RESUMO
We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Animais Geneticamente Modificados , Dano ao DNA , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Instabilidade Genômica , Células HeLa , Humanos , Modelos Biológicos , Mutação , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Basement membranes (BMs) are specialized extracellular scaffolds that influence behaviors of cells in epithelial, endothelial, muscle, nervous, and fat tissues. Throughout development and in response to injury or disease, BMs are fine-tuned with specific protein compositions, ultrastructure, and localization. These features are modulated through implements of the BM toolkit that is comprised of collagen IV, laminin, perlecan, and nidogen. Two additional proteins, peroxidasin and Goodpasture antigen-binding protein (GPBP), have recently emerged as potential members of the toolkit. In the present study, we sought to determine whether peroxidasin and GPBP undergo dynamic regulation in the assembly of uterine tissue BMs in early pregnancy as a tractable model for dynamic adult BMs. We explored these proteins in the context of collagen IV and laminin that are known to extensively change for decidualization. Electron microscopic analyses revealed: 1) a smooth continuous layer of BM in between the epithelial and stromal layers of the preimplantation endometrium; and 2) interrupted, uneven, and progressively thickened BM within the pericellular space of the postimplantation decidua. Quantification of mRNA levels by qPCR showed changes in expression levels that were complemented by immunofluorescence localization of peroxidasin, GPBP, collagen IV, and laminin. Novel BM-associated and subcellular spatiotemporal localization patterns of the four components suggest both collective pericellular functions and distinct functions in the uterus during reprogramming for embryo implantation.
Assuntos
Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Implantação do Embrião/genética , Proteínas da Matriz Extracelular/genética , Laminina/genética , Peroxidase/genética , Proteínas Serina-Treonina Quinases/genética , Útero/metabolismo , Animais , Colágeno Tipo IV/metabolismo , Implantação do Embrião/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Injeções , Laminina/metabolismo , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peroxidase/metabolismo , Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Óleo de Gergelim/administração & dosagem , Útero/efeitos dos fármacos , PeroxidasinaRESUMO
Basement membranes (BMs) are specialized extracellular scaffolds that provide architecture and modulate cell behaviors in tissues, such as fat, muscle, endothelium, endometrium, and decidua. Properties of BMs are maintained in homeostasis for most adult tissues. However, BM ultrastructure, composition, and localization are rapidly altered in select uterine tissues that are reprogrammed during pregnancy to enable early maternal-embryo interactions. Here, our data exhibit both static and dynamic BMs that were tracked in mouse uterine tissues during pre-, peri-, and postimplantation periods of pregnancy. The data exhibit spatial-temporal patterns of BM property regulation that coincide with the progression of adapted physiology. Further interpretation and discussion of these data in this article are described in the associated research article titled, "Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming" (C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, 2016) [1].