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ZnS@BSA Nanoclusters Potentiate Effectiveness involving Cancer malignancy Immunotherapy.
Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. SHIN1 These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system that is associated with leaky capillaries, and a predisposition to serious clinical conditions including intracerebral hemorrhage and seizures. Germline or sporadic mutations in the CCM1/KRIT1 gene are responsible for the majority of cases of CCM. In this article, we describe the original characterization of the CCM1/KRIT1 gene. This cloning was done through the use of a variant of the yeast two-hybrid screen known as the interaction trap, using the RAS-family GTPase KREV1/RAP1A as a bait. The partial clone of KRIT1 (Krev1 Interaction Trapped) initially identified was extended through 5'RACE and computational analysis to obtain a full-length cDNA, then used in a sequential screen to define the integrin-associated ICAP1 protein as a KRIT1 partner protein. We discuss how these interactions are relevant to the current understanding of KRIT1/CCM1 biology, and provide a protocol for library screening with the Interaction Trap.Cerebral cavernous malformation (CCM) is driven by changes in the cerebral microvascular endothelial cell population. Mouse models of CCM have successfully recapitulated the disease in vivo; however, dissection of the disease pathogenesis and molecular mechanism is challenging in vivo due to limited access to the involved tissue in live animals. Therefore, in vitro tissue culture models are required. This protocol is designed to facilitate the isolation of cerebral microvascular endothelial cells from whole murine brain tissue. The protocol utilizes papain for a shorter, single digestion step to maximize cell recovery and viability. Using this technique, we are able to isolate cells from a murine CCM model in which the absence of CCM proteins is driven by Cre-mediated recombination at birth, and results in CCM-like vascular malformations in adult animals.Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Neonatal mouse models of CCM disease have been established by deleting any one of the Ccm genes. These mouse models provide invaluable in vivo disease model to investigate molecular mechanisms and therapeutic approaches for the disease. Here, we describe detailed methodology to generate CCM disease in mouse models (Ccm1 and Ccm2-deficient) using inducible Cre/loxP recombination strategy.The use of vertebrate models allows researchers to investigate mechanisms of CCM pathogenesis in vivo, to investigate discrepancies between observations seen in the lab with in vitro experiments and how they translate into animal models; these in vivo models are more relevant in terms of CCM pathogenesis seen in humans than the in vitro counterparts. The use of CCM-deficient Zebrafish model offers advantages given their optical clarity during embryogenesis, short generation time, and high fecundity. When looking at the in vivo mouse model, gene conservation among CCM1, CCM2, and CCM3 is much higher among mammals (>92%), offering higher relevance in terms of similarities between what is seen in a mouse compared to human CCM pathogenesis. With both models, deficiencies in CCM1, CCM2, and CCM3 demonstrate perturbed cardiovascular development and underlying mechanisms of CCM pathogenesis at multiple stages seen in humans. The optimized methods described in this chapter allow researchers to benefit from both in vivo models, investigating impacts of deficiencies in CCM gene expression and its effect on angiogenesis and other signaling cascades, offering a much wider view of the molecular and cellular mechanisms in CCM progression.Our knowledge of the structure, localization, and interaction partners of cerebral cavernous malformations (CCM) proteins is mainly based on cell culture studies that lack the physiology of a three-dimensional multi-tissue environment. Uncovering the subcellular localization and the dynamic behavior of CCM proteins is an important aspect of characterizing the endothelial cell biology of CCM scaffold formation and for describing interactions with other protein complexes. However, the generation of specific antibodies to locate CCM scaffolds within cells has been challenging. To overcome the lack of functional antibodies, here, we describe the methodology involved in the generation of a construct for the expression of a fluorescently labeled CCM fusion construct and in the establishment of a transgenic zebrafish reporter line. The transgenic expression of fluorescently labeled CCM proteins within the developing zebrafish vasculature makes it possible to study the detailed subcellular localization and the dynamics of CCM proteins in vivo.
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