Eukaryotic double-ring chaperonin TRiC is an ATP-dependent protein-folding machine. Most of its substrates are known to form large ordered structures from multiple polypeptide chains. Since these ...structures are similar to fibrillar and oligomeric forms of amyloidogenic proteins, we hypothesized that TRiC may play a role in the development of neurodegenerative diseases of amyloid nature including prion diseases. Enzyme-linked immunosorbent assay showed that monomeric, oligomeric and fibrillar forms of prion protein (PrP) bind strongly to chaperonin TRiC, whereas glycation reduces the prion protein affinity for chaperonin. Nevertheless, dynamic light scattering, electron microscopy and thioflavin T fluorescence confirmed that all studied forms of PrP undergo an amyloid transformation after interaction with chaperonin, but different forms of prion protein are capable of having different effects on the functional state of TRiC. For example, prion protein monomers completely block its ability to reactivate the chaperonin's natural substrate - sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). At the same time, PrP oligomers and fibrils only partially prevent the reactivation of GAPDS upon the action of TRiC. The monomeric forms of prion protein glycated by methylglyoxal do not inhibit, but only slow down the chaperone-dependent reactivation of GAPDS. Thus, the interaction of amyloidogenic proteins with chaperonins could cause cell malfunction.
•All studied forms of prion protein (PrP) bind strongly to chaperonin TRiC.•All forms of PrP undergo an amyloid transformation using TRiC.•Glycation of prion protein reduces its affinity for chaperonin TRiC.•Only PrP monomers completely block TRiC chaperone activity.•TRiC may play a role in the development of neurodegenerative diseases.
Investigation of the chaperonin encoded by gene
146
of bacteriophage EL
Pseudomonas aeruginosa
that we characterized earlier has been continued. To reveal the mechanism of its functioning, new ...recombinant substrate proteins, fragments of gene product (gp) 183 containing the lysozyme domain were prepared. Their interaction with gp146 was studied. The influence of the phage chaperonin on the thermal aggregation of one of these gp183 fragments and endolysin (gp188) was investigated in both the presence and the absence of ATP by dynamic light scattering. In the absence of ATP, the phage chaperonin forms stable complexes with substrate proteins, thereby protecting them against thermal aggregation. Experimental data obtained for different substrate proteins are analyzed.
The roughly purified extract of
E. coli
proteins has been studied by cryoelectron microscopy, the class-sums containing 2D projections of two proteins (β-galactosidase and 2-oxoglutarate ...dehydrogenase complex catalytic domain (ODC-CD)), identified in an extract by tandem mass spectrometry, have been distinguished. The structures of these proteins have been solved at near-atomic resolution.
De novo
simulation of the ODC-CD structure yielded an atomic model that revealed differences in the positions of some amino acid residues of the active center, in comparison with the known crystal structures.
The complete DNA sequence of a new lytic T7-like bacteriophage φKMV is presented. It is the first genome sequence of a member of the
Podoviridae that infects
Pseudomonas aeruginosa. The linear G + ...C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand. Despite absence of homology at the DNA level, 11 of the 48 φKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins. Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins. Surprisingly, the major capsid protein completely lacks sequence homology to any known protein. Also, the strong virulence toward many clinical
P. aeruginosa isolates and a short replication time make φKMV attractive for phage therapy or a potential source for antimicrobial proteins.
Recombinant adenoviruses have enormous potential as vectors for gene therapy. They have evolved an efficient method of infection and a wide host range but this leads to concerns about the specificity ...of gene delivery. In order to target an adenovirus type 5-based vector we have investigated an antibody approach. A virus neutralising scFv antibody fragment was isolated from a phage library and a C-terminal fusion protein with epidermal growth factor (EGF) constructed. This fusion protein, or 'adenobody', bound both to the fibre protein of the adenovirus and to the EGF receptor (EGFR) on human cells, and was able to direct adenoviral binding to the new receptor. Using this system the efficiency of viral infection was markedly enhanced and was targeted to the EGFR. The adenobody-directed infection correlated with the level of EGF receptor expressed on the cells and could be blocked by competition with pure EGF. Peptide inhibition experiments suggest that infection is mediated directly through attachment to the EGFR and does not require penton-integrin interactions. This work shows that the 'adenobody' approach can enhance the efficiency as well as target adenoviral infection and has numerous potential applications for gene therapy.
Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A3 (CM). In the absence of the antibiotics, PG extracts from the nuclei ...about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this dif- ference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and s~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (s~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV- stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (s~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.
Background: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the
Escherichia coli host. The ...tail has a baseplate to whïch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell.
Results: The crystal structure of recombinant gp9, determined to 2.3 Å resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded
β sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel
β sandwich having some resemblance to ‘jelly-roll’ viral capsid protein structures.
Conclusions: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.
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