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The intestinal tract generates significant reactive oxygen species (ROS), but the role of T cell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C. rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.
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Glutationa , Interleucina 22 , Interleucinas , Mitocôndrias , Células Th17 , Animais , Interleucinas/metabolismo , Mitocôndrias/metabolismo , Glutationa/metabolismo , Células Th17/metabolismo , Células Th17/imunologia , Camundongos , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Citrobacter rodentium , Intestinos/patologia , Intestinos/imunologia , Inflamação/metabolismo , Inflamação/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/patologia , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologiaRESUMO
The human body hosts trillions of microorganisms throughout many diverse habitats with different physico-chemical characteristics. Among them, the oral cavity and the gut harbour some of the most dense and diverse microbial communities. Although these two sites are physiologically distinct, they are directly connected and can influence each other in several ways. For example, oral microorganisms can reach and colonize the gastrointestinal tract, particularly in the context of gut dysbiosis. However, the mechanisms of colonization and the role that the oral microbiome plays in causing or exacerbating diseases in other organs have not yet been fully elucidated. Here, we describe recent advances in our understanding of how the oral and intestinal microbiota interplay in relation to their impact on human health and disease.
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Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.
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The human microbiome significantly influences drug metabolism through the gut-liver axis, leading to modified drug responses and potential toxicity. Due to the complex nature of the human gut environment, the understanding of microbiome-driven impacts on these processes is limited. To address this, a multiorgan-on-a-chip (MOoC) platform that combines the human microbial-crosstalk (HuMiX) gut-on-chip (GoC) and the Dynamic42 liver-on-chip (LoC), mimicking the bidirectional interconnection between the gut and liver known as the gut-liver axis, is introduced. This platform supports the viability and functionality of intestinal and liver cells. In a proof-of-concept study, the metabolism of irinotecan, a widely used colorectal cancer drug, is imitated within the MOoC. Utilizing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), irinotecan metabolites are tracked, confirming the platform's ability to represent drug metabolism along the gut-liver axis. Further, using the authors' gut-liver platform, it is shown that the colorectal cancer-associated gut bacterium, Escherichia coli, modifies irinotecan metabolism through the transformation of its inactive metabolite SN-38G into its toxic metabolite SN-38. This platform serves as a robust tool for investigating the intricate interplay between gut microbes and pharmaceuticals, offering a representative alternative to animal models and providing novel drug development strategies.
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Microbioma Gastrointestinal , Irinotecano , Fígado , Humanos , Irinotecano/farmacocinética , Fígado/metabolismo , Microbioma Gastrointestinal/fisiologia , Dispositivos Lab-On-A-Chip , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodosRESUMO
Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.
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Glutamina , Piruvato Quinase , Piruvato Quinase/metabolismo , Glutamina/metabolismo , Glicólise , Carbono , Serina/metabolismoRESUMO
Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.
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Glioblastoma , Metabolismo dos Lipídeos , Humanos , Formiatos , Invasividade Neoplásica , Microambiente TumoralRESUMO
Although the intestinal tract is a major site of reactive oxygen species (ROS) generation, the mechanisms by which antioxidant defense in gut T cells contribute to intestinal homeostasis are currently unknown. Here we show, using T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that the ensuing loss of glutathione (GSH) impairs the production of gut-protective IL-22 by Th17 cells within the lamina propria. Although Gclc ablation does not affect T cell cytokine secretion in the gut of mice at steady-state, infection with C. rodentium increases ROS, inhibits mitochondrial gene expression and mitochondrial function in Gclc-deficient Th17 cells. These mitochondrial deficits affect the PI3K/AKT/mTOR pathway, leading to reduced phosphorylation of the translation repressor 4E-BP1. As a consequence, the initiation of translation is restricted, resulting in decreased protein synthesis of IL-22. Loss of IL-22 results in poor bacterial clearance, enhanced intestinal damage, and high mortality. ROS-scavenging, reconstitution of IL-22 expression or IL-22 supplementation in vivo prevent the appearance of these pathologies. Our results demonstrate the existence of a previously unappreciated role for Th17 cell-intrinsic GSH coupling to promote mitochondrial function, IL-22 translation and signaling. These data reveal an axis that is essential for maintaining the integrity of the intestinal barrier and protecting it from damage caused by gastrointestinal infection.
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Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
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Metilenotetra-Hidrofolato Desidrogenase (NADP) , Neoplasias , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , Tetra-HidrofolatosRESUMO
Blood analysis is one of the foundations of clinical diagnostics. In recent years, the analysis of proteins in blood samples by mass spectrometry has taken a jump forward in terms of sensitivity and the number of identified proteins. The recent development of parallel reaction monitoring with parallel accumulation and serial fragmentation (prm-PASEF) combines ion mobility as an additional separation dimension. This increases the proteome coverage while allowing the use of shorter chromatographic gradients. To demonstrate the method's full potential, we used an isotope-labeled synthetic peptide mix of 782 peptides, derived from 579 plasma proteins, spiked into blood plasma samples with a prm-PASEF measurement allowing the quantification of 565 plasma proteins by targeted proteomics. As a less time-consuming alternative to the prm-PASEF method, we describe guided data independent acquisition (dia)-PASEF (g-dia-PASEF) and compare its application to prm-PASEF for measuring blood plasma. To demonstrate both methods' performance in clinical samples, 20 patient plasma samples from a colorectal cancer (CRC) cohort were analyzed. The analysis identified 14 differentially regulated proteins between the CRC patient and control individual plasma samples. This shows the technique's potential for the rapid and unbiased screening of blood proteins, abolishing the need for the preselection of potential biomarker proteins.
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Peptídeos , Proteômica , Humanos , Proteômica/métodos , Peptídeos/análise , Espectrometria de Massas/métodos , Cromatografia Líquida , Proteoma , Proteínas SanguíneasRESUMO
The specific physiology and behaviour of children makes them particularly vulnerable to chemical exposure. Specific studies must therefore be conducted to understand the impact of pollution on children's health. Human biomonitoring is a reliable approach for exposure assessment, and hair, allowing the detection of parent chemicals and metabolites, and covering wider time windows than urine and blood is particularly adapted to study chronic exposure. The present study aims at assessing chemical exposure and investigating possible determinants of exposure in children living in Luxembourg. Hair samples were collected from 256 children below 13 y/o and tested for 153 compounds (140 pesticides, 4 PCBs, 7 BDEs and 2 bisphenols). Moreover, anthropometric parameters, information on diet, residence, and presence of pets at home was collected through questionnaires. Correlations, regressions, t-tests, PLS-DA and MANOVAs, were used to investigate exposure patterns. Twenty-nine to 88 (median = 61) compounds were detected per sample. The highest median concentration was observed for BPA (133.6 pg/mg). Twenty-three biomarkers were detected in ≥ 95% of the samples, including 13 in all samples (11 pesticides, BPA and BPS). Exposure was higher at younger ages (R2 = 0.57), and boys were more exposed to non-persistent pesticides than girls. Presence of persistent organic pollutants in most children suggests that exposure is still ongoing. Moreover, diet (e.g. imazalil: 0.33 pg/mg in organic, 1.15 pg/mg in conventional, p-value < 0.001), residence area (e.g. imidacloprid: 0.29 pg/mg in urban, 0.47 pg/mg in countryside, p-value = 0.03), and having pets (e.g. fipronil: 0.32 pg/mg in pets, 0.09 pg/mg in no pets, p-value < 0.001) were identified as determinants of exposure. The present study demonstrates that children are simultaneously exposed to multiple pollutants from different chemical classes, and confirms the suitability of hair to investigate exposure. These results set the basis for further investigations to better understand the determinants of chemical exposure in children.
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Expossoma , Praguicidas , Criança , Monitoramento Ambiental/métodos , Feminino , Análise do Cabelo , Humanos , Luxemburgo , Masculino , Praguicidas/análiseRESUMO
Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.
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Neoplasias da Mama , Antagonistas do Ácido Fólico , Neoplasias da Mama/metabolismo , Ciclo do Carbono , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Mitocôndrias/metabolismo , Serina/metabolismoRESUMO
The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression.
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Neoplasias Colorretais , Microbioma Gastrointestinal , Animais , Bactérias , Neoplasias Colorretais/metabolismo , Formiatos , Fusobacterium nucleatum , Humanos , Camundongos , Microambiente TumoralRESUMO
The emergence and spread of antimicrobial resistance (AMR) represent an ever-growing healthcare challenge worldwide. Nevertheless, the mechanisms and timescales shaping this resistome remain elusive. Using an antibiotic cocktail administered to a murine model along with a longitudinal sampling strategy, we identify the mechanisms by which gut commensals acquire antimicrobial resistance genes (ARGs) after a single antibiotic course. While most of the resident bacterial populations are depleted due to the treatment, Akkermansia muciniphila and members of the Enterobacteriaceae, Enterococcaceae, and Lactobacillaceae families acquire resistance and remain recalcitrant. We identify specific genes conferring resistance against the antibiotics in the corresponding metagenome-assembled genomes (MAGs) and trace their origins within each genome. Here we show that, while mobile genetic elements (MGEs), including bacteriophages and plasmids, contribute to the spread of ARGs, integrons represent key factors mediating AMR in the antibiotic-treated mice. Our findings suggest that a single course of antibiotics alone may act as the selective sweep driving ARG acquisition and incidence in gut commensals over a single mammalian lifespan.
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Antibacterianos , Genes Bacterianos , Animais , Antibacterianos/farmacologia , Bactérias/genética , Humanos , Mamíferos/genética , Metagenoma , Camundongos , PlasmídeosRESUMO
Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.
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Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.
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Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Quimiocina CCL2/genética , Colo/metabolismo , Neoplasias Colorretais/genética , Receptores de Superfície Celular/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Diferenciação Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Colo/patologia , Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Células Mieloides/metabolismo , Células Mieloides/patologia , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Metabolism is considered to be the core of all cellular activity. Thus, extensive studies of metabolic processes are ongoing in various fields of biology, including cancer research. Cancer cells are known to adapt their metabolism to sustain high proliferation rates and survive in unfavorable environments with low oxygen and nutrient concentrations. Hence, targeting cancer cell metabolism is a promising therapeutic strategy in cancer research. However, cancers consist not only of genetically altered tumor cells but are interwoven with endothelial cells, immune cells and fibroblasts, which together with the extracellular matrix (ECM) constitute the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs), which are linked to poor prognosis in different cancer types, are one important component of the TME. CAFs play a significant role in reprogramming the metabolic landscape of tumor cells, but how, and in what manner, this interaction takes place remains rather unclear. This review aims to highlight the metabolic landscape of tumor cells and CAFs, including their recently identified subtypes, in different tumor types. In addition, we discuss various in vitro and in vivo metabolic techniques as well as different in silico computational tools that can be used to identify and characterize CAF-tumor cell interactions. Finally, we provide our view on how mapping the complex metabolic networks of stromal-tumor metabolism will help in finding novel metabolic targets for cancer treatment.
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Fibroblastos Associados a Câncer/metabolismo , Contagem de Células , Humanos , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. Here, we describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, we detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is highly reproducible between injections and enables accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.
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Peptídeos/análise , Proteômica , Isótopos de Carbono , Células HeLa , Humanos , Espectrometria de Mobilidade Iônica , Isótopos de Nitrogênio , Peptídeos/síntese química , Fatores de TempoRESUMO
The view that myosins, which are actin based molecular motors, are only driving muscle contraction evolved a lot during the last decades. Nowadays, it is known that they reshape the actin skeleton, anchor or transport vesicles, organelles as well as protein complexes. Here, we review how their role in cell division, polarization, migration and death is related to the cancer phenotype. We will further focus our attention on recent evidences suggesting that these central roles make them prime biomarker candidates for the prognosis of various cancers. Finally, we will discuss emerging evidences raising myosins as new therapeutic targets to fight malignant tumors.