The C-terminal cysteine annulus participates in auto-chaperone function for Salmonella phage P22 tailspike folding and assembly

Elongated trimeric adhesins are a distinct class of proteins employed by phages and viruses to recognize and bind to their host cells, and by bacteria to bind to their target cells and tissues. The tailspikes of E. coli phage K1F and Bacillus phage Ø29 exhibit auto-chaperone activity in their trimeric C-terminal domains. The P22 tailspike is structurally homologous to those adhesins. Though there are no disulfide bonds or reactive cysteines in the native P22 tailspikes, a set of C-terminal cysteines are very reactive in partially folded intermediates, implying an unusual local conformation in the domain. This is likely to be involved in the auto-chaperone function. We examined the unusual reactivity of C-terminal tailspike cysteines during folding and assembly as a potential reporter of auto-chaperone function. Reaction with IAA blocked productive refolding in vitro, but not off-pathway aggregation. Two-dimensional PAGE revealed that the predominant intermediate exhibiting reactive cysteine side chains was a partially folded monomer. Treatment with reducing reagent promoted native trimer formation from these species, consistent with transient disulfide bonds in the auto-chaperone domain. Limited enzymatic digestion and mass spectrometry of folding and assembly intermediates indicated that the C-terminal domain was compact in the protrimer species. These results indicate that the C-terminal domain of the P22 tailspike folds itself and associates prior to formation of the protrimer intermediate, and not after, as previously proposed. The C-terminal cysteines and triple β-helix domains apparently provide the staging for the correct auto-chaperone domain formation, needed for alignment of P22 tailspike native trimer

Increased occurrence of protein kinase CK2 in astrocytes in Alzheimer’s disease pathology

Background Alzheimer’s disease (AD) is the most common neurodegenerative disease. In addition to the occurrence of amyloid deposits and widespread tau pathology, AD is associated with a neuroinflammatory response characterized by the activation of microglia and astrocytes. Protein kinase 2 (CK2, former casein kinase II) is involved in a wide variety of cellular processes. Previous studies on CK2 in AD showed controversial results, and the involvement of CK2 in neuroinflammation in AD remains elusive. Methods In this study, we used immunohistochemical and immunofluorescent staining methods to investigate the localization of CK2 in the hippocampus and temporal cortex of patients with AD and non-demented controls. We compared protein levels with Western blotting analysis, and we investigated CK2 activity in human U373 astrocytoma cells and human primary adult astrocytes stimulated with IL-1β or TNF-α. Results We report increased levels of CK2 in the hippocampus and temporal cortex of AD patients compared to non-demented controls. Immunohistochemical analysis shows CK2 immunoreactivity in astrocytes in AD and control cases. In AD, the presence of CK2 immunoreactive astrocytes is increased. CK2 immunopositive astrocytes are associated with amyloid deposits, suggesting an involvement of CK2 in the neuroinflammatory response. In U373 cells and human primary astrocytes, the selective CK2 inhibitor CX-4945 shows a dose-dependent reduction of the IL-1β or TNF-α induced MCP-1 and IL-6 secretion. Conclusions This data suggests that CK2 in astrocytes is involved in the neuroinflammatory response in AD. The reduction in pro-inflammatory cytokine secretion by human astrocytes using the selective CK2 inhibitor CX-4945 indicates that CK2 could be a potential target to modulate neuroinflammation in AD

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context

Compensatory changes in GroEL/Gp31 affinity as a mechanism for allele-specific genetic interaction

Previous work has shown that the GroEL-GroES interaction is primarily mediated by the GroES mobile loop. In bacteriophage T4 infection, GroES is substituted by the gene 31-encoded cochaperonin, Gp31. Using a genetic selection scheme, we have identified a new set of mutations in gene 31 that affect interaction with GroEL; all mutations result in changes in the mobile loop of Gp31. Biochemical analyses reveal that the mobile loop mutations alter the affinity between Gp31 and GroEL, most likely by modulating the stability of the GroEL-bound hairpin conformation of the mobile loop. Surprisingly, mutations in groEL that display allele-specific interactions with mutations in gene 31 alter residues in the GroEL intermediate domain, distantly located from the mobile loop binding site. The observed patterns of genetic and biochemical interaction between GroES or Gp31 and GroEL point to a mechanism of genetic allele specificity based on compensatory changes in affinity of the protein-protein interaction. Mutations studied in this work indirectly alter affinity by modulating a folding transition in the Gp31 mobile loop or by modulating a hinged conformational change in GroEL

Isolation and expression of a plastid alpha chaperonin cDNA sequence from "triticum aestivum"

This thesis describes the discovery of a new class of related proteins which has been named the chaperonins (Hemmingsen, Woolford, van der Vies, Tilly, Dennis, Georgopoulos, Hendrix & Ellis, Nature 333, 330-334, 1988). The proteins in this highly conserved class are structurally and immunologically related and ubiquitous in their occurrence in plastids, mitochondria and bacteria. The chaperonins comprise one class of the larger family of molecular chaperones since their function of assisting in the folding and assembly of other polypeptides without being components of the final structure meets the criteria suggested for molecular chaperones (Ellis, Nature 328, 378-379, 1987). The chaperonin class of proteins was discovered during studies on the assembly of the hexadecameric enzyme ribulose-1.5-bisphosphate carboxylase-oxygenase (Rubisco); this enzyme is found in the chloroplasts of plants where it catalyzes the first step in the pathways of both photosynthesis and photorespiration. The assembly of Rubisco in vivo had been proposed to require the activity of another chloroplast protein, originally known as the Rubisco large subunit binding protein because it binds to Rubisco large subunits newly- synthesized in isolated chloroplasts of Pisum sativum (Barraclough & Ellis, Biochim. Biophys, Acta 608, 19-31, 1980). Newly-imported Rubisco small subunits have since been shown to bind to the same chloroplast protein, which has therefore been renamed the Rubisco subunit binding protein (abbreviated to binding protein), or the plastid chaperonin. Antibodies raised against the plastid chaperonin purified from P.sativum recognize two subunit polypeptides with an apparent Mr of 61 500 and 60 500 (termed alpha and beta respectively) in extracts of Triticum aestivum leaves. With the aid of these antibodies a cDNA fragment has been isolated and sequenced from a lambda gtl 1 expression library of cDNA from leaves of Triticum aestivum. The cDNA fragment of 1834 bp encodes the entire mature plastid chaperonin alpha subunit plus two amino acids of the presequence. The amino acid sequence of the T.aestivum alpha chaperonin shows 46% identity to a protein from Escherichia coli known as the groEL protein; this protein had previously been shown to be essential for cell viability and is required for the assembly of bacteriophage capsids. An identity of 59% is found between the wheat alpha chaperonin and a groEL-like protein present in Mycobacterium leprae and M. tuberculosis. Immunologically related proteins were also detected in a variety of prokaryotes including cyanobacteria and Prochlorothrix hollandica, the eukaryote Chlamydomonas reinhardii and in mitochondrial fractions from P.sativum leaves and Solarum tuberosum tubers. All these related proteins comprise the new class of chaperonins. Amino acid sequences of all the known chaperonins from plastids, mitochondria and bacteria were compared and show 41%-58% amino acid identity. The chloroplast alpha chaperonin is as closely related to the chloroplast beta chaperonin (amino acid identity is 50%), as it is to the bacterial and mitochondrial chaperonins. The most interesting finding to emerge from the analysis of the deduced amino acid sequences is the presence of a possible dinucleotide binding site. A highly conserved region of 36 amino acids shows 9 out of 11 matches reported for the dinucleotide binding sit fingerprint (Wierenga, Terpstra & Holl, J. Mol. Biol. 187, 101-107, 1986), but only when the chaperonin sequence is read from the carboxy to the aminoterminus and two additional amino acids are allowed. Other proteins such as the Ca2+-ATPase of the sarcoplasmic reticulum and the ecdysone-induced protein Eip 28129 of Drosophila melanogaster also contain this reversed dinucleotide binding site sequence. This finding raises the novel possibility that a given binding site can be constructed from a set of amino acids running in either direction along the polypeptide chain; this possibility should be tested for other consensus sequences. When the T.aestivum chloroplast alpha chaperonin is synthesized in E.coli cells, it forms a hybrid oligomeric complex with the host chaperonin. The T.aestivum Rubisco large subunits that are synthesized in E.coli are found associated with either the E.coli chaperonin or with the hybrid chaperonin complex, whereas co-synthesized Rubisco small subunits bind neither to the large subunits nor to the chaperonin complexes. The T.aestivum Rubisco subunits fail to assemble into an enzymically active oligomer when synthesized in the presence of the T.aestivum chloroplast alpha chaperonin. This work is discussed in light of the conclusion emerging from studies in several laboratories that chaperonins function in many processes within the cell, the common feature of which is the requirement to prevent folding and assembly occurring between transiently exposed interactive protein surfaces