ABSTRACT
KRIT1 loss-of-function mutations underlie the pathogenesis of Cerebral Cavernous Malformation (CCM), a major vascular disease affecting the central nervous system (CNS). However, KRIT1 is also expressed outside the CNS and modulates key regulators of metabolic and oxy-inflammatory pathways, including the master transcription factor FoxO1, suggesting a widespread functional significance. Herein, we show that the KRIT1/FoxO1 axis is implicated in liver metabolic functions and antioxidative/antiglycative defenses. Indeed, by performing comparative studies in KRIT1 heterozygous (KRIT1+/-) and wild-type mice, we found that KRIT1 haploinsufficiency resulted in FoxO1 expression/activity downregulation in the liver, and affected hepatic FoxO1-dependent signaling pathways, which are markers of major metabolic processes, including gluconeogenesis, glycolysis, mitochondrial respiration, and glycogen synthesis. Moreover, it caused sustained activation of the master antioxidant transcription factor Nrf2, hepatic accumulation of advanced glycation end-products (AGEs), and abnormal expression/activity of AGE receptors and detoxifying systems. Furthermore, it was associated with an impairment of food intake, systemic glucose disposal, and plasma levels of insulin. Specific molecular alterations detected in the liver of KRIT1+/- mice were also confirmed in KRIT1 knockout cells. Overall, our findings demonstrated, for the first time, that KRIT1 haploinsufficiency affects glucose homeostasis and liver metabolic and antioxidative/antiglycative functions, thus inspiring future basic and translational studies.
Subject(s)
Insulins , NF-E2-Related Factor 2 , Animals , Antioxidants , Glucose , Glycogen , KRIT1 Protein , Liver , Mice , NF-E2-Related Factor 2/genetics , Oxidative Stress/geneticsABSTRACT
BACKGROUND: Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. METHODS AND RESULTS: We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. CONCLUSIONS: By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.
Subject(s)
Central Nervous System Neoplasms/drug therapy , Disease Models, Animal , Drug Repositioning/methods , Hemangioma, Cavernous, Central Nervous System/drug therapy , Animals , Cells, Cultured , Central Nervous System Neoplasms/pathology , Cholecalciferol/pharmacology , Cholecalciferol/therapeutic use , Drug Screening Assays, Antitumor/methods , Endothelial Cells/drug effects , Endothelial Cells/pathology , Free Radical Scavengers/pharmacology , Free Radical Scavengers/therapeutic use , Hemangioma, Cavernous, Central Nervous System/pathology , Humans , Mice , Mice, Knockout , Mice, Transgenic , Treatment OutcomeABSTRACT
Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker's yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.
Subject(s)
Chlorogenic Acid/metabolism , Cynara scolymus/genetics , Cynara scolymus/metabolism , Plants/enzymology , Plants/metabolism , 3-Hydroxyanthranilic Acid/metabolism , Amides/metabolism , Animals , Coumaric Acids , Esters/metabolism , Genes , Mice , Phenols/metabolism , Plants/genetics , Propionates , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Nicotiana/genetics , Nicotiana/metabolism , Yeasts/genetics , Yeasts/metabolismABSTRACT
KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.
Subject(s)
Central Nervous System Vascular Malformations/physiopathology , Hemangioma, Cavernous, Central Nervous System/physiopathology , Microtubule-Associated Proteins/physiology , Protein Interaction Domains and Motifs/physiology , Proto-Oncogene Proteins/physiology , Animals , COS Cells , Cell Line , Central Nervous System Vascular Malformations/genetics , Chlorocebus aethiops , Computer Simulation , HeLa Cells , Hemangioma, Cavernous, Central Nervous System/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , KRIT1 Protein , Mice , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Point Mutation , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/physiology , Protein Structure, Secondary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Two-Hybrid System Techniques , rap1 GTP-Binding Proteins/chemistry , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolismABSTRACT
The development of distinct cellular and animal models has allowed the identification and characterization of molecular mechanisms underlying the pathogenesis of cerebral cavernous malformation (CCM) disease. This is a major cerebrovascular disorder of proven genetic origin, affecting 0.5% of the population. Three disease genes have been identified: CCM1/KRIT1, CCM2, and CCM3. These genes encode for proteins implicated in the regulation of major cellular structures and mechanisms, such as cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, and endothelial-to-mesenchymal transition, suggesting that they may act as pleiotropic regulators of cellular homeostasis. Indeed, accumulated evidence in cellular and animal models demonstrates that emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses. In particular, we demonstrated that KRIT1 loss-of-function affects master regulators of cellular redox homeostasis and responses to oxidative stress, including major redox-sensitive transcriptional factors and antioxidant proteins, and autophagy, suggesting that altered redox signaling and oxidative stress contribute to CCM pathogenesis, and opening novel preventive and therapeutic perspectives.In this chapter, we describe materials and methods for isolation of mouse embryonic fibroblast (MEF) cells from homozygous KRIT1-knockout mouse embryos, and their transduction with a lentiviral vector encoding KRIT1 to generate cellular models of CCM disease that contributed significantly to the identification of pathogenetic mechanisms.
Subject(s)
Fibroblasts/metabolism , Hemangioma, Cavernous, Central Nervous System/genetics , Hemangioma, Cavernous, Central Nervous System/metabolism , KRIT1 Protein/genetics , Animals , Disease Models, Animal , Gene Order , Gene Targeting , Genetic Loci , Genetic Vectors/genetics , Hemangioma, Cavernous, Central Nervous System/diagnosis , Homologous Recombination , Homozygote , Mice , Mice, Knockout , Mice, Transgenic , Transduction, GeneticABSTRACT
Oat (Avena sativa) is a cereal known since antiquity as a useful grain with abundant nutritional and health benefits. It contains distinct molecular components with high antioxidant activity, such as tocopherols, tocotrienols, and flavanoids. In addition, it is a unique source of avenanthramides, phenolic amides containing anthranilic acid and hydroxycinnamic acid moieties, and endowed with major beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. In this review, we report on the biological activities of avenanthramides and their derivatives, including analogs produced in recombinant yeast, with a major focus on the therapeutic potential of these secondary metabolites in the treatment of aging-related human diseases. Moreover, we also present recent advances pointing to avenanthramides as interesting therapeutic candidates for the treatment of cerebral cavernous malformation (CCM) disease, a major cerebrovascular disorder affecting up to 0.5% of the human population. Finally, we highlight the potential of foodomics and redox proteomics approaches in outlining distinctive molecular pathways and redox protein modifications associated with avenanthramide bioactivities in promoting human health and contrasting the onset and progression of various pathologies. The paper is dedicated to the memory of Adelia Frison.
Subject(s)
Cerebrovascular Disorders/drug therapy , Cerebrovascular Disorders/prevention & control , Protective Agents/therapeutic use , Skin/drug effects , ortho-Aminobenzoates/therapeutic use , Animals , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Humans , Protective Agents/chemistry , Protective Agents/pharmacology , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacologyABSTRACT
KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3-0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies.
Subject(s)
Brain/pathology , Central Nervous System Neoplasms/genetics , Endothelial Cells/physiology , Hemangioma, Cavernous, Central Nervous System/genetics , KRIT1 Protein/genetics , Mutation/genetics , Oxidative Stress/genetics , Animals , Apoptosis , Autophagy/genetics , Cells, Cultured , Central Nervous System Neoplasms/metabolism , HSP27 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Hemangioma, Cavernous, Central Nervous System/metabolism , Homeostasis , Humans , KRIT1 Protein/metabolism , Lactoylglutathione Lyase/metabolism , Mice , Mice, Knockout , NF-E2-Related Factor 2/metabolism , Oxidation-Reduction , Protein Processing, Post-Translational , Pyruvaldehyde/metabolismABSTRACT
The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF-α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.
Subject(s)
Endothelium/metabolism , KRIT1 Protein/deficiency , NADPH Oxidases/metabolism , Oxidation-Reduction , Signal Transduction , Animals , Antioxidants/metabolism , Capillary Permeability/drug effects , Capillary Permeability/genetics , Gene Expression Regulation , KRIT1 Protein/genetics , KRIT1 Protein/metabolism , Mice , Mice, Knockout , NADPH Oxidase 4/genetics , NADPH Oxidase 4/metabolism , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Cerebral Cavernous Malformation (CCM) is a major cerebrovascular disease of proven genetic origin affecting 0.3-0.5% of the general population. It is characterized by abnormally enlarged and leaky capillaries, which predispose to seizures, focal neurological deficits and intracerebral hemorrhage. Causative loss-of-function mutations have been identified in 3 genes, KRIT1 (CCM1), CCM2 and PDCD10 (CCM3). While providing new options for the development of pharmacological therapies, recent advances in knowledge of the functions of these genes have clearly indicated that they exert pleiotropic effects on several biological pathways. Recently, we found that defective autophagy is a common feature of loss-of-function mutations of the 3 known CCM genes, and underlies major phenotypic signatures of CCM disease, including endothelial-to-mesenchymal transition and enhanced ROS production, suggesting a unifying pathogenetic mechanism and reconciling the distinct therapeutic approaches proposed so far. In this invited review, we discuss autophagy as a possible unifying mechanism in CCM disease pathogenesis, and new perspectives and avenues of research for disease prevention and treatment, including novel potential drug repurposing and combination strategies, and identification of genetic risk factors as basis for development of personalized medicine approaches.
ABSTRACT
BACKGROUND: Familial Cerebral Cavernous Malformation type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and characterized by multiple brain lesions. CCM lesions manifest across a range of different phenotypes, including wide differences in lesion number, size and susceptibility to intracerebral hemorrhage (ICH). Oxidative stress plays an important role in cerebrovascular disease pathogenesis, raising the possibility that inter-individual variability in genes related to oxidative stress may contribute to the phenotypic differences observed in CCM1 disease. Here, we investigated whether candidate oxidative stress-related cytochrome P450 (CYP) and matrix metalloproteinase (MMP) genetic markers grouped by superfamilies, families or genes, or analyzed individually influence the severity of CCM1 disease. METHODS: Clinical assessment and cerebral susceptibility-weighted magnetic resonance imaging (SWI) were performed to determine total and large (≥5mm in diameter) lesion counts as well as ICH in 188 Hispanic CCM1 patients harboring the founder KRIT1/CCM1 'common Hispanic mutation' (CCM1-CHM). Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We analyzed 1,122 genetic markers (both single nucleotide polymorphisms (SNPs) and insertion/deletions) grouped by CYP and MMP superfamily, family or gene for association with total or large lesion count and ICH adjusted for age at enrollment and gender. Genetic markers bearing the associations were then analyzed individually. RESULTS: The CYP superfamily showed a trend toward association with total lesion count (P=0.057) and large lesion count (P=0.088) in contrast to the MMP superfamily. The CYP4 and CYP8 families were associated with either large lesion count or total lesion count (P=0.014), and two other families (CYP46 and the MMP Stromelysins) were associated with ICH (P=0.011 and 0.007, respectively). CYP4F12 rs11085971, CYP8A1 rs5628, CYP46A1 rs10151332, and MMP3 rs117153070 single SNPs, mainly bearing the above-mentioned associations, were also individually associated with CCM1 disease severity. CONCLUSIONS: Overall, our candidate oxidative stress-related genetic markers set approach outlined CYP and MMP families and identified suggestive SNPs that may impact the severity of CCM1 disease, including the development of numerous and large CCM lesions and ICH. These novel genetic risk factors of prognostic value could serve as early objective predictors of disease outcome and might ultimately provide better options for disease prevention and treatment.
Subject(s)
Brain/metabolism , Cytochrome P-450 Enzyme System/genetics , Hemangioma, Cavernous, Central Nervous System/genetics , Matrix Metalloproteinases/genetics , Oxidative Stress/genetics , Adult , Aged , Brain/diagnostic imaging , Brain/pathology , Female , Genotype , Hemangioma, Cavernous, Central Nervous System/diagnostic imaging , Hemangioma, Cavernous, Central Nervous System/pathology , Heterozygote , Humans , KRIT1 Protein , Magnetic Resonance Imaging , Male , Microtubule-Associated Proteins/genetics , Middle Aged , Multigene Family/genetics , Mutation , Phenotype , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins/genetics , Severity of Illness IndexABSTRACT
Saccharomyces cerevisiae has been proven to be a valuable tool for the expression of plant metabolic pathways. By engineering a S. cerevisiae strain with two plant genes (4cl-2 from tobacco and hct from globe artichoke) we previously set up a system for the production of two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, Yav I) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, Yav II). These compounds have a structural similarity with a class of bioactive oat compounds called avenanthramides. By developing a fermentation process for the engineered S. cerevisiae strain, we obtained a high-yield production of Yav I and Yav II. To examine the biological relevance of these compounds, we tested their potential antioxidant and antiproliferative properties upon treatment of widely used cell models, including immortalized mouse embryonic fibroblast cell lines and HeLa cancer cells. The outcomes of our experiments showed that both Yav I and Yav II enter the cell and trigger a significant up-regulation of master regulators of cell antioxidant responses, including the major antioxidant protein SOD2 and its transcriptional regulator FoxO1 as well as the down-regulation of Cyclin D1. Intriguingly, these effects were also demonstrated in cellular models of the human genetic disease Cerebral Cavernous Malformation, suggesting that the novel phenolic compounds Yav I and Yav II are endowed with bioactive properties relevant to biomedical applications. Taken together, our data demonstrate the feasibility of biotechnological production of yeast avenanthramides and underline a biologically relevant antioxidant activity of these molecules.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antioxidants/pharmacology , Genes, Plant , Reactive Oxygen Species/antagonists & inhibitors , Saccharomyces cerevisiae/genetics , ortho-Aminobenzoates/pharmacology , Animals , Antineoplastic Agents, Phytogenic/biosynthesis , Antineoplastic Agents, Phytogenic/isolation & purification , Antioxidants/isolation & purification , Antioxidants/metabolism , Biological Transport , Cell Line, Transformed , Cyclin D1/antagonists & inhibitors , Cyclin D1/genetics , Cyclin D1/metabolism , Cynara scolymus/chemistry , Cynara scolymus/genetics , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Forkhead Box Protein O1 , Forkhead Transcription Factors/agonists , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , HeLa Cells , Hemangioma, Cavernous, Central Nervous System/drug therapy , Hemangioma, Cavernous, Central Nervous System/genetics , Hemangioma, Cavernous, Central Nervous System/metabolism , Humans , Metabolic Engineering , Mice , Models, Biological , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Nicotiana/chemistry , Nicotiana/genetics , Transgenes , ortho-Aminobenzoates/isolation & purification , ortho-Aminobenzoates/metabolismABSTRACT
Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3-0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.
Subject(s)
Autophagy , Hemangioma, Cavernous, Central Nervous System/genetics , Hemangioma, Cavernous, Central Nervous System/pathology , Microtubule-Associated Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Endothelial Cells/physiology , Gene Knockdown Techniques , Humans , KRIT1 Protein , Mice , Mice, Knockout , Microtubule-Associated Proteins/genetics , Proto-Oncogene Proteins/geneticsABSTRACT
The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumor progression, and metastasis. Recent findings state the molecular mechanisms underlying the fine-balanced relationship between cadherins and integrins. In particular, some of the novel results recently obtained raise the possibility of a pivotal role for the small GTPase Rap1 in the functional crosstalk between cadherins and integrins. Considering the importance of the molecular signalling triggered by Rap1, here we provide protocols to study this small GTPase in signalling pathways involving cadherins and integrins.
Subject(s)
Cadherins/metabolism , Integrins/metabolism , Microscopy/methods , rap1 GTP-Binding Proteins/metabolism , Animals , Calcium/metabolism , Cell Adhesion , Cell Count , Cell Line , Endocytosis , Enzyme Inhibitors/pharmacology , Extracellular Matrix/metabolism , Focal Adhesions/metabolism , Glutathione/metabolism , Humans , Intercellular Junctions , Protein Tyrosine Phosphatases/antagonists & inhibitors , Time FactorsABSTRACT
The Ras-related GTPase Rap has been implicated in multiple cellular functions. In particular, Rap1 is a crucial regulator of both inside-out integrin activation and outside-in E-cadherin-mediated signaling. Thus, Rap1 was proposed as a fundamental regulator of the cross talk between cadherins and integrins. We provide microscopic techniques to study subcellular localization of Rap1 protein in the crosstalk between integrins and cadherins.
Subject(s)
Intracellular Space/metabolism , Microscopy, Fluorescence/methods , rap1 GTP-Binding Proteins/metabolism , Cadherins/metabolism , Cell Line , Cell Proliferation , Fluorescent Antibody Technique , Integrins/metabolism , Protein TransportABSTRACT
The Ras superfamily of small GTPases is composed of more than 150 members, which share a conserved structure and biochemical properties, acting as binary molecular switches turned on by binding GTP and off by hydrolyzing GTP to GDP. However, despite considerable structural and biochemical similarities, these proteins play multiple and divergent roles, being versatile and key regulators of virtually all fundamental cellular processes. Conversely, their dysfunction plays a crucial role in the pathogenesis of serious human diseases, including cancer and developmental syndromes. Fuelled by the original identification in 1982 of mutationally activated and transforming human Ras genes in human cancer cell lines, a variety of powerful experimental techniques have been intensively focused on discovering and studying structure, biochemistry, and biology of Ras and Ras-related small GTPases, leading to fundamental research breakthroughs into identification and structural and functional characterization of a huge number of Ras superfamily members, as well as of their multiple regulators and effectors. In this review we provide a general overview of the major milestones that eventually allowed to unlock the secret treasure chest of this large and important superfamily of proteins.
Subject(s)
ras Proteins , Animals , Genetic Techniques , Humans , Protein Processing, Post-Translational , ras Proteins/chemistry , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
Redox agents have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that mammalian cells can rapidly respond to ligand stimulation with a change in intracellular ROS thus indicating that the production of intracellular redox agents is tightly regulated and that they serve as intracellular signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some members of the Ras GTPase superfamily appear to regulate the production of redox agents and that oxidants can function as effector molecules for the small GTPases, thus contributing to their overall biological function. In addition, many of the Ras superfamily small GTPases have been shown to be redox sensitive, thanks to the presence of redox-sensitive sequences in their primary structure. The action of redox agents on these redox-sensitive GTPases is similar to that of guanine nucleotide exchange factors in that they perturb GTPase nucleotide-binding interactions that result in the enhancement of the guanine nucleotide exchange of small GTPases. Thus, Ras GTPases may act both as upstream regulators and downstream effectors of redox agents. Here we overview current understanding concerning the interplay between Ras GTPases and redox agents, also taking into account pathological implications of misregulation of this cross talk and highlighting the potentiality of these cellular pathways as new therapeutical targets for different pathologies.
Subject(s)
ras Proteins/metabolism , Animals , Humans , Molecular Targeted Therapy , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
Loss-of-function mutations in the KRIT1 gene (CCM1) have been associated with the pathogenesis of cerebral cavernous malformations (CCM), a major cerebrovascular disease. However, KRIT1 functions and CCM pathogenetic mechanisms remain incompletely understood. Indeed, recent experiments in animal models have clearly demonstrated that the homozygous loss of KRIT1 is not sufficient to induce CCM lesions, suggesting that additional factors are necessary to cause CCM disease. Previously, we found that KRIT1 is involved in the maintenance of the intracellular reactive oxygen species (ROS) homeostasis to prevent ROS-induced cellular dysfunctions, including a reduced ability to maintain a quiescent state. Here, we show that KRIT1 loss of function leads to enhanced expression and phosphorylation of the redox-sensitive transcription factor c-Jun, as well as induction of its downstream target COX-2, in both cellular models and human CCM tissues. Furthermore, we demonstrate that c-Jun upregulation can be reversed by either KRIT1 re-expression or ROS scavenging, whereas KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. Taken together with the reported role of c-Jun in vascular dysfunctions triggered by oxidative stress, our findings shed new light on the molecular mechanisms underlying KRIT1 function and CCM pathogenesis.
Subject(s)
Hemangioma, Cavernous, Central Nervous System/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Microtubule-Associated Proteins/genetics , Oxidative Stress/genetics , Proto-Oncogene Proteins/genetics , Animals , Gene Expression Regulation , Hemangioma, Cavernous, Central Nervous System/metabolism , Hemangioma, Cavernous, Central Nervous System/pathology , Humans , JNK Mitogen-Activated Protein Kinases/genetics , KRIT1 Protein , Mutation , Reactive Oxygen Species/metabolismABSTRACT
The members of the RasGTPase superfamily are involved in various signaling networks responsible for fundamental cellular processes. Their activity is determined by their guanine nucleotide-bound state. Recent evidence indicates that some of these proteins may be regulated by redox agents. Reactive oxygen species (ROSs) and reactive nitrogen species (RNSs) have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that the intracellular production of ROS is tightly regulated and that these redox agents serve as signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some Ras GTPases appear to regulate ROS production and that oxidants function as effector molecules for the small GTPases, thus contributing to their overall biological function. Thus, redox agents may act both as upstream regulators and as downstream effectors of Ras GTPases. Here we discuss current understanding concerning mechanisms and physiopathological implications of the interplay between GTPases and redox agents.
ABSTRACT
The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumor progression, and metastasis. However, the molecular mechanisms underlying the fine-tuned functional communication between cadherins and integrins are still elusive. This paper focuses on recent findings towards the involvement of reactive oxygen species (ROS) in the regulation of cell adhesion and signal transduction functions of integrins and cadherins, pointing to ROS as emerging strong candidates for modulating the molecular crosstalk between cell-matrix and cell-cell adhesion receptors.
ABSTRACT
Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.