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DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding
Get PDFThe cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement
Structures of an active-site mutant of a plant 1,3-beta-glucanase in complex with oligosaccharide products of hydrolysis
Get PDFPlant endo-1,3-beta-glucanases are involved in important physiological processes such as defence mechanisms, cell division and flowering. They hydrolyze (13)-beta-glucans, with very limited activity towards mixed (13,14)-beta-glucans and branched
(13,16)-beta-glucans. Here, crystal structures of the potato (Solanum tuberosum) endo-1,3-beta-glucanase GLUB20-2 with the nucleophilic Glu259 residue substituted by alanine (E259A) are reported. Despite this active-site mutation, the protein retained residual endoglucanase activity and when incubated in the crystallization buffer with a linear hexameric substrate derived from (13)-beta-glucan (laminarahexose) cleaved it in two different ways, generating trisaccharides and tetrasaccharides, as confirmed by mass spectrometry. The trisaccharide (laminaratriose) shows higher binding affinity and was found to fully occupy the -1, -2 and -3 sites of the active-site cleft, even at a low molar excess of the substrate. At elevated substrate concentration the tetrasaccharide molecule (laminaratetrose) also occupies the active site, spanning the opposite sites +1, +2, +3 and +4 of the cleft. These are the first crystal structures of a plant glycoside hydrolase family 17 (GH17) member to reveal the protein–saccharide interactions and were determined at resolutions of 1.68 and 1.55 A˚ ,respectively. The geometry of the active-site cleft clearly precludes any (14)-beta-glucan topology at the subsites from -3 to +4 and could possibly accommodate beta-1,6-branching only at subsites +1 and +2. The glucose units at subsites -1 and -2 interact with highly conserved protein residues. In contrast, subsites -3, +3 and +4 are variable, suggesting that the mode of glucose binding at these sites may vary between different plant endo-1,3-beta-glucanases. Low substrate affinity is observed at subsites +1 and +2, as manifested by disorder of the glycosyl units there
HMG CoA reductase inhibition by Simvastatin gets rat β-Myosin heavy chain disappeared: A statin paradox.
Get PDFA Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5.
Get PDFLactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors
Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins
Get PDFHopQ1 (Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv. phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean. However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to co-purify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using FRET-FLIM techniques. Moreover, mass spectrometric (LC-MS-MS/MS) analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, pS denotes phosphoserine) located in the N-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector
Redox Active Dipyrromethene-Cu(II) Monolayer for Oriented Immobilization of His-Tagged RAGE Domains - the Base of Electrochemical Biosensor for Determination of Aβ16-23′
No full textCulturable bacteria community development in postglacial soils of Ecology Glacier, King George Island, Antarctica
Get PDFGlacier forelands are excellent sites in which to study microbial succession because conditions change
rapidly in the emerging soil. Development of the bacterial community was studied along two transects on lateral
moraines of Ecology Glacier, King George Island, by culture-dependent and culture-independent approaches
(denaturating gradient gel electrophoresis, DGGE). Environmental conditions such as cryoturbation and soil
composition affected both abundance and phylogenetic diversity of bacterial communities. Microbiocenosis
structure along transect 1(severe cryoturbation) differed markedly from that along transect 2 (minor
cryoturbation). Soil physical and chemical factors changed along the chronosequence (time since exposure) and
influenced the taxonomic diversity of cultivated bacteria (CB), particularly along transect 2. Arthrobacter spp.
played a pioneer role, and were present in all soil samples, but were most abundant along transect 1. Cultivated
bacteria isolated from transect 2 were taxonomically more diverse than those cultivated from transect 1; those
from transect 1 tended to express a broader range of enzyme and assimilation activities. Our data suggest that
cryoturbation is a major factor in controlling bacterial community development in postglacial soils, shed light on
microbial succession in glacier forelands, and add a new parameter to models that describe succession
phenomena
The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ ATP Carrier to Bongkrekate in Yeast
Get PDFThe ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains
of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC
containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of DAAC2 lethality, SAL1, was also
inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACDsal1D strain was lethal. In both strains
ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia
and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both
modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of
ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide
exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing
media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of
yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner
Complete Genome Sequence of the Probiotic Strain Lactobacillus casei (Formerly Lactobacillus paracasei) LOCK919.
Get PDFLactobacillus casei is usually regarded as a bacterium that lives naturally in the human intestinal tract, where it can contribute to host health and well-being. We describe here the complete genome sequence of L. casei LOCK919, a strain with probiotic properties isolated from child feces. The genome consists of a 3.11-Mb chromosome and a 29,768-bp plasmid