Biology subjects

Caldwell, S.

Publications and source records attributed to Caldwell, S..

10 recordsLinked to original sources

bioRxiv2026

Cancer cells acquire cysteine directly from a reduced tumor microenvironment

The metabolic environment of a tumor is shaped by the nutrients available in its interstitial fluid, yet the redox composition of that fluid is poorly defined. Measuring sulfur metabolites in the interstitial fluid of tumors, matched healthy organs, and serum across three mouse models of cancer, we find that the tumor microenvironment (TME) is a chemically reduced compartment in which free cysteine predominates over its oxidized form, cystine. This is especially pronounced in the lung TME, where cystine is nearly absent. We further show that lung cancer cells take up cysteine and cystine with comparable efficiency and proliferate on cysteine alone, and that the abundant glutathione in the TME helps keep cysteine reduced through disulfide exchange. In a medium formulated to match the sulfur composition of the lung tumor interstitial fluid, cells become independent of cystine import through xCT. Cells cultured in this medium were resistant to glutaminase inhibitors, oxidants and ferroptosis inducers, and better maintained NADPH levels under oxidative challenge. These findings suggest that standard culture conditions overestimate the dependence of cancer cells on cystine uptake and their vulnerability to oxidative stress-targeting therapies.

cancer biology↗

bioRxiv2026

PHGDH is a targetable driver of PDAC progression

Pancreatic ductal adenocarcinoma (PDAC) arises in a nutrient-deprived microenvironment through progressive stages from pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma. While serine metabolism supports tumor growth across multiple cancer types, the stage-specific role of de novo serine synthesis in PDAC evolution remains undefined. Here, we show that expression of phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of serine biosynthesis, increases progressively from PanIN to invasive PDAC in human and mouse specimens. Using genetically engineered mouse models with inducible PHGDH knockdown, we found that PHGDH loss delayed PDAC development. Unexpectedly, PHGDH-deficient tumors did not increase reliance on exogenous serine, and dietary serine/glycine manipulation had no effect on tumor development. Instead, stable isotope tracing and metabolomic profiling revealed that PHGDH loss suppressed mTOR signaling, reduced expression of the glutamine transporter ASCT2, and impaired glutamine uptake and utilization. Leveraging this metabolic liability, we demonstrated that PHGDH-deficient tumors exhibited selective sensitivity to the glutamine antagonist DRP-104, whereas PHGDH-intact tumors were resistant. These findings reveal an unanticipated connection between serine biosynthesis and glutamine metabolism in PDAC and identify a therapeutic vulnerability that may be exploited through combined metabolic targeting. Statement of significancePHGDH supports PDAC progression not primarily through serine provision, but by maintaining glutamine metabolism and mTOR signaling. This unanticipated metabolic crosstalk creates a synthetic lethal vulnerability to glutamine antagonism in PHGDH-deficient tumors, providing a rationale for combining serine synthesis pathway inhibitors with glutamine-targeting therapies in pancreatic cancer.

cancer biology↗

bioRxiv2025

A simple, anesthesia-free infusion technique for in vivo metabolic tracing

Accurate metabolic flux analysis requires tracer delivery that preserves physiological metabolism. Current methods may distort metabolism through anesthesia, surgical stress, or complex procedures. We demonstrate that isoflurane anesthesia profoundly alters serum and tissue metabolism across multiple pathways. Glycolytic and TCA cycle intermediates, sulfur and aromatic amino acid metabolites, acylcarnitines, and nucleotide pools decreased, while branched-chain amino acids, their ketoacids, ketone bodies, and fatty acids increased. These coordinated changes were suggestive of mitochondrial complex I inhibition and reduced oxidative catabolism, leading to shifts in metabolite pool sizes that compromise isotopologue-based flux interpretation. We established a tail vein catheterization method completed in minutes under brief anesthesia that enables multi-hour tracer infusion in awake, freely moving mice. This method achieved steady-state labeling of cystine and downstream products comparable to jugular infusion without supraphysiologic cystine accumulation. This platform provides a practical, physiologically accurate method for in vivo steady-state isotope tracing.

biochemistry↗

bioRxiv2024

Distinct Roles for the Thioredoxin and Glutathione Antioxidant Systems in Nrf2-Mediated Lung Tumor Initiation and Progression

AbstractRedox regulators are emerging as critical mediators of lung tumorigenesis. NRF2 and its negative regulator KEAP1 are commonly mutated in human lung cancers, leading to NRF2 accumulation and constitutive expression of NRF2 target genes, many of which are at the interface between antioxidant function and anabolic processes that support cellular proliferation. Nrf2 activation promotes lung tumor initiation and early progression in murine models of lung cancer, but which Nrf2 targets mediate these phenotypes is unknown. Nrf2 regulates two parallel antioxidant systems mediated by thioredoxin reductase 1 (TXNRD1) and glutathione reductase (GSR), which promote the reduction of protein antioxidant thioredoxin (TXN) and tripeptide antioxidant glutathione (GSH), respectively. We deleted TXNRD1 and GSR alone, or in combination, in lung tumors harboring mutations in KrasG12D and Nrf2D29H. We found that tumor initiation was promoted by expression of GSR, but not TXNRD1, regardless of Nrf2 status. In contrast, Nrf2D29H tumors, but not Nrf2WT, were dependent on TXNRD1 for tumor progression, while GSR was dispensable. Simultaneous deletion of GSR and TXNRD1 reduced initiation and progression independent of Nrf2 status, but surprisingly did not completely abrogate tumor formation. Thus, the thioredoxin and glutathione antioxidant systems play unique roles in tumor initiation and progression.

cancer biology↗

bioRxiv2023

Local structural flexibility drives oligomorphism in computationally designed protein assemblies

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

bioengineering↗

bioRxiv2023

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well-known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro, and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

microbiology↗

bioRxiv2022

The Origin Of Cysteine and its Catabolism in Mammalian Tissues and Tumors

Cysteine plays critical roles in cellular biosynthesis, enzyme catalysis, and is an essential contributor to redox metabolism. While cultured cells are highly dependent on exogenous cystine for proliferation and survival, how diverse tissues obtain and use cysteine in vivo has not been characterized. We comprehensively interrogated cysteine metabolism in normal murine tissues and the cancer that arise from them using stable isotope 13C-serine and 13C-cystine tracing. We found that de novo cysteine synthesis was highest in normal liver and pancreas and absent in lung tissue. In tumors, cysteine synthesis was either inactive or downregulated during tumorigenesis. By contrast, cystine uptake and metabolism to downstream metabolites was a universal feature of normal tissues and tumors. Differences in cysteine catabolism were evident across tumor types, including glutathione synthesis. Thus, cystine is a major contributor to the cysteine pool in tumors and cysteine catabolic pathways are differentially active across tumor types.

cancer biology↗

bioRxiv2022

Distinct Nrf2 Signaling Thresholds Mediate Lung Tumor Initiation and Progression

Mutations in the KEAP1-NRF2 pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases and often confer resistance to therapy and poor outcomes. Here, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and comprehensively interrogated their impact on tumor initiation and progression. Chronic Nrf2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors p53 or Lkb1. When combined with KrasG12D/+, constitutive Nrf2 activation promoted lung tumor initiation and early progression of hyperplasia to low-grade tumors but impaired their progression to advanced-grade tumors, which was reversed by Nrf2 deletion. Finally, NRF2 overexpression in KEAP1 mutant NSCLC cell lines was detrimental to cell proliferation, viability, and anchorage-independent colony formation. Collectively, our results establish the context-dependence and activity threshold for NRF2 during the lung tumorigenic process. SignificanceThis study reports murine lung cancer models harboring mutations in the Keap1/Nrf2 pathway and highlights the context-dependent and diverse roles of Nrf2 during lung tumor initiation and progression.

cancer biology↗

bioRxiv2021

Chronic Presence of Blood Circulating Anti-NMDAR1 Autoantibodies Impairs Cognitive Function in Mice

High titers of anti-NMDAR1 autoantibodies in brain cause anti-NMDAR1 encephalitis that displays psychiatric symptoms of schizophrenia and/or other psychiatric disorders in addition to neurological symptoms. Low titers of anti-NMDAR1 autoantibodies are reported in the blood of a subset of the general human population and psychiatric patients. Since [~]0.1-0.2% of blood circulating antibodies cross the blood-brain barriers and antibodies can persist for months and years in human blood, it is important to investigate whether chronic presence of these blood circulating anti- NMDAR1 autoantibodies may impair human cognitive functions and contribute to the development of psychiatric symptoms. Here, we generated mice carrying low titers of anti-NMDAR1 autoantibodies in blood against a single antigenic epitope of mouse NMDAR1. Mice carrying the anti-NMDAR1 autoantibodies are healthy and display no differences in locomotion, sensorimotor gating, and contextual memory compared to controls. Chronic presence of the blood circulating anti-NMDAR1 autoantibodies, however, is sufficient to specifically impair T-maze spontaneous alternation in the integrity of blood-brain barriers across all 3 independent mouse cohorts, indicating a robust cognitive deficit in spatial working memory and/or novelty detection. Our studies implicate that chronic presence of low titers of blood circulating anti-NMDAR1 autoantibodies may impair cognitive functions in both the general healthy human population and psychiatric patients.

neuroscience↗

bioRxiv2017

Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis

Pathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts and Mycobacterium tuberculosis (Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infection in mammals. While the uptake of cholesterol by Mtb is mediated by the Mce4 transporter, the route(s) of uptake of fatty acids remain unknown. Here, we demonstrate that an uncharacterized protein LucA, integrates the assimilation of both cholesterol and fatty acids in Mtb. LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters. We also demonstrate that Mce1 functions as an important fatty acid transporter in Mtb and we determine that the integration of cholesterol and fatty acid transport by LucA is required for full bacterial virulence in vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key role for LucA in coordinating both transport activities.

microbiology↗