RESUMO
The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.
Assuntos
Células Epiteliais , Proteínas de Membrana , Núcleosídeo-Fosfato Quinase , Proteínas de Junções Íntimas , Animais , Camundongos , Citoesqueleto de Actina , Actinas , Citoesqueleto , Citosol , Núcleosídeo-Fosfato Quinase/genética , Proteínas de Membrana/genéticaRESUMO
The G-protein coupled estrogen receptor (GPER), a proposed tumor suppressor, relays short-term non-genomic responses in target cells and tissues. It frequently undergoes down-modulation in primary tumors of the breast, ovary, and endometrium. Liu and co-workers recently reported loss of GPER expression in colorectal cancer and attributed it to DNA methylation-dependent silencing. We hypothesized that GPER expression is inversely correlated with methylation in the upstream CpG island (upCpGi) in the GPER locus. Methylation in the upCpGi was analysed by bisulfite sequencing and correlated with GPER expression in a panel of colon cancer cell lines. Eight downstream CpGs of the upCpGi was differentially methylated across the cell lines. Methylation in this differentially methylated region (DMR) correlated inversely with GPER expression. Two cell lines, namely SW620 and COLO-320DM, were compared in terms of their viability in response to varying concentrations of G1, a GPER specific agonist. SW-620 cells, which had the least methylated DMR and the highest level of GPER expression, showed significant loss of viability with 1 µM G1. COLO-320DM, which had the most methylated DMR and the lowest level of GPER expression, did not show a significant response to 1 µM G1. At 5 µM G1, SW620 cells showed a greater reduction in viability than COLO-320DM cells. DNA methylation in the DMR is inversely correlated with GPER expression. DNA methylation-dependent silencing of GPER may be, at least in part, the underlying reason behind the loss of estrogen's oncoprotective effect via GPER in the colon.
Assuntos
Neoplasias do Colo/metabolismo , Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Proteínas de Neoplasias/biossíntese , Receptores de Estrogênio/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA de Neoplasias/genética , Células HCT116 , Células HT29 , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Several post-translational protein modifications lie predominantly within regions of disorder: the biased localization has been proposed to expand the binding versatility of disordered regions. However, investigating a representative dataset of 500 human N-glycoproteins, we observed the sites of N-linked glycosylations or N-glycosites, to be predominantly present in the regions of predicted order. When compared with disordered stretches, ordered regions were not found to be enriched for asparagines, serines and threonines, residues that constitute the sequon signature for conjugation of N-glycans. We then investigated the basis of mutual exclusivity between disorder and N-glycosites on the basis of amino acid distribution: when compared with control ordered residue stretches without any N-glycosites, residue neighborhoods surrounding N-glycosites showed a depletion of bulky, hydrophobic and disorder-promoting amino acids and an enrichment for flexible and accessible residues that are frequently found in coiled structures. When compared with control disordered residue stretches without any N-glycosites, N-glycosite neighborhoods were depleted of charged, polar, hydrophobic and flexible residues and enriched for aromatic, accessible and order-promoting residues with a tendency to be part of coiled and ß structures. N-glycosite neighborhoods also showed greater phylogenetic conservation among amniotes, compared with control ordered regions, which in turn were more conserved than disordered control regions. Our results lead us to propose that unique primary structural compositions and differential propensities for evolvability allowed for the mutual spatial exclusion of N-glycosite neighborhoods and disordered stretches.
