Gotowa bibliografia na temat „WD40-repeat proteins”

A yeast two-hybrid approach was used to discern possible new effectors for the βγ subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gβ, each exhibiting the WD40 repeat motif. Two of these proteins, thereceptor foractivatedCkinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gβγ using an anti-Gβ antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gβ1γ1but also unexpectedly with the transducin heterotrimer Gαtβ1γ1. Gαtalone does not interact, but it must contribute to the interaction because the apparent EC50value of RACK1 for Gαtβ1γ1is 3-fold greater than that for Gβ1γ1(0.1versus0.3 μm). RACK1 is a scaffold that interacts with several proteins, among which are activated βIIPKC and dynamin-1 (1). βIIPKC and dynamin-1 compete with Gβ1γ1and Gαtβ1γ1for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gβγ interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

Abstract In planta, a vital regulatory complex, MYB–basic helix–loop–helix (bHLH)–WD40 (MBW), is involved in trichome development and synthesis of anthocyanin and proanthocyanin in Arabidopsis. Usually, WD40 proteins provide a scaffold for protein–protein interaction between MYB and bHLH proteins. Members of subgroup 9 of the R2R3 MYB transcription factors, which includes MYBMIXTA-Like (MML) genes important for plant cell differentiation, are unable to interact with bHLH. In this study, we report that a cotton (Gossypium hirsutum) seed trichome or lint fiber-related GhMML factor, GhMML4_D12, interacts with a diverged WD40 protein (GhWDR) in a process similar to but different from that of the MBW ternary complex involved in Arabidopsis trichome development. Amino acids 250–267 of GhMML4_D12 and the first and third WD40 repeat domains of GhWDR determine their interaction. GhWDR could rescue Arabidopsis ttg1 to its wild type, confirming its orthologous function in trichome development. Our findings shed more light towards understanding the key role of the MML and WD40 families in plants and in the improvement of cotton fiber production.

Coatomer is required for the retrieval of proteins from an early Golgi compartment back to the endoplasmic reticulum. The WD40 domain of α-COP is required for the recruitment of KKTN-tagged proteins into coatomer-coated vesicles. However, lack of the domain has only minor effects on growth in yeast. Here, we show that the WD40 domain of β′-COP is required for the recycling of the KTKLL-tagged Golgi protein Emp47p. The protein is degraded more rapidly in cells with a point mutation in the WD40 domain of β′-COP (sec27-95) or in cells lacking the domain altogether, whereas a point mutation in the Clathrin Heavy Chain Repeat (sec27-1) does not affect the turnover of Emp47p. Lack of the WD40 domain of β′-COP has only minor effects on growth of yeast cells; however, absence of both WD40 domains of α- and β′-COP is lethal. Two hybrid studies together with our analysis of the maturation of KKTN-tagged invertase and the turnover of Emp47p in α- and β′-COP mutants suggest that the two WD40 domains of α- and β′-COP bind distinct but overlapping sets of di-lysine signals and hence both contribute to recycling of proteins with di-lysine signals.

WD40-repeat (WDR) proteins are a family of proteins that are characterized by widespread occurrence, low level of sequence conservation, common structural conformation (β propeller structure) and functional diversity. They act as scaffolds for multi-protein complex assembly during cellular processes like DNA repair, cell division, apoptosis, etc. Chapter 1 introduces WDR proteins and reviews the various features that characterize this family of proteins. The functions and the significance of WDR proteins have been described and the importance of characterizing the WDR proteins of unknown function, which have been implicated in human disorders, is discussed. Specifically, the link between a putative missense mutation in a relatively unstudied WDR protein, WDR8 and isolated Microspherophakia is elaborated on. Microspherophakia is a congenital, autosomal recessive disorder in humans characterized by the presence of a smaller, more spherical lens. The clinical, phenotypic and genotypic characteristics of this developmental disorder are explained. Chapter 2 lists the various protocols and the experimental techniques and methods used in this study. Chapter 3 details the results regarding the function of WDR8 in zebrafish eye development. Morpholino-mediated knockdown of WDR8 during development caused a decrease in cell numbers in the lens and retinal layers, ultimately resulting in a reduction in eye size and lens size, without affecting the gross morphology of the eye. When embryos were supplied with exogenous WDR8 lacking the morpholino-binding site, this reduction in eye size was rescued proving that the phenotype was due to the knock down of WDR8. Further, this phenotype was specific to WDR8, since knock down of another centrosomal WDR protein, WDR62 (mutations in which cause Microcephaly), did not affect the eye size in the embryos. Cell cycle analysis of whole embryos at 24 hpf (hours-post-fertilization) and the retinal cells at 48 hpf revealed cell cycle arrest selectively in the retina of the WDR8 morphants. The results discussed in this chapter also reveal an abnormal persistence of Phospho-Histone H3 (PH3+, a marker for mitosis) positive cells in the eyes of morphants, suggesting mitotic arrest in the retinal cells. Moreover, WDR8 morphants showed an increase in the PH3+ retinal cells undergoing programmed cell death, indicating the removal of the cells arrested during mitosis by apoptosis. Results from flow-cytometric analysis of co-stained retinal cells showed that the cells undergoing cell cycle arrest in the WDR8 morphants were predominantly PAX6+. Paired Box 6 (PAX6) is a major transcriptional regulator of early eye development and differentiation. Interestingly, it is also shown that unlike the knock down, the over-expression of WDR8 affected cell division ubiquitously, resulting in extensive apoptosis and decreased survival of the embryos. Thus, the results from the knock down experiments showed that WDR8 is involved in the regulation of cell division in the eye during zebrafish development. Chapter 3 also contains the results of the experiments aimed at understanding the mechanism by which a putative mutation (p.Pro383Leu) in WDR8 could affect the function of the protein and contribute to Microspherophakia. The results presented reveal that, when the WDR8 morphants were injected with the plasmid expressing either the human wild type or mutant WDR8 in order to compensate for the deficiency in zebrafish WDR8, the wild type human WDR8 could suppress the morphant phenotype and rescue the cell cycle defect and the increased apoptosis. However, the mutant human WDR8 failed to suppress the reduction in eye size and restore the level of cell division in the retina, suggesting that the mutation indeed abrogated the protein function. Further, Chapter 3 also presents the findings from the expression of either the human wild type or mutant WDR8 in HeLa cells that was carried out in order to identify differences between the wild type and mutant proteins in terms of the localization and the interaction with binding partners. The centrosomal localization of the mutant protein was found to be unaltered in the presence of the endogenous wild type protein. Also, the over-expression of either the wild type or the mutant WDR8 resulted in cell cycle arrest in HeLa cells. Importantly, co-immunoprecipitation experiments showed that the mutation interferes with the interaction of the WDR8 protein with its binding partners, such as OFD1 (oral-facial-digital syndrome 1), a centrosomal protein. Chapter 4 discusses the findings and the conclusions of the present study. Based on the results explained in Chapter 3, the function of WDR8 during eye development and its role in the causation of microspherophakia are explained. The present study offers the following insights: 1. WDR8 plays an important role in the cell-cycle progression in the precursor cells of the developing optic vesicle. Thus, WDR8 is required in the developing eye for attaining the optimal cell numbers in the lens and retina of zebrafish. 2. The missense mutation (p.Pro383Leu) in WDR8 diminishes its interaction ability and affects its function. Thus, homozygous missense mutation in WDR8 can abrogate its function, leading to the disease phenotype suggesting that WDR8 is a causative gene for Microspherophakia.