Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.     

Effects of mutation at a conserved N-glycosylation site in the bovine retinal cyclic nucleotide-gated ion channel

Bovine retinal cyclic nucleotide-gated (CNG) ion channel contains an evolutionary conserved N-glycosylation site in the external loop between the fifth transmembrane segment and the pore-forming region. The effect of tunicamycin treatment and the site-specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N-glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N-glycosylation site did not affect expression and functionality of the CNG channel in oocytes.     

Prmt5, which forms distinct homo-oligomers, is a member of the protein-arginine methyltransferase family

We found that JBP1, known as a human homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the hepatitis C virus in a yeast two-hybrid screen. Amino acid sequence analysis revealed that Skb1Hs/JBP1 contains conserved motifs of S-adenosyl-l-methionine-dependent protein-arginine methyltransferases (PRMTs). Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family. Recombinant PRMT5 protein purified from human cells methylated myelin basic protein, histone, and the amino terminus of fibrillarin fused to glutathione S-transferase. Myelin basic protein methylated by PRMT5 contained monomethylated and dimethylated arginine residues. Recombinant glutathione S-transferase-PRMT5 protein expressed in Escherichia coli also contained the catalytic activity. Sedimentation analysis of purified PRMT5 on a sucrose density gradient indicated that PRMT5 formed distinct homo-oligomeric complexes, including a dimer and tetramer, that comigrated with the enzyme activity. The PRMT5 homo-oligomers were dissociated into a monomer in the presence of a reducing agent, whereas a monomer, dimer, and multimer were detected in the absence or at low concentrations of a reducing agent. The results indicate that both covalent linkage by a disulfide bond and noncovalent association are involved in the formation of PRMT5 homo-oligomers. Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract. PRMT5 appears to have lower specific enzyme activity than PRMT1. Although PRMT1 is known to be mainly located in the nucleus, human PRMT5 is predominantly localized in the cytoplasm.     

Mixotrophy in the sand-dwelling dinoflagellate Thecadinium kofoidii

Thecadinium kofoidii is a marine sand-dwelling dinoflagellate that sometimes forms dense blooms. This species was previously thought to be an exclusively autotrophic dinoflagellate, and its mixotrophic ability has not been explored yet. By investigating its ecophysiology, its trophic mode should be revealed. We explored the mixotrophic ability of T. kofoidii by examining its protoplasm under light and transmission electron microscopes with diverse algal prey species. Furthermore, the feeding mechanism of T. kofoidii and prey species on which it feeds were investigated. In addition, the growth and ingestion rates of T. kofoidii as a function of prey concentration were determined when feeding on the benthic cryptophyte Rhodomonas salina. Thecadinium kofoidii was able to feed on R. salina and the dinoflagellate Symbiodinium voratum, which had equivalent spherical diameters (ESDs) ≤ 10.1?µm, while it did not feed on the benthic dinoflagellates Levanderina fissa, Prorocentrum concavum or Ostreopsis cf. ovata, which had ESDs ≥ 15?µm. Thecadinium kofoidii fed on the edible prey cells using the peduncle. The maximum ingestion rate of T. kofoidii on R. salina was 1.3 cells predator^?1 d^?1. However, feeding on R. salina did not significantly increase the growth rate of T. kofoidii. The low ingestion rate of T. kofoidii on R. salina may have partially resulted in the lack of significant increase in its growth rate due to mixotrophy. The present study discovered predator–prey relationships between T. kofoidii and R. salina and S. voratum, which may change our view of the energy flow and carbon cycling in marine benthic food webs.     

Record of Blue tilapia Oreochromis aureus (Steindachner, 1864) in the Eerste River catchment,Western Cape province,South Africa

Oreochromis aureus was imported from Israel into South Africa in 1959 but data on its current status in South Africa are lacking. Genomic DNA was extracted and the COI gene amplified at the South African Institute for Aquatic Biodiversity. The identity of the sequences and specimens was determined using the Barcode of Life Data Systems and GenBank. Morphological and genetic assessment demonstrated that 11 specimens collected from two farm dams in the Eerste River System, Western Cape province, were Oreochromis aureus. A MaxEnt model compiled using global distribution, rainfall and temperature data predicted that large areas of southern Africa were climatically suitable for this species, indicating considerable invasion debt in southern Africa. As a result, surveys to assess for the extent of the invasion in South Africa and eradication of existing populations, if feasible, are recommended management actions.     

Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium

Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.     

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Background

Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders'' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers'' targets.

Results

We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.

Conclusions

We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.