Identification of PatL1, a human homolog to yeast P body component Pat1

In yeast, the activators of mRNA decapping, Pat1, Lsm1 and Dhh1, accumulate in processing bodies (P bodies) together with other proteins of the 5'-3'-deadenylation-dependent mRNA decay pathway. The Pat1 protein is of particular interest because it functions in the opposing processes of mRNA translation and mRNA degradation, thus suggesting an important regulatory role. In contrast to other components of this mRNA decay pathway, the human homolog of the yeast Pat1 protein was unknown. Here we describe the identification of two human PAT1 genes and show that one of them, PATL1, codes for an ORF with similar features as the yeast PAT1. As expected for a protein with a fundamental role in translation control, PATL1 mRNA was ubiquitously expressed in all human tissues as were the mRNAs of LSM1 and RCK, the human homologs of yeast LSM1 and DHH1, respectively. Furthermore, fluorescence-tagged PatL1 protein accumulated in distinct foci that correspond to P bodies, as they co-localized with the P body components Lsm1, Rck/p54 and the decapping enzyme Dcp1. In addition, as for its yeast counterpart, PatL1 expression was required for P body formation. Taken together, these data emphasize the conservation of important P body components from yeast to human cells.     

Inhibition of eukaryotic translation initiation by the marine natural product pateamine A

Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, we report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.     

Cyanobacterial response regulator PatA contains a conserved N-terminal domain (PATAN) with an alpha-helical insertion

The cyanobacterium Anabaena (Nostoc) PCC 7120 responds to starvation for nitrogen compounds by differentiating approximately every 10th cell in the filament into nitrogen-fixing cells called heterocysts. Heterocyst formation is subject to complex regulation, which involves an unusual response regulator PatA that contains a CheY-like phosphoacceptor (receiver, REC) domain at its C-terminus. PatA-like response regulators are widespread in cyanobacteria; one of them regulates phototaxis in Synechocystis PCC 6803. Sequence analysis of PatA revealed, in addition to the REC domain, a previously undetected, conserved domain, which we named PATAN (after PatA N-terminus), and a potential helix-turn-helix (HTH) domain. PATAN domains are encoded in a variety of environmental bacteria and archaea, often in several copies per genome, and are typically associated with REC, Roadblock and other signal transduction domains, or with DNA-binding HTH domains. Many PATAN domains contain insertions of a small additional domain, termed alpha-clip, which is predicted to form a four-helix bundle. PATAN domains appear to participate in protein-protein interactions that regulate gliding motility and processes of cell development and differentiation in cyanobacteria and some proteobacteria, such as Myxococcus xanthus and Geobacter sulfurreducens.     

Hepatitis C virus infection alters P-body composition but is independent of P-body granules

Processing bodies (P-bodies) are highly dynamic cytoplasmic granules conserved among eukaryotes. They are present under normal growth conditions and contain translationally repressed mRNAs together with proteins from the mRNA decay and microRNA (miRNA) machineries. We have previously shown that the core P-body components PatL1, LSm1, and DDX6 (Rck/p54) are required for hepatitis C virus (HCV) RNA replication; however, how HCV infection affects P-body granules and whether P-body granules per se influence the HCV life cycle remain unresolved issues. Here we show that HCV infection alters P-body composition by specifically changing the localization pattern of P-body components that are required for HCV replication. This effect was not related to an altered expression level of these components and could be reversed by inhibiting HCV replication with a polymerase inhibitor. Similar observations were obtained with a subgenomic replicon that supports only HCV translation and replication, indicating that these early steps of the HCV life cycle trigger the P-body alterations. Finally, P-body disruption by Rap55 depletion did not affect viral titers or HCV protein levels, demonstrating that the localization of PatL1, LSm1, and DDX6 in P-bodies is not required for their function on HCV. Thus, the HCV-induced changes on P-bodies are mechanistically linked to the function of specific P-body components in HCV RNA translation and replication; however, the formation of P-body granules is not required for HCV infection.     

pilG Gene cluster and split pilL genes involved in pilus biogenesis,motility and genetic transformation in the cyanobacterium Synechocystis sp. PCC 6803

The unicellular motile cyanobacterium Synechocystis sp. PCC 6803 exhibits phototactic motility that depends on the type IV-like thick pilus structure. By gene disruption analysis, we showed that a gene cluster of slr1041, slr1042, slr1043 and slr1044, whose predicted products are homologous to PatA, CheY, CheW and MCP, respectively, was more or less required for pilus assembly, motility and natural transformation competency with extraneous DNA. By sequence homology, the missing cheA-like gene in this cluster was identified as novel split genes, slr0073 and slr0322, at separate loci on the genome. This was confirmed by non-motile phenotype of their disruptants. Unique hyperpiliation was observed in the slr1042 and slr0073 disruptants, suggestive of their specific interaction with pilT1. The genes, thus identified as pil genes in this study, were designated pilG (slr1041), pilH (slr1042), pilI (slr1043), pilJ (slr1044), pilL-N (slr0073) and pilL-C (slr0322).     

Translational repression by the oocyte-specific protein P100 in Xenopus

The translational regulation of maternal mRNAs is one of the most important steps in the control of temporal-spatial gene expression during oocyte maturation and early embryogenesis in various species. Recently, it has become clear that protein components of mRNPs play essential roles in the translational regulation of maternal mRNAs. In the present study, we investigated the function of P100 in Xenopus oocytes. P100 exhibits sequence conservation with budding yeast Pat1 and is likely the orthologue of human Pat1a (also called PatL2). P100 is maternally expressed in immature oocytes, but disappears during oocyte maturation. In oocytes, P100 is an RNA binding component of ribosome-free mRNPs, associating with other mRNP components such as Xp54, xRAP55 and CPEB. Translational repression by overexpression of P100 occurred when reporter mRNAs were injected into oocytes. Intriguingly, we found that when P100 was overexpressed in the oocytes, the kinetics of oocyte maturation was considerably retarded. In addition, overexpression of P100 in oocytes significantly affected the accumulation of c-Mos and cyclin B1 during oocyte maturation. These results suggest that P100 plays a role in regulating the translation of specific maternal mRNAs required for the progression of Xenopus oocyte maturation.     

De‐O‐acetylation of peptidoglycan regulates glycan chain extension and affects in vivo survival of Neisseria meningitidis

Peptidoglycan O‐acetylation is a modification found in many bacteria. In Gram‐positive pathogens, it contributes to virulence by conferring resistance to host lysozyme. However, in Gram‐negative pathogens, its contribution to physiology and virulence is unknown. We examined the contribution of patA, patB and ape1 to peptidoglycan O‐acetylation in the major human pathogen Neisseria meningitidis (Nm). Using genetic expression of all possible combinations of the three genes in Escherichia coli and Nm, we confirmed that PatA and PatB were required for PG O‐acetylation, while ApeI removed the O‐acetyl group. ApeI was active on all O‐acetylated muropeptides produced by PatA and PatB during heterologous expression in E. coli and was also active on several PG structures in vitro. Interestingly, in Nm, ApeI was found to preferentially de‐O‐acetylate muropeptides with tripeptide stems (GM3), suggesting that its activity is highly regulated. Accordingly, de‐O‐acetylation of GM3 regulated glycan chain elongation and cell size. Additionally, the virulence of Nm lacking ApeI was drastically reduced suggesting that regulation of glycan chain length by O‐acetylation contributes to bacterial fitness in the host. Altogether, our results suggest that ApeI represents an attractive target for new drug development.     

Substrate Specificity and Kinetic Characterization of Peptidoglycan O-Acetyltransferase B from Neisseria gonorrhoeae

The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.     

HOX genes in embryo- and phylogenesis

This review is concerned with the structure and function of the protein products and homeobox genes of the HOX complex. We also trace a relationship between morphological evolution and the evolution of the homeotic complex.