously unknown BVRA-Nrf2 axis controls an essential pathway of redox signaling in neuroprotection. Our findings establish BVRA as a dual-function integrator of antioxidant defenses in both the lipophilic and hydrophilic subcellular compartments, bridging these two distinct and critical cellular protection mechanisms in the brain. This advancement in understanding the endogenous antioxidant system of the brain positions the BVRA-Nrf2 axis as a promising therapeutic target for neurodegenerative disease. Significance StatementWe show a non-canonical role for biliverdin reductase A (BVRA), classically known as the biosynthetic enzyme for bilirubin, in nonenzymatic modulation of antioxidant neuroprotective nuclear factor erythroid-derived factor-like 2 (Nrf2) signaling in the brain. Both BVRA and Nrf2 signaling are compromised in neurodegenerative diseases such as Alzheimers disease, and the BVRA-Nrf2 axis offers a new direction for developing neuroprotective therapies.

neuroscience↗

bioRxiv2025

FUNCTIONAL ANALYSIS OF BIPARTITE NRF2 ACTIVATORS THAT OVERCOME FEEDBACK REGULATION FOR AGE-RELATED CHRONIC DISEASES

Activating Nrf2 with small molecules is a promising strategy for countering aging, oxidative stress, inflammation, and various disorders, including neurodegeneration. The primary regulator of Nrf2 protein stability is Keap1, a redox sensor protein and an adapter in the Cullin III ubiquitin ligase complex, which labels Nrf2 for proteasomal degradation. The known Nrf2 activators either chemically modify sensor thiols in Keap1 or competitively displace Nrf2 from the ubiquitin ligase complex. The latter approach is considered the most suitable for continuous administration, as non-specific chemical modifiers of Keap1 thiols also modify active thiols on other proteins, thus causing side effects. However, when transitioning from homogeneous to cell-based assays, genuine displacement activators show a significant loss in potency by several orders of magnitude. As we demonstrate here, this offset is due to the presence of high micromolar concentrations of Keap1 in both the cell lines and brain tissue. A potential solution could involve targeted delivery of an alkylating agent to Keap1 to achieve the desired specificity. Transcriptomic analysis of a cell-permeable Nrf2 peptide bearing an alkylating fumarate moiety indicates selective activation of the Nrf2 genetic program, confirming the high specificity of this approach. The Nrf2-triggered genetic program has a feedback regulation mechanism through the activation of Bach1, an Nrf2 transcriptional repressor, which is elevated in age-related neurodegeneration. Thus, a benign bipartite Nrf2 activator with Bach1 inhibition properties is needed for maximal benefits. The recently developed heterocyclic carboxamide, HPPE, shows overlap with the Nrf2 pathway activated by the fumarate-linked Nrf2 peptide and with zinc and tin protoporphyrins, which are recognized inhibitors of Bach1. Therefore, HPPE presents a promising and unique combination of the two desired activities that could be further optimized to treat age-related neurodegeneration. HighlightsO_LIThe decrease in potency for reversible displacement activators of Nrf2 in biological assays is attributed to high micromolar concentrations of Keap1 and competition with endogenous Keap1 client proteins. C_LIO_LINrf2 activators specific for Keap1 should combine a displacement scaffold with a substitution that undergoes intracellular conversion into active pro-oxidant or alkylating species. C_LIO_LICell-permeable fumarate-linked Nrf2 peptide solely activates the Nrf2 antioxidant genetic program, as demonstrated by transcriptomic analysis. C_LIO_LIHPPE, a small bipartite molecule, exhibits properties of both Nrf2 activation and Bach1 inhibition, to bypass feedback regulation by targeting both Keap1 and Bach1. C_LI

biochemistry↗

bioRxiv2025

Ex vivo high-resolution nano-computed tomography imaging reveals spatial architecture of the adult male mouse lower urogenital tract

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms/dysfunction (LUTS/LUTD) in aging men. Over the past 30 years, the prevalence of BPH has increased by 122%, rising from 50.7 million cases in 1990 to 112.5 million in 2021. It is expected that this number will continue to rise over the next 15 years with the global aging population. Although mouse models are invaluable for studying human disease, gross anatomical differences between human and murine prostates complicate their translational relevance for BPH/LUTS research. The purpose of this study was to develop and validate a nanoCT-based imaging approach to enable detailed anatomical analysis of the murine lower urinary tract, with the dual goals of advancing tools for LUTD research and improving the translational relevance of mouse models to human prostate disease. Advancements in nano-computed tomography (nanoCT) have enabled high-resolution characterization of murine organ anatomy, providing new insights into how morphological differences contribute to pathology. To accomplish this, whole lower urogenital tracts from 8-week-old healthy male C57BL/6J mice and microdissected urethras were imaged on a v|tome|x M or nanotom M nano-CT system, respectively. Images were processed for 2D segmentation and subsequent 3D reconstruction using DragonFly 3D World software. Our approach enabled high-resolution visualization and characterization of the murine lower urogenital tract, including the urinary bladder, prostate lobes, seminal vesicles, and ductus deferens, as well as the microscopic ductal architecture of the prostatic urethra. The resulting 3D reconstructions preserved native anatomical relationships and allowed for comparisons between murine and human prostate anatomy. Together, these findings establish a method that can be used for assessing anatomical and morphological changes associated with LUTD development, while also highlighting key anatomical similarities that enhance the translational relevance of mouse models for human prostate disease.

pathology↗

bioRxiv2025

Tissue resident memory T cells populate the human uveal tract

The current concept is that the eye is an immune privileged site endowed with innate immune regulatory networks to maintain organ function. We now have evidence that resident T cells occupy intraocular tissues. In immune-mediated inflammatory diseases, such as psoriasis and rheumatoid arthritis, tissue resident T cells trigger disease flares in the skin and joints. This suggests resident T cells in the uvea may have similar functions in non-infectious immune-mediated uveitis, a collective term for autoinflammatory and autoimmune diseases of the uveal tract causing intraocular inflammation. Here, we demonstrate by spectral cytometry and immunofluorescence imaging that non-inflamed uveal tissue contains multiple T cell subtypes including CD8+ CD103+ tissue resident memory T (TRM) cells. Using single cell RNA & T cell receptor (TCR) sequencing to profile aqueous humour cells from donors with acute, active uveitis, we identify clonally expanded T cells which are enriched for TRM -associated genes. We further show that in donors with active uveitis, CD8+ CD103+ T cells persist within tissue in the uveal tract. Using bulk RNA sequencing and weighted gene co-expression network analysis (WGCNA) we show that quiescent iris tissue from donors with a history of uveitis are enriched for genes associated with T cell activation and antigen presentation. Finally, we demonstrate that TRM cells persist in the anterior uvea in mice following resolution of experimental autoimmune uveoretinitis (EAU). Our results show that the human eye contains T cells both in health and during active inflammation. Our findings challenge the dogma that the eye is devoid of lymphocytes and supports the concept of resident T cell involvement in the pathogenesis of non-infectious immune-mediated uveitis and as promising targets for therapy. One Sentence SummaryT cells infiltrate aqueous humour during intraocular inflammation and have capacity to migrate into uveal tissue where they remain long-lived.

immunology↗

bioRxiv2023

The new mega dataset combined with a deep neural network makes progress in predicting the impact of single mutations on protein stability

Prediction of proteins stability change ({Delta}{Delta}G) due to single mutation is important for biotechnology, medicine, and our understanding of physics underlying protein folding. Despite the recent tremendous success in 3D protein structure prediction, the apparently simpler problem of predicting the effect of mutations on protein stability has been hampered by the low amount of experimental data. With the recent high-throughput measurements of mutational effects in mega experiment for ~850,000 mutations [Tsuboyama et al., bioRxiv, 2022] it becomes possible to apply the state-of-the-art deep learning methods. Here we explore the ability of ESM2 deep neural network architecture with added Light Attention mechanism to predict the change of protein stability due to single mutations. The resulting method ABYSSAL predicts well the data from the mega experiment (Pearson correlation 0.85) while the prediction of {Delta}{Delta}G values from previous experiments is more modest (Pearson correlation 0.50). ABYSSAL also shows a perfect satisfaction of the antisymmetry property. The ABYSSAL training demonstrated that the dataset should contain around ~100,000 data points for taking advantage of the state-of-the-art deep learning methods. Overall, our study shows great perspectives for developing the deep learning {Delta}{Delta}G predictors.

bioinformatics↗

bioRxiv2022

PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

Phosphatidylinositol 3-phosphate (PI3P) and Phosphatidylinositol 5-phosphate (PI5P) are low abundant phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P, in vitro. In this study, we report a novel role for PIP4K in regulating PI3P levels in Drosophila tissues. Loss-of-function mutants of the only PIP4K gene in Drosophila (dPIP4K29) show reduced cell size in larval salivary glands. We find that PI3P levels are elevated in dPIP4K29 tissues and that reverting PI3P levels back towards wild type, without changes in PI5P levels, can also rescue the reduced cell size phenotype. dPIP4K29 mutants also show an upregulation in autophagy and the reduced cell size can be reverted by decreasing Atg8a, that is required for autophagosome maturation. Lastly, increasing PI3P levels in wild type salivary glands can phenocopy the reduction in cell size and associated upregulation of autophagy seen in dPIP4K29. Thus, our study reports for the first time, a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size regulation that may explain the reported role of PIP4K in regulating neurodegeneration and tumour growth.

cell biology↗

bioRxiv2022

Follicular helper T cells expressing Blimp1 are specialized for plasma cell differentiation

B cells differentiate into antibody-producing plasma cells (PC) and germinal center (GC) B cells under the guidance of specialized CD4+ follicular helper T (TFH) cells. Here, we demonstrate that CD4 T cells require Prdm1 expression for both early PC differentiation and post-GC PC formation. Using dual Blimp1/Foxp3 reporter mice and single cell-indexed analysis, we segregate persistent compartments and expressed transcriptional programs of Blimp1+ CXCR5+PD1hi TFH (referred to here as PC-TFH) from canonical Blimp1- Bcl6+ TFH (GC-TFH) and Blimp1+Foxp3+ TFR immune regulators. Antigen recall expands localized PC-TFH compartments with rapidly divergent antigen-specific memory PC-TFH and GC-TFH programs. Thus, Blimp1 is a central mediator of PC-TFH function producing specialized TFH subsets that co-ordinate with GC-TFH function to establish high-affinity long-lasting protective immunity to vaccines and infection. One-Sentence SummaryBlimp1 expressing TFH cells express unique transcriptional programs to control PC formation RESEARCH ARTICLE SUMMARYO_ST_ABSIntroductionC_ST_ABSAdaptive B cell immunity rapidly emerges to form plasma cells (PC) for antibody production and non-PC that enter germinal centers (GC) to evolve higher affinity B cell receptors. Both pathways are essential to long-term high-affinity immune protection. The early PC to GC cell fate division is driven by B cell expression of mutually antagonist transcriptional repressors Blimp1 and Bcl6. This dichotomous B cell outcome is orchestrated through antigen-specific contact by follicular helper T (TFH) cells that express Bcl6 to upregulate CXCR5, localize into B cell regions and express transcriptional programs that influence B cell fate and function. It remains unclear what TFH cell mechanisms differentially impact these divergent B cell pathways. RationaleBlimp1 is found in Foxp3+ follicular regulatory T (TFR) cells known to impact GC B cell outcomes and play a role controlling antibody-mediated autoimmunity. In the context of infection, induced Blimp1 expression in CD4 T cells is expressed by conventional non-TFH effector cell compartments. Blimp1 segregates with emigrant CD4 T cells that leave the reactive lymphoid tissue to control innate immune function at the site of antigen entry. Conversely, Bcl6 is predominantly expressed in the GC regulating TFH pathway and is demonstrated to suppress Blimp1 expression. Germline ablation of Bcl6 exaggerates type 2 effector TH cell functions that promote excessive antibody production in the absence of the GC reaction. Similarly, loss of Bcl6 in CD4 T cells abrogates GC formation and post-GC PC responses, however multiple recent reports indicate continued support for antibody production without a Bcl6+ TFH compartment. To reconcile these findings, we propose a division of TFH function with separable pathways to regulate PC and GC differentiation. We hypothesize a central role for persistent CD4 T cell expressed Blimp1 that segregates early TFH transcriptional control to create an effector cell program that selectively targets PC differentiation. ResultsDirect intracellular staining for protein, confirmed with single Blimp1 and dual (Foxp3) reporter mice, identified Blimp1 expressing CXCR5+PD1hi TFH and TFR subsets within the spleen, bone marrow and other lymphoid tissues at steady-state. Conditional deletion of Prdm1 in CD4 T cells and adoptive transfer into immunodeficient hosts with splenic B cells, truncated both early pre-GC and late post-GC formation of PC providing a causal link to both pathways of differentiation in vivo. Across steady-state splenic T cells, in vitro activated Blimp1+CD25- CD4 T cells in T-B cell co-cultures correlated with significant levels of PC induction. Integrated single cell-indexed strategies segregate the transcriptional programs of Blimp1 expressing TFH cells (referred to here as PC-TFH) from canonical GC-inducing Bcl6+ TFH cells (GC-TFH), both distinct from Blimp1+ TFR cell programs in the steady-state. Immunization and recall produce follicular localized PC-TFH with pMHCII-tetramer binding memory response TFH cells that segregate across PC-TFH and GC-TFH compartments re-iterating the dichotomous transcriptome seen at steady-state. ConclusionThis study identifies Blimp1 as a key mediator of PC-TFH cells that sub-specialize as inducers of PC differentiation and bifurcate from the Bcl6+ GC-TFH cell pathway and functions. Persistent PC-TFH compartments assort across multiple lymphoid tissues at steady-state and are distinct from Foxp3+Blimp1+ TFR immune regulators. While PC TFH cells alone are required for early and rapid antibody responses, both TFH sub-classes are essential to the generation of high-affinity long-lived and memory response PC compartments. Cellular organization and molecular components of the PC-TFH transcriptional program indicate functional sub-specialization that can be separately targeted for immunotherapeutic purposes and adjuvant design in future vaccines. Sub-specialized Blimp1+ PC-TFH cells control PC differentiationAdaptive immune protection requires balancing the evolution of BCR affinity within germinal center (GC) B cells and the differentiation of plasma cells (PC) for production of antibodies. Both functional B cell pathways require the antigen-specific induction of specialized CD4+ follicular T (TFH) cells. Within GC-inducing TFH cells, Bcl6 is required to drive the formation and function of GC B cells. Here, we segregate PC-inducing TFH cells that require Blimp1 as a key mediator of antigen-specific PC differentiation. The Blimp1+ PC-TFH transcriptional program diverges from Bcl6+ GC-TFH compartment and Blimp1+Foxp3+ follicular regulatory T (TFR) compartments. Antigen-specific PC-TFH emerge and segregate rapidly from GC-TFH after priming and recall to co-operatively induce effective long-term adaptive immunity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/486642v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@b96936org.highwire.dtl.DTLVardef@aa8be3org.highwire.dtl.DTLVardef@c6095aorg.highwire.dtl.DTLVardef@1900ec7_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Redundant function of Ets1 and Ets2 in regulating M-phase progression in post-natal angiogenesis

Angiogenesis is a highly orchestrated process involving complex crosstalk between several endothelial cell processes including cell cycle, cell survival and migration. Transcription factors ETS1 and ETS2 are required for EC functions necessary for embryonic angiogenesis. Here, by utilizing endothelial cell-specific deletion of Ets1 and Ets2 on post-natal angiogenesis we demonstrate that ETS1 and ETS2 have a redundant regulatory role in controlling the expression of key G2/M regulators and anti-apoptotic gene modules by recruiting the transcriptional activator CBP/p300 to the enhancers of these genes. Further, cultured aortic ECs lacking both ETS1 and ETS2 demonstrated G2/M phase arrest and increased apoptosis. In vivo, EC infiltration and invasion was attenuated when matrigel admixed with mouse mammary tumor cells was injected into adult mice with EC-specific ablation of Ets1 and Ets2. These results demonstrate that deletion of Ets1 and Ets2 in endothelial cells inhibits angiogenesis by altering cell cycle progression and decreasing cell survival.

genomics↗