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Iron is an essential element for life owing to its ability to participate in a diverse array of oxidation-reduction reactions. However, misregulation of iron-dependent redox cycling can also produce oxidative stress, contributing to cell growth, proliferation, and death pathways underlying aging, cancer, neurodegeneration, and metabolic diseases. Fluorescent probes that selectively monitor loosely bound Fe(II) ions, termed the labile iron pool, are potentially powerful tools for studies of this metal nutrient; however, the dynamic spatiotemporal nature and potent fluorescence quenching capacity of these bioavailable metal stores pose challenges for their detection. Here, we report a tandem activity-based sensing and labeling strategy that enables imaging of labile iron pools in live cells through enhancement in cellular retention. Iron green-1 fluoromethyl (IG1-FM) reacts selectively with Fe(II) using an endoperoxide trigger to release a quinone methide dye for subsequent attachment to proximal biological nucleophiles, providing a permanent fluorescent stain at sites of elevated labile iron. IG1-FM imaging reveals that degradation of the major iron storage protein ferritin through ferritinophagy expands the labile iron pool, while activation of nuclear factor-erythroid 2-related factor 2 (NRF2) antioxidant response elements (AREs) depletes it. We further show that lung cancer cells with heightened NRF2 activation, and thus lower basal labile iron, have reduced viability when treated with an iron chelator. By connecting labile iron pools and NRF2-ARE activity to a druggable metal-dependent vulnerability in cancer, this work provides a starting point for broader investigations into the roles of transition metal and antioxidant signaling pathways in health and disease.
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Elementos de Resposta Antioxidante , Ferro , Humanos , Ferro/metabolismo , Corantes Fluorescentes/química , Fator 2 Relacionado a NF-E2/metabolismo , Ferritinas/metabolismo , Estresse Oxidativo , Oxirredução , Linhagem Celular Tumoral , Antioxidantes/metabolismoRESUMO
Glioblastoma is the most common and lethal central nervous system malignancy with a median survival after progression of only 6-9 months. Major biochemical mechanisms implicated in glioblastoma recurrence include aberrant molecular pathways, a recurrence-inducing tumor microenvironment, and epigenetic modifications. Contemporary standard-of-care (surgery, radiation, chemotherapy, and tumor treating fields) helps to control the primary tumor but rarely prevents relapse. Cytoreductive treatment such as surgery has shown benefits in recurrent glioblastoma; however, its use remains controversial. Several innovative treatments are emerging for recurrent glioblastoma, including checkpoint inhibitors, chimeric antigen receptor T cell therapy, oncolytic virotherapy, nanoparticle delivery, laser interstitial thermal therapy, and photodynamic therapy. This review seeks to provide readers with an overview of (1) recent discoveries in the molecular basis of recurrence; (2) the role of surgery in treating recurrence; and (3) novel treatment paradigms emerging for recurrent glioblastoma.
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Neoplasias Encefálicas , Glioblastoma , Recidiva Local de Neoplasia , Glioblastoma/terapia , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Recidiva Local de Neoplasia/terapia , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Microambiente Tumoral , Terapia Viral Oncolítica/métodos , AnimaisRESUMO
Angela Casini (Technical University of Munich, Germany), Hui Chao (Sun Yat-Sen University, China), Hongzhe Sun (University of Hong Kong, China), and Christopher J. Chang (University of California, Berkeley, United States) introduce the themed collection on 'Chemical biology of metals'.
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Copper plays critical roles as a metal active site cofactor and metalloallosteric signal for enzymes involved in cell proliferation and metabolism, making it an attractive target for cancer therapy. In this study, we investigated the efficacy of polydopamine nanoparticles (PDA NPs), classically applied for metal removal from water, as a therapeutic strategy for depleting intracellular labile copper pools in triple-negative breast cancer models through the metal-chelating groups present on the PDA surface. By using the activity-based sensing probe FCP-1, we could track the PDA-induced labile copper depletion while leaving total copper levels unchanged and link it to the selective MDA-MB-231 cell death. Further mechanistic investigations revealed that PDA NPs increased reactive oxygen species (ROS) levels, potentially through the inactivation of superoxide dismutase 1 (SOD1), a copper-dependent antioxidant enzyme. Additionally, PDA NPs were found to interact with the mitochondrial membrane, resulting in an increase in the mitochondrial membrane potential, which may contribute to enhanced ROS production. We employed an in vivo tumor model to validate the therapeutic efficacy of PDA NPs. Remarkably, in the absence of any additional treatment, the presence of PDA NPs alone led to a significant reduction in tumor volume by a factor of 1.66 after 22 days of tumor growth. Our findings highlight the potential of PDA NPs as a promising therapeutic approach for selectively targeting cancer by modulating copper levels and inducing oxidative stress, leading to tumor growth inhibition as shown in these triple-negative breast cancer models.
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Cobre , Indóis , Nanopartículas , Polímeros , Espécies Reativas de Oxigênio , Neoplasias de Mama Triplo Negativas , Cobre/química , Cobre/farmacologia , Polímeros/química , Polímeros/farmacologia , Indóis/química , Indóis/farmacologia , Humanos , Animais , Camundongos , Nanopartículas/química , Feminino , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Oxirredução , Nanomedicina , Proliferação de Células/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Superóxido Dismutase-1/metabolismoRESUMO
Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.
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Formaldehyde (FA) is both a highly reactive environmental genotoxin and an endogenously produced metabolite that functions as a signaling molecule and one-carbon (1C) store to regulate 1C metabolism and epigenetics in the cell. Owing to its signal-stress duality, cells have evolved multiple clearance mechanisms to maintain FA homeostasis, acting to avoid the established genotoxicity of FA while also redirecting FA-derived carbon units into the biosynthesis of essential nucleobases and amino acids. The highly compartmentalized nature of FA exposure, production, and regulation motivates the development of chemical tools that enable monitoring of transient FA fluxes with subcellular resolution. Here we report a mitochondrial-targeted, activity-based sensing probe for ratiometric FA detection, MitoRFAP-2, and apply this reagent to monitor endogenous mitochondrial sources and sinks of this 1C unit. We establish the utility of subcellular localization by showing that MitoRFAP-2 is sensitive enough to detect changes in mitochondrial FA pools with genetic and pharmacological modulation of enzymes involved in 1C and amino acid metabolism, including the pervasive, less active genetic mutant aldehyde dehydrogenase 2*2 (ALDH2*2), where previous, non-targeted versions of FA sensors are not. Finally, we used MitoRFAP-2 to comparatively profile basal levels of FA across a panel of breast cancer cell lines, finding that FA-dependent fluorescence correlates with expression levels of enzymes involved in 1C metabolism. By showcasing the ability of MitoRFAP-2 to identify new information on mitochondrial FA homeostasis, this work provides a starting point for the design of a broader range of chemical probes for detecting physiologically important aldehydes with subcellular resolution and a useful reagent for further studies of 1C biology.
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In the era of big data in human genetics, large-scale biobanks aggregating genetic data from diverse populations have emerged as important for advancing our understanding of human health and disease. However, the computational and storage demands of whole genome sequencing (WGS) studies pose significant challenges, especially for researchers from underfunded institutions or developing countries, creating a disparity in research capabilities. We introduce new approaches that significantly enhance computational efficiency and reduce data storage requirements for WGS studies. By developing algorithms for compressed storage of genetic data, focusing particularly on optimizing the representation of rare variants, and designing regression methods tailored for the scale and complexity of WGS data, we significantly lower computational and storage costs. We integrate our approach into PLINK 2.0. The implementation demonstrates considerable reductions in storage space and computational time without compromising analytical accuracy, as evidenced by the application to the AllofUs project data. We improve runtime of an exome-wide association analysis of 19.4 million variants and a single phenotype from 695.35 minutes (approximately 11.5 hours) on a single machine to 1.57 minutes using 30Gb of memory and 50 threads (8.67 minutes using 4 threads). Similarly, we generalize to multi-phenotype analysis. We anticipate that our approach will enable researchers across the globe to unlock the potential of population biobanks, accelerating the pace of discoveries that can improve our understanding of human health and disease.
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Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.
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Autoanticorpos , Lúpus Eritematoso Discoide , Lúpus Eritematoso Sistêmico , Pele , Humanos , Lúpus Eritematoso Discoide/imunologia , Lúpus Eritematoso Discoide/patologia , Feminino , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Autoanticorpos/imunologia , Autoanticorpos/sangue , Pele/patologia , Pele/imunologia , Pele/metabolismo , Adulto , Pessoa de Meia-Idade , Alelos , Antígenos HLA/genética , Antígenos HLA/imunologia , Adulto Jovem , MultiômicaRESUMO
Although transition metals constitute less than 0.1% of the total mass within a human body, they have a substantial impact on fundamental biological processes across all kingdoms of life. Indeed, these nutrients play crucial roles in the physiological functions of enzymes, with the redox properties of many of these metals being essential to their activity. At the same time, imbalances in transition metal pools can be detrimental to health. Modern analytical techniques are helping to illuminate the workings of metal homeostasis at a molecular and atomic level, their spatial localization in real time, and the implications of metal dysregulation in disease pathogenesis. Fluorescence microscopy has proven to be one of the most promising non-invasive methods for studying metal pools in biological samples. The accuracy and sensitivity of bioimaging experiments are predominantly determined by the fluorescent metal-responsive sensor, highlighting the importance of rational probe design for such measurements. This review covers activity- and binding-based fluorescent metal sensors that have been applied to cellular studies. We focus on the essential redox-active metals: iron, copper, manganese, cobalt, chromium, and nickel. We aim to encourage further targeted efforts in developing innovative approaches to understanding the biological chemistry of redox-active metals.
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Corantes Fluorescentes , Oxirredução , Corantes Fluorescentes/química , Humanos , Metais/química , Metais/metabolismo , Animais , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Microscopia de FluorescênciaRESUMO
Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.
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NAD , Neoplasias , Humanos , Hidrogenação , NAD/metabolismo , Carbono , Formiatos/químicaRESUMO
Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.
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Cátions , Ciclização , Indicadores e Reagentes , Proteínas , Triptofano , Cátions/química , Indicadores e Reagentes/química , Oxirredução , Proteoma/química , Triptofano/química , Peptídeos/química , Química Click , Proteínas/químicaRESUMO
Formaldehyde is commonly thought of as an environmental toxin or laboratory fixation reagent, but there is a growing appreciation for its broader physiological contributions as a naturally generated one-carbon metabolite across all kingdoms of life. In this In Focus article, we summarize emerging advances in the field that show how formaldehyde plays diverse roles as a one-carbon signal in DNA damage, one-carbon metabolism, and epigenetic regulation.
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Carbono , Epigênese Genética , Carbono/metabolismo , Metilação , Dano ao DNA , Formaldeído/metabolismo , Metilação de DNARESUMO
Docetaxel has been the standard first-line chemotherapy for lethal metastatic prostate cancer (mPCa) since 2004, but resistance to docetaxel treatment is common. The molecular mechanisms of docetaxel resistance remain largely unknown and could be amenable to interventions that mitigate resistance. We have recently discovered that several docetaxel-resistant mPCa cell lines exhibit lower uptake of cellular copper and uniquely express higher levels of a copper exporter protein ATP7B. Knockdown of ATP7B by silencing RNAs (siRNA) sensitized docetaxel-resistant mPCa cells to the growth-inhibitory and apoptotic effects of docetaxel. Importantly, deletions of ATP7B in human mPCa tissues predict significantly better survival of patients after their first chemotherapy than those with wild-type ATP7B (P = 0.0006). In addition, disulfiram (DSF), an FDA-approved drug for the treatment of alcohol dependence, in combination with copper, significantly enhanced the in vivo antitumor effects of docetaxel in a docetaxel-resistant xenograft tumor model. Our analyses also revealed that DSF and copper engaged with ATP7B to decrease protein levels of COMM domain-containing protein 1 (COMMD1), S-phase kinase-associated protein 2 (Skp2), and clusterin and markedly increase protein expression of cyclin-dependent kinase inhibitor 1 (p21/WAF1). Taken together, our results indicate a copper-dependent nutrient vulnerability through ATP7B exporter in docetaxel-resistant prostate cancer for improving the therapeutic efficacy of docetaxel.
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Adenosina Trifosfatases , Proteínas de Transporte de Cátions , ATPases Transportadoras de Cobre , Cobre , Dissulfiram , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata , Taxoides , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Humanos , Dissulfiram/farmacologia , Dissulfiram/uso terapêutico , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Taxoides/farmacologia , Taxoides/uso terapêutico , Animais , Linhagem Celular Tumoral , Camundongos , Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacosRESUMO
Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.
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This cross-sectional study examines federal funding, registered clinical trials, and publications to quantify trends in firearm injury prevention research in the US from 1985 to 2022.
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Armas de Fogo , Ferimentos por Arma de Fogo , Humanos , Pesquisa sobre Serviços de Saúde , Estados Unidos , Ferimentos por Arma de Fogo/epidemiologia , Ferimentos por Arma de Fogo/prevenção & controle , Ensaios Clínicos como AssuntoRESUMO
Copper (Cu) is an essential trace element, however an excess is toxic due to its redox properties. Cu homeostasis therefore needs to be tightly regulated via cellular transporters, storage proteins and exporters. An imbalance in Cu homeostasis has been associated with neurodegenerative disorders such as Wilson's disease, but also Alzheimer's or Parkinson's disease. In our current study, we explored the utility of using Caenorhabditis elegans (C. elegans) as a model of Cu dyshomeostasis. The application of excess Cu dosing and the use of mutants lacking the intracellular Cu chaperone atox-1 and major Cu storage protein ceruloplasmin facilitated the assessment of Cu status, functional markers including total Cu levels, labile Cu levels, Cu distribution and the gene expression of homeostasis-related genes. Our data revealed a decrease in total Cu uptake but an increase in labile Cu levels due to genetic dysfunction, as well as altered gene expression levels of Cu homeostasis-associated genes. In addition, the data uncovered the role ceruloplasmin and atox-1 play in the worm's Cu homeostasis. This study provides insights into suitable functional Cu markers and Cu homeostasis in C. elegans, with a focus on labile Cu levels, a promising marker of Cu dysregulation during disease progression.
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Estimating the abundance of cell-free DNA (cfDNA) fragments shed from a tumor (i.e., circulating tumor DNA (ctDNA)) can approximate tumor burden, which has numerous clinical applications. We derived a novel, broadly applicable statistical method to quantify cancer-indicative methylation patterns within cfDNA to estimate ctDNA abundance, even at low levels. Our algorithm identified differentially methylated regions (DMRs) between a reference database of cancer tissue biopsy samples and cfDNA from individuals without cancer. Then, without utilizing matched tissue biopsy, counts of fragments matching the cancer-indicative hyper/hypo-methylated patterns within DMRs were used to determine a tumor methylated fraction (TMeF; a methylation-based quantification of the circulating tumor allele fraction and estimate of ctDNA abundance) for plasma samples. TMeF and small variant allele fraction (SVAF) estimates of the same cancer plasma samples were correlated (Spearman's correlation coefficient: 0.73), and synthetic dilutions to expected TMeF of 10-3 and 10-4 had estimated TMeF within two-fold for 95% and 77% of samples, respectively. TMeF increased with cancer stage and tumor size and inversely correlated with survival probability. Therefore, tumor-derived fragments in the cfDNA of patients with cancer can be leveraged to estimate ctDNA abundance without the need for a tumor biopsy, which may provide non-invasive clinical approximations of tumor burden.