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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.

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.

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.

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., Graphical abstract Schematic models representing adaptive redox responses associated with KRIT1 loss-of-function. KRIT1 loss-of-function causes a persistent activation of the redox-sensitive transcription factors c-Jun and Nrf2 and consequent upregulation of downstream targets, including cycloxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and glyoxalase 1 (GLO1). While the c-Jun/COX-2 axis promotes pro-oxidant and pro-inflammatory effects, the Nrf2/HO-1 and Nrf2/GLO1 pathways mediate adaptive antioxidant responses that counteract these effects by limiting ROS* and MG intracellular accumulation, thus contributing to reduce a vicious cycle of oxidative stress and providing an adaptive defense for long term cell survival. However, this sustained adaptive redox homeostasis occurs at the expense of other cytoprotective mechanisms, including the MG-dependent formation of cytoprotective AP-Hsp70 and AP-Hsp27 protein adducts, leading to enhanced cell susceptibility to oxidative DNA damage and apoptosis, and sensitizing cells to additional stressful insults. Inter-individual differences in Nrf2-mediated adaptive defense mechanisms might influence susceptibility to CCM disease onset and progression. *The generic ROS term refers to O2•−and H2O2as well as to putative secondary oxidative products that might be implicated without certainty.fx1, Highlights • KRIT1 loss causes a chronic adaptive redox response based on the JNK-Nrf2-Glo1 axis. • Phospho-JNK, Nrf2 and Glo1 are upregulated in endothelial cells lining human CCMs. • Defective autophagy contributes to the sustained upregulation of the Nrf2-Glo1 axis. • Nrf2-Glo1 upregulation causes a drop of AP-modified Hsp70 and Hsp27 proteins. • Sustained Nrf2-Glo1 activation sensitizes cells to oxidative stress and apoptosis.

Cerebral Cavernous Malformation (CCM) is a major cerebrovascular disease of genetic origin affecting 0.3-0.5% of the population and still awaiting therapies other than neurosurgery. It is characterized by abnormally enlarged and leaky capillaries, which predispose to seizures, neurological deficits and intracerebral hemorrhage (ICH), and may occur sporadically or is dominantly inherited with incomplete penetrance and variable expressivity. Three disease genes have been identified, KRIT1 (CCM1), CCM2 and PDCD10 (CCM3), whose loss-of-function mutations are major pathogenic determinants, accounting for the main phenotypic hallmarks of CCM disease, including destabilization of endothelial cell-cell junctions and increased vascular permeability [1]. However, accumulating evidence in animal models clearly demonstrate that homozygous loss of CCM genes is not fully sufficient to cause CCM lesion formation and disease progression, suggesting the necessary contribution of additional determinants, including microenvironmental stress events [1]. Indeed, the clinical behavior in individual patients, including development of numerous and large lesions, and risk of ICH, remains highly unpredictable, while novel pharmacological strategies are particularly needed to limit disease progression and severity in susceptible individuals [1]. Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins. Previously, we found that CCM proteins, including KRIT1, play an important role in maintaining intracellular redox homeostasis through the modulation of master regulators of ROS production/detoxification and cell responses to oxidative stress, thereby limiting altered redox signaling and oxidative damage, and preserving cellular resistance to oxidative stress [1-3]. Consistently, recently we demonstrated that KRIT1 loss-of-function causes upregulation of NADPH oxidase-mediated redox signaling, leading to enhanced endothelial cell sensitivity to oxidative stress and inflammation, and decreased microvessel barrier function, further suggesting that altered redox signaling and oxidative stress contribute to CCM pathogenesis [4]. Moreover, preliminary results indicate that these events involve also carbonyl compounds generated through the lipid peroxidation process, and a sustained upregulation of Nrf2, suggesting a complex but unifying pathogenic mechanism that reconciles both the pleiotropic functions of CCM proteins and the distinct therapeutic approaches proposed so far. In addition, we identified genetic modifiers influencing disease severity, including polymorphisms in genes related to inter-individual variability in susceptibility to oxidative stress [5]. Taken together, our findings point to a major role for altered redox signaling in CCM pathogenesis and indicate that inter-individual variability in cell responses to oxidative stress may impact disease onset, progression and severity, suggesting novel preventive and therapeutic approaches.

Cerebral Cavernous Malformation (CCM) is a major cerebrovascular disease with a prevalence of 0.3–0.5% in the general population and highly variable clinical expressivity. It may cause various symptoms at any age, including recurrent headaches, neurological deficits, seizures, and intracerebral hemorrhage, and has been associated with loss-of-function mutations in three CCM genes, CCM1 (KRIT1), CCM2 and CCM3. However, growing evidence in animal models demonstrates that loss of CCM genes is not sufficient to cause CCM disease, suggesting the contribution of additional triggers occurring locally in the neurovascular microenvironment, including stress factors. Indeed, we found that both cellular and animal models of CCM disease show an enhanced sensitivity to oxidative stress and inflammatory insults due to defects in mechanisms involved in cellular defense against these stressful conditions, including antioxidant responses and autophagy. Furthermore, we provide novel insights into the identification of risk factors and biomarkers associated with CCM disease progression and severity, including the accumulation of major glycation and oxidation products, which may serve as early objective predictors of disease outcome and provide better options for disease prevention and treatment.

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.

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., Graphical abstract, Highlights • KRIT1 loss of function leads to the upregulation of c-Jun. • c-Jun upregulation occurs in human cerebral cavernous malformation (CCM) lesions. • KRIT1 loss-dependent c-Jun upregulation can be reversed by ROS scavenging. • KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. • A novel mechanism for CCM pathogenesis is suggested.

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.

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.

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 (≥5 mm 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., Graphical abstract, Highlights • Inter-individual variability in oxidative stress-related genes may impact CCM1 disease severity. • CYP4 and CYP8 families were associated with either large lesion or total lesion count, while CYP46 and MMP Stromelysin families were associated with ICH. • Single SNPs in CYP4F8, CYP4F11, CYP4F12, CYP8A1, CYP19A1, CYP27A1, CYP27B1, CYP46A1 and CYP51A1 genes showed significant associations with at least one disease severity phenotype. • A single SNP in the MMP3 gene was strongly associated with ICH. • CYP and MMP SNPs associated with CCM1 disease severity could serve as early objective predictors of disease susceptibility and outcome.

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.

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K-Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three-gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at-risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified,nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255-4delGTA; c.1682-1683 delTA in CCM1; c.48A > G; c.82-83dupAG in CCM2; and c.395 + 1G > A in CCM3 genes [corrected].The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at-risk relatives.

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 D 3 ) 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.

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.

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.

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.

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.

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.

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.

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.

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.

Cerebral cavernous malformations (CCMs) are a diffuse cerebrovascular disease affecting approximately 0.5% of the population. A CCM is characterized by abnormally enlarged and leaky capillaries arranged in mulberry-like structures with no clear flow pattern. The lesion might predispose to seizures, focal neurological deficits or fatal intracerebral hemorrhage. However, a CCM can also remain neurologically silent. It might either occur sporadically or as an inherited disorder with incomplete penetrance and variable expressivity. Due to advances in imaging techniques, the incidence of CCM diagnoses are increasing, and the patient must be managed on a multidisciplinary basis: genetic counselling, treatment if needed, and follow-up. Advances have been made using radiological and pathological correlates of CCM lesions adding to the accumulated knowledge of this disease, although management of these patients is very variable among centers. This review is aimed at providing an update in genetic and molecular insights of this condition. Included are implications for genetic counselling, and possible approaches to prevention and treatment that derive from the understanding of pathogenetic mechanisms.

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.

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K‐Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three‐gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at‐risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified, nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255‐4delGTA; c.1681‐1682delTA in CCM1; c.48A > G; c.82‐83insAG in CCM2; and c.396G > A in CCM3 genes. The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at‐risk relatives.

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.

KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We 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. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1(-/-) cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1(-/-) cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45alpha, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions.