Project description:Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.
Project description:SecA facilitates bacterial protein translocation by its association with presecretory or membrane proteins and the SecYEG translocon channel. Once assembled, SecA ATPase undergoes cycles of membrane insertion and retraction at SecYEG that drive protein translocation in a stepwise fashion. SecA exists in equilibrium between a monomer and dimer, and association with its translocation ligands shifts this equilibrium dramatically. Here, we examined the proposal that protein translocation can occur by means of a SecA monomer. We produced a mutant SecA protein lacking residues 2-11, which was found to exist mostly as a monomer, and it was unable to complement a conditional-lethal secA mutant, was inactive for in vitro protein translocation, and was poorly active for translocation ATPase activity. Furthermore, we developed a technique termed membrane trapping, where wild-type SecA subunits became trapped within the membrane by overproduction of membrane-stuck mutant SecA proteins, and, in one case, a membrane-associated SecA heterodimer was demonstrated. Finally, we examined both endogenous and reconstituted membrane-bound SecA and found a significant level of SecA dimer in both cases, as assessed by chemical crosslinking. Collectively, our results strongly suggest that membrane-bound SecA dimer is critical for the protein translocation cycle, although these results cannot exclude participation of SecA monomer at some stage in the translocation process. Our findings have important implications regarding SecA motor function and translocon assembly and activation.
Project description:Dmbx1 is a paired-class homeodomain transcription factor. We show here that mice deficient in Dmbx1 exhibit severe leanness associated with hypophagia and hyperactivity and that isolation of a Dmbx1(-/-) mouse from its cohabitants induces self-starvation, sometimes leading to death, features similar to those of anorexia nervosa in humans. Interestingly, overexpression of agouti in Dmbx1(-/-) mice failed to induce aspects of the A(y)/a phenotype, including hyperphagia, obesity, and diabetes mellitus. In Dmbx1(-/-) mice, administration of agouti-related protein increased cumulative food intake for the initial 6 h but significantly decreased it over 24- and 48-h periods. In addition, Dmbx1 was shown to be expressed at embryonic day 15.5 in the lateral parabrachial nucleus, the rostral nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus, and the reticular nucleus in the brainstem, all of which receive melanocortin signaling, indicating involvement of Dmbx1 in the development of the neural network for the signaling. Thus, Dmbx1 is essential for various actions of agouti-related protein and plays a role in normal regulation of energy homeostasis and behavior.
Project description:The signal recognition particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.
Project description:Essential genes, those critical for the survival of an organism under certain conditions, play a significant role in pharmaceutics and synthetic biology. Knowledge of protein localization is invaluable for understanding their function as well as the interaction of different proteins. However, systematical examination of essential genes from the aspect of the localizations of proteins they encode has not been explored before. Here, a comprehensive protein localization analysis of essential genes in 27 prokaryotes including 24 bacteria, 2 mycoplasmas and 1 archaeon has been performed. Both statistical analysis of localization information in these genomes and GO (Gene Ontology) terms enriched in the essential genes show that proteins encoded by essential genes are enriched in internal location sites, while exist in cell envelope with a lower proportion compared with non-essential ones. Meanwhile, there are few essential proteins in the external subcellular location sites such as flagellum and fimbrium, and proteins encoded by non-essential genes tend to have diverse localizations. These results would provide further insights into the understanding of fundamental functions needed to support a cellular life and improve gene essentiality prediction by taking the protein localization and enriched GO terms into consideration.
Project description:Argonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes. The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA. We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes.Ago2 was deleted specifically in the growing oocytes. Although the Ago2-deficient oocytes are able to develop to mature oocytes, they have abnormal spindles and chromosomes that are unable to cluster together properly. This phenotype is very similar to the phenotype of Dicer-deficient oocytes. We examined the microRNA expression profile in the Ago2-deficient oocyte and found that the expression of most microRNAs was reduced by more than 80%. To determine the downstream genes that are regulated by Ago2, we used microarray analysis on Ago2-deficient oocytes and found that 512 genes were upregulated and 1,073 genes were downregulated (FC > 2, P < 0.05).Our study shows that Ago2 has a key function in the mouse oocyte through global regulation of microRNA stability, and through this mechanism it affects gene expression in developing oocytes.
Project description:Cleft palate is among the most common birth defects. Currently, only 30% of cases have identified genetic causes, whereas the etiology of the majority remains to be discovered. We identified a new regulator of palate development, protein arginine methyltransferase 1 (PRMT1), and demonstrated that disruption of PRMT1 function in neural crest cells caused complete cleft palate and craniofacial malformations. PRMT1 is the most highly expressed of the protein arginine methyltransferases, enzymes responsible for methylation of arginine motifs on histone and nonhistone proteins. PRMT1 regulates signal transduction and transcriptional activity that affect multiple signal pathways crucial in craniofacial development, such as the BMP, TGFβ, and WNT pathways. We demonstrated that Wnt1-Cre;Prmt1 fl/fl mice displayed a decrease in palatal mesenchymal cell proliferation and failure of palatal shelves to reach the midline. Further analysis in signal pathways revealed that loss of Prmt1 in mutant mice decreased BMP signaling activation and reduced the deposition of H4R3me2a mark. Collectively, our study demonstrates that Prmt1 is crucial in palate development. Our study may facilitate the development of a better strategy to interrupt the formation of cleft palate through manipulation of PRMT1 activity.
Project description:We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p.
Project description:Essential genes are a group of genes that are indispensable for cell survival and cell fertility. Studying human essential genes helps scientists reveal the underlying biological mechanisms of a human cell but also guides disease treatment. Recently, the publication of human essential gene data makes it possible for researchers to train a machine-learning classifier by using some features of the known human essential genes and to use the classifier to predict new human essential genes. Previous studies have found that the essentiality of genes closely relates to their properties in the protein-protein interaction (PPI) network. In this work, we propose a novel supervised method to predict human essential genes by network embedding the PPI network. Our approach implements a bias random walk on the network to get the node network context. Then, the node pairs are input into an artificial neural network to learn their representation vectors that maximally preserves network structure and the properties of the nodes in the network. Finally, the features are put into an SVM classifier to predict human essential genes. The prediction results on two human PPI networks show that our method achieves better performance than those that refer to either genes' sequence information or genes' centrality properties in the network as input features. Moreover, it also outperforms the methods that represent the PPI network by other previous approaches.