Identification of Krit1B: a novel alternative splicing isoform of cerebral cavernous malformation gene-1

Retta, Saverio Francesco; Avolio, Maria; Francalanci, Floriana; Procida, Simone; Balzac, Fiorella; Degani, Simona; Tarone, Guido; Silengo, Lorenzo

doi:10.1016/j.gene.2003.09.046

Cited by 18 publications

(15 citation statements)

References 71 publications

(104 reference statements)

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“…A KRIT1A-expresssing lentiviral construct was generated from the HIV-derived self-inactivating transfer construct pCCLsin.PPT.PGK.EGFP.Wpre (provided by L. Naldini, HSR-TIGET) by replacing the GFP cassette with the murine KRIT1A cDNA [127]. Lentiviral vector particles were produced in 293T packaging cells, transiently cotransfected with a mix of transfer, envelope, and core-packaging constructs, as described previously [31].…”

Section: Methodsmentioning

confidence: 99%

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

et al. 2010

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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 Gadd45α, 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.

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Section: Methodsmentioning

confidence: 99%

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

et al. 2010

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“…pLVX-KRIT1 overexpression vector was obtained by subcloning the KRIT1 cDNA cassette [24] into the pLVX lentiviral expression vector (Clontech Laboratories, Mountain View, CA). The human miR-21 gene was amplified from HeLa genomic DNA and cloned into the pWPT lentivirus vector (Addgene, Cambridge, MA) to obtain pWPT-miR-21.The luciferase reporter vector containing a partial portion (973 nts, positions 340–1313) of the human KRIT1 3′UTR was generated by PCR amplification from HeLa genomic DNA, cloned downstream of the luciferase coding sequence into the pMIR-REPORT™ vector (Ambion, Austin, TX).…”

Section: Methodsmentioning

confidence: 99%

miR-21 coordinates tumor growth and modulates KRIT1 levels

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Balzac

Marino

et al. 2013

Biochemical and Biophysical Research Communications

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“…In a previous work we identified a novel KRIT1 isoform, KRIT1B, characterized by the alternative splicing of the 15th coding exon, which results in a 117-bp in-frame deletion accounting for the exclusion of 39 aa in a region close to C-terminus of full-length KRIT1A [24]. The alternative splicing affects the β sandwich of the F3/PTB lobe of the KRIT1 FERM domain.…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, we previously identified a second KRIT1 isoform, KRIT1B, characterized by the alternative splicing of the 15th coding exon, which results in a shorter protein lacking 39 aa in the C-terminus [24]. We found that the alternative splicing strongly affects the structure of the F3/PTB lobe of the KRIT1 FERM domain with important functional implications, including the loss of the ability to undertake head-to-tail intramolecular interaction, binding to Rap1 and nucleocytoplasmic shuttling.…”

Section: Introductionmentioning

confidence: 99%

Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

et al. 2012

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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.

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Identification of Krit1B: a novel alternative splicing isoform of cerebral cavernous malformation gene-1

Cited by 18 publications

References 71 publications

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

miR-21 coordinates tumor growth and modulates KRIT1 levels

Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

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