Contribution of protein-protein interactions to the endothelial barrier-stabilizing function of KRIT1

Krev-interaction trapped 1 (KRIT1) is an endothelial scaffold protein that promotes adherens junction (AJ) stability. The precise mechanism by which KRIT1 promotes barrier stabilization is unclear. We tested the ability of a panel of KRIT1 constructs containing mutations that inhibit Rap1 binding, ICAP1 binding, disrupt KRIT1's protein tyrosine binding domain (PTB), or direct KRIT1 to the plasma membrane, either alone or in combination, to restore barrier function in KRIT1-deficient endothelial cells. We found that ablating the 192NPAY195 motif or disrupting the PTB domain was sufficient to restore AJ protein localization and barrier function to control levels, irrespective of the junctional localization of KRIT1 or Rap1 binding. The ability of our KRIT1 constructs to rescue AJ/barrier function in KRIT1 depleted endothelial cells correlated with decreased 1 integrin activity and maintenance of cortical actin fibers. Together, our findings indicate that Rap1 binding, ICAP1 binding, and junctional localization are not required for the ability of KRIT1 to stabilize endothelial contacts, and suggest that the ability of KRIT1 to limit integrin activity may be involved in barrier stabilization.

In silico Analysis of Nonsynonymous Genomic Variants within CCM2 Gene Reaffirm the Existence of Dual Cores within Typical PTB Domain

Purpose: The objective of this study is to validate the existence of dual cores within the typical phosphotyrosine binding (PTB) domain and to identify potentially damaging and pathogenic nonsynonymous coding single nuclear polymorphisms (nsSNPs) in the canonical PTB domain of the CCM2 gene that causes cerebral cavernous malformations (CCMs). Methods: The nsSNPs within the coding sequence for PTB domain of human CCM2 gene, retrieved from exclusive database search, were analyzed for their functional and structural impact using a series of bioinformatic tools. The effects of the mutations on tertiary structure of the PTB domain in human CCM2 protein were predicted to examine the effect of the nsSNPs on tertiary structure on PTB Cores. Results: Our mutation analysis, through alignment of protein structures between wildtype CCM2 and mutant, indicated that the structural impacts of pathogenic nsSNPs is biophysically limited to only the spatially adjacent substituted amino acid site with minimal structural influence on the adjacent core of the PTB domain, suggesting both cores are independently functional and essential for proper CCM2 function. Conclusion: Utilizing a combination of protein conservation and structure-based analysis, we analyzed the structural effects of inherited pathogenic mutations within the CCM2 PTB domain. Our results indicated that the pathogenic amino acid substitutions lead to only subtle changes locally confined to the surrounding tertiary structure of the PTB core within which it resides, while no structural disturbance to the neighboring PTB core was observed, reaffirming the presence of dual functional cores in the PTB domain.

Differential expression of GULP1 in cancer of the vulva.

In these brief notes we document work using published microarray data (1, 2) to pioneer integrative transcriptome analysis comparing vulvar carcinoma to its tissue of origin, the vulva. We report the differential expression of GULP, engulfment adaptor PTB domain containing 1, encoded by GULP1, in cancer of the vulva. GULP1 may be of pertinence to understanding transformation and disease progression in vulvar cancer (3).

Differential expression of GULP, engulfment adaptor PTB domain containing 1 in human epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer (1). We performed discovery of genes associated with epithelial ovarian cancer and of the high-grade serous ovarian cancer (HGSC) subtype, using published microarray data (2, 3) to compare global gene expression profiles of normal ovary or fallopian tube with that of primary tumors from women diagnosed with epithelial ovarian cancer or HGSC. We identified the gene encoding GULP, engulfment adaptor PTB domain containing 1, GULP1, as among the genes whose expression was most different in epithelial ovarian cancer as compared to the normal fallopian tube. GULP1 expression was significantly lower in high-grade serous ovarian tumors relative to normal fallopian tube. GULP1 expression correlated with overall survival in patients with ovarian cancer. These data indicate that expression of GULP1 is perturbed in epithelial ovarian cancers broadly and in ovarian cancers of the HGSC subtype. GULP1 may be relevant to pathways underlying ovarian cancer initiation (transformation) or progression.

Novel CCM2 missense variants abrogating the CCM1–CCM2 interaction cause cerebral cavernous malformations

BackgroundCerebral cavernous malformations (CCMs) are vascular malformations mostly located within the central nervous system. Most deleterious variants are loss of function mutations in one of the three CCM genes. These genes code for proteins that form a ternary cytosolic complex with CCM2 as a hub. Very few CCM2 missense variants have been shown to be deleterious by modifying the ternary CCM complex stability.ObjectivesTo investigate the causality of novel missense CCM2 variants detected in patients with CCM.MethodsThe three CCM genes were screened in 984 patients referred for CCM molecular screening. Interaction between CCM1 and CCM2 proteins was tested using co-immunoprecipitation experiments for the CCM2 missense variants located in the phosphotyrosine binding (PTB) domain.Results11 distinct CCM2 rare missense variants were found. Six variants predicted to be damaging were located in the PTB domain, four of them were novel. When co-transfected with CCM1 in HEK293T cells, a loss of interaction between CCM1 and CCM2 was observed for all six variants.ConclusionWe showed, using co-immunoprecipitation experiments, that CCM2 missense variants located in the PTB domain were actually damaging by preventing the normal interaction between CCM1 and CCM2. These data are important for diagnosis and genetic counselling, which are challenging in patients harbouring such variants.

Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein

Cerebral cavernous malformations (CCMs) is a microvascular disorder in the central nervous system. Despite tremendous efforts, the causal genetic mutation in some CCM patients has not be identified, raising the possibility of an unknown CCM locus. The CCM2/MGC4607 gene has been identified as one of three known genes causing CCMs. In this report, we defined a total of 29 novel exons and 4 novel promoters in CCM2 genomic structure and subsequently identified a total of 50 new alternative spliced isoforms of CCM2 which eventually generated 22 novel protein isoforms. Genetic analysis of CCM2 isoforms revealed that the CCM2 isoforms can be classified into two groups based on their alternative promoters and alternative start codon exons. Our data demonstrated that CCM2 isoforms not only are specific in their subcellular compartmentation but also have distinct cellular expression patterns among various tissues and cells, indicating the pleiotropic cellular roles of CCM2 through their multiple isoforms. In fact, the complexity of the CCM2 genomic structure was reflected by the multiple layers of regulation of CCM2 expression patterns. At the transcriptional level, it is accomplished by alternative promoters, alternative splicing, and multiple transcriptional start sites and termination sites; while at the translational level, it is carried out with various cellular functions with a distinguishable CCM2 protein group pattern, specified abundance and composition of selective isoforms in a cell and tissue specific fashion. Through experimentation, we discovered a unique phosphotyrosine binding (PTB) domain, namely atypical phosphotyrosine binding (aPTB) domain. Some long CCM2 isoform proteins contain both classes of PTB domains, making them a dual PTB domain-containing protein. Both CCM1 and CCM3 can bind competitively to this aPTB domain, indicating CCM2 as the cornerstone for CCM signaling complex (CSC).