Biology subjects

Das, N. K.

Publications and source records attributed to Das, N. K..

10 recordsLinked to original sources

bioRxiv2025

Myoglobin leaching into the serum of IDA mice is driven by the high activation of sGC under anemic conditions which induces myoglobin expression

Our study reveals that status of the sGC heterodimer or its subsequent activation aligns with active erythropoiesis, and this heterodimer also correlates with the expression of myoglobin (Mb) or HO1. In this study we found that Mb expression which is driven by iron restriction and high sGC activation in iron deficiency anemia (IDA) leaches out more into the serum relative to non-anemic WTs. Tissues from IDA mice of both models developed either by nutritional iron deprivation or by ablation of ferroportin (Fpn) gene or from iron refractory iron deficiency anemia (IRIDA) mice found that Mb expression follows a variable pattern in different tissues but always correlates to the status of the sGC heterodimer or its subsequent activation. Here higher Mb expression happening in anemic (IDA, Fe<5 ppm or IDA, Fpn) or non-anemic WT mice is both due to iron restriction and an elevated sGC heterodimer that corroborated with greater sGC activation. More importantly we find significant leaching of Mb into the serum of these anemic (IDA) mice from both models and our spectral data suggests that this Mb is heme-free. This Mb leaching in anemia is a cumulative impact of Mb secreting out from various tissues including lungs, spleen, skeletal or cardiac muscles where Mb is expressed and not just in the skeletal muscles where Mb expression is low. Based on these findings we construct a working model of anemia, where high activation of sGC under anemic conditions (Fpn ablation or restricted Fe diet) induces Apo-Mb or heme-free Mb expression which can then leach out into the serum. Our findings of Mb leaching are novel and can find further application as a diagnostic strategy in anemia.

biochemistry↗

bioRxiv2025

Aspartate transaminases are required for blood development

Red blood cells (RBCs) have a limited lifespan of approximately 120 days. This necessitates continuous RBC production, resulting in [~]200 billion new RBCs made per day to maintain oxygen delivery. Despite this enormous biosynthetic demand, the metabolic pathways supporting erythropoiesis are poorly understood. We profiled metabolites across four independent models of elevated erythropoiesis and a consistent increase in aspartate levels emerged when compared to controls. This suggested a potential role for aspartate metabolism in RBC production. To test this, we deleted the aspartate aminotransferases Got1 or Got2 globally or in an erythroid-specific manner. Loss of either enzyme resulted in anemia, with Got2 deficiency producing a more severe phenotype. Individual loss of either Got or dual Got deletion led to an erythroid defect, where early progenitors accumulated. In human and mouse models of erythropoiesis, GOT1 and GOT2 loss had opposing impacts on aspartate despite exhibiting similar anemic phenotypes, suggesting an aspartate-independent function for these enzymes. GOT1 and GOT2 are also components of the malate-aspartate shuttle (MAS), which regulates NAD(H) homeostasis. However, conditional deletion of another MAS enzyme, Mdh1, did not cause anemia, and alleviating NADH reductive stress in GOT2-deficient cells with cytoplasmic bacterial NADH oxidase (LbNOX) failed to restore erythropoiesis. Instead, transcriptomic and epigenetic analyses revealed dysregulation of chromatin histone modifications in GOT-deficient erythroid cells, implicating epigenetic dysfunction as a driver of defective erythropoiesis. Collectively, these findings identify a previously unrecognized role for GOT1 and GOT2 as a central link between metabolism and epigenetic regulation during erythroid development. These insights may inform the development of new therapeutic strategies for anemia.

biochemistry↗

bioRxiv2025

Intraoperative arteriovenous patient sampling to assess in situ non-small cell lung cancer metabolism

We performed intraoperative arteriovenous sampling in participants undergoing surgical resection for non-small cell lung cancer (NSCLC) to characterize in situ tumor metabolism, directly measuring metabolite consumption and secretion in tumor-bearing lung versus normal lung and the systemic circulation. Healthy lung tissue secreted lower levels of lactate, pyruvate, and tricarboxylic acid (TCA) intermediates and consumed less glucose compared to the systemic circulation. In contrast, tumor-bearing lung demonstrated elevated lactate secretion, along with increased efflux of succinate, fumarate, glycine, and aspartate, despite similar glucose uptake. Lactate secretion correlated with tumor PET avidity but not size, and overall metabolic profiles distinguished cancerous from normal lung tissue. These findings confirm enhanced glycolysis in NSCLC in vivo, while also revealing context-dependent patterns of TCA metabolite accumulation and amino acid secretion. Our results demonstrate the utility of intraoperative sampling to uncover metabolic features of human tumors.

cancer biology↗

bioRxiv2025

Labile iron pool dynamics do not drive ferroptosis potentiation in colorectal cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC tumors exhibit aberrant iron accumulation, which supports tumor cell proliferation through multiple metabolic pathways. However, the oncogenic benefits of elevated iron must be counterbalanced by its potential to catalyze oxidative damage via reactive oxygen species generated from labile, redox-active iron. Ferroptosis is a regulated, non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. This process is tightly controlled by the selenoenzyme glutathione peroxidase 4 (GPX4), which reduces lipid peroxides and can be pharmacologically inhibited by agents such as RSL3 and JKE1674. A key source of redox-active iron is the labile iron pool (LIP), yet its role in regulating ferroptosis remains incompletely defined. To examine this, we supplemented CRC cells with exogenous iron following pharmacologic induction of ferroptosis. Iron supplementation significantly reduced cell viability, suggesting that expansion of the LIP potentiates ferroptotic cell death. However, whether ferroptosis is accompanied by dynamic changes in the LIP, and if such changes are mechanistically required for its potentiation, were unknown. To further characterize this response, we profiled the expression of iron regulatory genes under ferroptotic conditions and observed no change in transcriptional response in iron homeostasis genes. Using a reactivity-based probe of labile iron, we found that the LIP did not measurably increase during ferroptosis induction with GPX4 inhibition or inhibition of the SLC7A11 cysteine/glutamate antiporter. These findings suggest that the LIP does not expand upon pharmacological initiated ferroptosis, despite the potentiating effect of exogenous iron supplementation.

cancer biology↗

bioRxiv2024

Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer

Ferroptosis is a non-apoptotic form of cell death resulting from the iron-dependent accumulation of lipid peroxides. Colorectal cancer (CRC) cells accumulate high levels of intracellular iron and reactive oxygen species (ROS) and are thus particularly sensitive to ferroptosis. The compound (S)-RSL3 ([1S,3R]-RSL3) is a commonly used ferroptosis inducing compound that is currently characterized as a selective inhibitor of the selenocysteine containing enzyme (selenoprotein) Gluathione Peroxidase 4 (GPx4), an enzyme that utilizes glutathione to directly detoxify lipid peroxides. However, through chemical controls utilizing the (R) stereoisomer of RSL3 ([1R,3R]-RSL3) that does not bind GPx4, combined with inducible genetic knockdowns of GPx4 in CRC cell lines, we revealed that GPx4 dependency does not always align with (S)-RSL3 sensitivity, questioning the current characterization of GPx4 as the central regulator of ferroptosis. Utilizing affinity pull-down mass spectrometry with chemically modified (S)-RSL3 probes we discovered that the effects of (S)-RSL3 extend far beyond GPx4 inhibition, revealing that (S)-RSL3 is a broad and non-selective inhibitor of selenoproteins. To further investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed additional chemical and genetic approaches. We found that the selenoprotein inhibitor auranofin, an FDA approved gold-salt, chemically induced oxidative cell death and ferroptosis in both in-vitro and in-vivo models of CRC. Consistent with these data, we found that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translation of selenoproteins, is essential for the in-vitro growth and xenograft survival of CRC cell lines. In summary, these findings recharacterize the mechanism of action of the most commonly used ferroptosis inducing molecule, (S)-RSL3, and reveal that broad inhibition of selenoproteins is a promising novel therapeutic angle for the treatment of CRC.

cancer biology↗

bioRxiv2023

A diet of oxidative stress-adapted bacteria improves stress resistance and lifespan in C. elegans via p38-MAPK.

Organisms across taxa are exposed to stresses such as variable temperature, redox imbalance, and xenobiotics. Successfully responding to stress and restoring homeostasis is crucial for viability of the organism. During aging, the ability to effectively respond to stress declines, contributing to development of disease. In many multicellular animals, aging also coincides with changes in the microbiome that can contribute to disease-states. Because animals and their microbiota coexist in the same broad environment, they each must adapt to similar stresses. However, the short generation time of microbes leads to faster evolution, allowing the possibility that microbial stress adaptation may influence host physiology. Here we leverage a simplified model involving the nematode C. elegans and its bacterial diet. Our work highlights how bacterial adaptation to oxidative stress impacts the hosts lifespan and response to stress. Intriguingly, our findings reveal that worms fed with bacteria adapted to withstand oxidative stress exhibit enhanced stress resistance and an extended lifespan. Through whole genome sequencing, genetic assays, and metabolic analysis, this study underscores the pivotal role of the bacterial iron-sulfur pathway in governing host stress resistance and lifespan. We further find that iron in the stress-evolved bacteria boost the worms stress resistance and lifespan through activation of the mitogen-activated protein kinase (MAPK) pathway. In conclusion, this study provides evidence that understanding the evolutionary path of microbial adaptation during stress could be leveraged to slow aging and mitigate age-related decline in health.

molecular biology↗

bioRxiv2022

PINK1 supports colorectal cancer growth by regulating the labile iron pool.

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. We show that PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth following nutrient stress. However, the mechanism by which PINK1 maintains colon cancer growth remains equivocal. Inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited colon cancer cell proliferation, whereas disruption of other mitophagy receptors did not similarly impact cellular proliferation. Mechanistically, we observed a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione following PINK1 KD. Mitochondria are important hubs for storing iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. An increase iron storage protein ferritin and a decrease labile iron pool was observed in PINK1 KD cells. However, neither total cellular iron nor markers of iron starvation/overload were affected. Cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). Overexpressing nuclear receptor coactivator 4 (NCOA4), a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. We demonstrate that PINK1 regulates intracellular iron availability by integrating mitophagy to ferritinophagy. In conclusion, these results indicate that PINK1 is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

cancer biology↗

bioRxiv2022

Microenvironmental Ammonia Enhances T cell Exhaustion in Colorectal Cancer

Effective therapies are lacking for patients with advanced colorectal cancer (CRC). The CRC tumor microenvironment has elevated metabolic waste products due to altered metabolism and proximity to the microbiota. The role of metabolite waste in tumor development, progression, and treatment resistance is unclear. We generated an autochthonous metastatic mouse model of CRC and unbiased multi-omic analyses in this model reveals a robust accumulation of tumoral ammonia. The high ammonia levels induce T cell metabolic reprogramming, increase exhaustion and decrease proliferation. CRC patients have increased serum ammonia, and our ammonia-related gene signature correlates with altered T cell response, adverse patient outcomes, and lack of response to immune checkpoint blockade. We demonstrate that enhancing ammonia clearance reactivates T cells, decreases tumor growth, and extends survival. Moreover, decreasing tumor-associated ammonia enhances anti-PD-L1 efficacy. Our findings indicate that ammonia detoxification can reactivate T cells, highlighting a new approach to enhance the efficacy of immunotherapies. Statement of SignificanceWe demonstrate that ammonia accumulates in the microenvironment of colorectal cancer. Ammonia alters T-cells redox singling leading to a decrease in T cell proliferation and an increase in T cell exhaustion. Enhancing ammonia clearance reduces tumor size, increases survival, and increases the efficacy to immunotherapies.

cancer biology↗

bioRxiv2020

Microbiota metabolic exchange is critical for colorectal cancer redox homeostasis and growth

Intestinal microbiota play a fundamental role in human health and disease. Microbial dysbiosis is a hallmark of colorectal cancer (CRC) as tumor stage-specific shifts potentiate tumor growth, influence the inflammatory microenvironment, and alter response to therapy. Recent work has demonstrated a critical role for microbial metabolite exchange in host response. However, the role of most microbial metabolites in colon cancer growth is unclear. To better understand how metabolic exchange between the microbiota and tumor epithelium alter CRC growth, a screen of the most abundant bacterially derived metabolites was assessed. Several metabolites were found to alter CRC growth, but reuterin most significantly suppressed CRC cell proliferation. Reuterin is a bifunctional metabolite containing both hydroxy and aldehyde functional groups. Reuterin is primarily synthesized from glycerol by Lactobacillus reuteri, a commensal bacterium found throughout the gastrointestinal tract. We found that reuterin suppresses growth via alterations to the redox balance of CRC cells. Mechanistically, reuterin potentiates reactive oxygen species (ROS) which leads to irreversible cysteine oxidation and enhanced cell death. Supplementation of either antioxidants or hydrogen sulfide fully rescued growth, suggesting that reuterin is suppressing CRC growth through protein oxidation. These studies demonstrate the potential of reuterin to act as a potent chemotherapeutic for treating colorectal cancers.

cancer biology↗

bioRxiv2020

Intestinal ferritinophagy is regulated by HIF-2 and is essential for systemic iron homeostasis

Iron is critical for many processes including oxygen transport and erythropoiesis. Transcriptomic analysis demonstrates that HIF-2 regulates over 90% of all transcripts induced following iron deficiency in the intestine. However, beyond divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1) and duodenal cytochrome b (Dcytb), no other genes/pathways have been critically assessed with respects to their importance in intestinal iron absorption. Ferritinophagy is associated with cargo specific autophagic breakdown of ferritin and subsequent release of iron. We show here that nuclear receptor co-activator 4 (NCOA4)-mediated intestinal ferritinophagy is integrated to systemic iron demand via HIF-2. Duodenal NCOA4 expression is regulated by HIF-2 during high systemic iron demands. Moreover, overexpression of intestinal HIF-2 is sufficient to activate NCOA4 and promote lysosomal degradation of ferritin. Promoter analysis revealed NCOA4 as a direct HIF-2 target. To demonstrate the importance of intestinal HIF-2/ferritinophagy axis in systemic iron homeostasis, whole body and intestine-specific NCOA4-null mouse lines were assessed. These analyses demonstrate an iron sequestration in the enterocytes, and significantly high tissue ferritin levels in the dietary iron deficiency and acute hemolytic anemia models. Together, our data suggests efficient ferritinophagy is critical for intestinal iron absorption and systemic iron homeostasis.

physiology↗