Hsp20 is one of the newly described members of the mammalian small heat‐shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat ...heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far‐ultraviolet circular dichroism spectra as the most closely related sHsp, αB‐crystallin, but was less heat stable, denaturing upon heating to 50 +C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43‐kDa dimers and 470‐kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone‐like activity than αB‐crystallin, as indicated by its relatively poor capacity to diminish the reduction‐induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C‐terminus and less polar than other sHsps, but 1H‐NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C‐terminal extension, may contribute to the less effective chaperone‐like activity.
Abstract
We report on the first PGD performed for the m.14487 T>C mitochondrial DNA (mtDNA) mutation in the MT-ND6 gene, associated with Leigh syndrome. The female carrier gave birth to a healthy ...baby boy at age 42. This case adds to the successes of PGD for mtDNA mutations.
The reporting of suspected adverse drug reactions by health care professionals to monitoring agencies 1 and to medical journals 2 has been important in alerting doctors to drug safety problems, but ...Mitchell et al suggested that the time lag to the first reports of adverse reactions might be shortened if patients themselves reported adverse events. 3 For a newly introduced antidepressant we compared the time to reporting of adverse drug reactions by patients and by health care professionals. The information available from patients in the telephone service was, however, often crude and incomplete in comparison with professional adverse drug reaction reports. ...the telephone service cannot be relied on as an independent reporting system; it might generate too many false alarms.
The rudimentary eyes of the mole rat Spalax ehrenbergi have lost their visual function, but are still required for the control of circadian rhythms. It has previously been found that αA-crystallin, a ...major eye lens protein in other mammals, evolved much faster in the mole rat than in rodents with normal vision. Yet, although mole rat αA-crystallin seems superfluous as a lens protein, its rate of change is still much slower than that of pseudogenes, suggesting some remaining function. The authors therefore studied the structure and function of recombinant mole rat αA-crystallin. Circular dichroism (CD), tryptophan fluorescence and gel permeation analyses indicated that the overall structure and stability of mole rat αA-crystallin are comparable to that of rat αA-crystallin. However, the chaperone-like activity of mole rat αA-crystallin is considerably lower than that of its rat orthologue. Two-dimensional NMR spectroscopy of mole rat αA-crystallin suggests that this may be in part due to a diminished flexibility of the C-terminal extension, which is thought to be important for the chaperoning capacity. Overall, mole rat αA-crystallin appears to still be a viable protein, confirming that it has some as yet elusive role, despite the loss of its primary lens function.
The rudimentary eyes of the mole rat Spalax ehrenbergi have lost their visual function, but are still required for the control of circadian rhythms. It has previously been found that ...alphaA-crystallin, a major eye lens protein in other mammals, evolved much faster in the mole rat than in rodents with normal vision. Yet, although mole rat alphaA-crystallin seems superfluous as a lens protein, its rate of change is still much slower than that of pseudogenes, suggesting some remaining function. The authors therefore studied the structure and function of recombinant mole rat alphaA-crystallin. Circular dichroism (CD), tryptophan fluorescence and gel permeation analyses indicated that the overall structure and stability of mole rat alphaA-crystallin are comparable to that of rat alphaA-crystallin. However, the chaperone-like activity of mole rat alphaA-crystallin is considerably lower than that of its rat orthologue. Two-dimensional NMR spectroscopy of mole rat alphaA-crystallin suggests that this may be in part due to a diminished flexibility of the C-terminal extension, which is thought to be important for the chaperoning capacity. Overall, mole rat alphaA-crystallin appears to still be a viable protein, confirming that it has some as yet elusive role, despite the loss of its primary lens function.
α‐Crystallins are members of the family of small heat‐shock proteins. The conformation and mode of action of these ‘junior chaperones’ are unknown. To investigate the structure and chaperone‐like ...activity, four mutants of bovine αA‐crystallin were generated by site‐directed mutagenesis. In comparison with wild‐type αA‐crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat‐protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat‐protective capacity were observed. Although it is assumed that small heat‐shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in α‐crystallin can also influence the efficiency of substrate binding.
The amine-donor substrate specificity of tissue-type transglutaminase has been studied in a series of recombinant alpha A-crystallin mutants. These mutant proteins have been provided with a potential ...substrate lysine residue, flanked by different amino acid residues, in the C-terminal extended arm of alpha A-crystallin. A biotinylated amine-acceptor hexapeptide was used as a probe for labelling the amine-donor sites. Wild-type bovine alpha A-crystallin does not function as an amine-donor substrate for tissue-type transglutaminase. Yet, upon introduction of a lysine residue at the C-terminal or penultimate position, all mutant alpha A-crystallins act as amine-donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine-donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.
α-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens,
where it serves a structural role, and at low levels in several ...non-lenticular tissues. Like other members of the small heat
shock protein family, α-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins
in vitro . Apart from the major αA- and αB-subunits, α-crystallin of rodents contains an additional minor subunit resulting from alternative
splicing, αA -crystallin. This polypeptide is identical to normal αA-crystallin except for an insert peptide of 23 residues. To explore
the structural and functional consequences of this insertion, we have expressed rat αA- and αA -crystallin in Escherichia coli . The multimeric particles formed by αA are larger and more disperse than those of αA, but they are native-like and display a similar thermostability and morphology,
as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared
with αA, the αA -particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of β -crystallin. Our experiments indicate that αA -multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size
and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant αA -crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.
α-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the
mechanism of this chaperone-like activity is unknown, the affinity of ...α-crystallin for aggregation-prone proteins is probably
based on hydrophobic interactions. α-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they
are very stable and highly soluble. An explanation for this paradox may be that α-crystallin subunits have a polar and unstructured
C-terminal extension that functions as a sort of solubilizer. In this paper we have described five αA-crystallins in which
charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate
does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes
functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements
show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the
presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction
of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity
of the C-terminal extension of αA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural
and functional integrity.
α‐Crystallins are members of the family of small heat‐shock proteins. The conformation and mode of action of these ‘junior chaperones’ are unknown. To investigate the structure and chaperone‐like ...activity, four mutants of bovine αA‐crystallin were generated by site‐directed mutagenesis. In comparison with wild‐type αA‐crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat‐protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat‐protective capacity were observed. Although it is assumed that small heat‐shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in α‐crystallin can also influence the efficiency of substrate binding.
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