The role of small heat shock proteins (sHsps) as molecular chaperones is still poorly understood. We therefore investigated the effect of proteasomal inhibition on sHsps in the rat cardiac myoblast ...cell line H9c2. Proteasomes are responsible for controlled degradation of intracellular proteins. Inhibition of their activities leads to accumulation of unfolded proteins, which can form insoluble “aggresomes” together with proteasomes and heat shock proteins Hsp70 and Hsp90. We here report that upon proteasome inhibition, α B-crystallin and Hsp25 translocate from the detergent-soluble cytosolic fraction to the detergent-insoluble nuclear/cytoskeletal fraction. Although phosphorylation of both α B-crystallin and Hsp25 is induced, this does not seem to be essential for the translocation. Immunocytochemistry revealed that α B-crystallin and Hsp25, which show a diffuse cytoplasmic staining in unstressed H9c2 cells, colocalize with F-actin upon proteasomal inhibition. After transfection in H9c2 cells, other sHsps (α A-crystallin, Hsp20, HspB2 and HspB3) showed similar translocation to the actin cytoskeleton. The redistribution of sHsps upon proteasomal inhibition may reflect a mechanism by which cells are protected from damaged intracellular proteins by sequestering them on the cytoskeleton.
Site-specific racemization in aging α-crystallin Groenen, Patricia J.T.A.; van den Ijssel, Paul R.L.A.; Voorter, Christina E.M. ...
FEBS letters,
August 20, 1990, Letnik:
269, Številka:
1
Journal Article
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Of all aspartyl residues in bovine αA-crystallin, only Asp-151 exhibits pronounced racemization. Asp-151 is also one of the sites where peptide bond cleavage occurs in in vivo aging αA-crystallin. ...This aspartyl residue is followed by an alanyl residue and resides in a flexible carboxyl terminal extension of α-crystallin. Both in vivo and in vitro racemization studies indicate that the pronounced and site-specific racemization of Asp-151 proceeds via formation of a succinimide intermediate. The in vivo racemization of aspartyl residues in αA-crystallin is discussed with regard to the proposed tertiary structure of α-crystallin.
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.
Two members of the small heat shock protein family, αB-crystallin and hsp25, occur at high levels in the mammalian heart. To try and understand any differences in functioning, we compared their ...properties in cultured rat neonatal cardiac myocytes. Both proteins are stress-inducible, but the level of hsp25 is only slightly increased in cultured cardiac myocytes subjected to hyperthermic stress, while αB-crystallin levels even remain unchanged. Phosphorylation of αB-crystallin and to a lesser extent also of hsp2S is induced after the heat shock. Directly after heat stress, αB-crystallin and hsp25 are partly found in detergent-insoluble fractions, representing cytoskeletal/nuclear structures. Additionally, we show by confocal laser scanning microscopy that αB-crystallin and hsp25 become associated with sarcomeric structures directly after the heat shock, indicating a cytoskeletal protective function. Four to six hours after the heat shock, both proteins reoccupy their original positions in the cytoplasm again. In contrast to αB-crystallin, hsp25 not only translocates to the cytoskeleton but also migrates to positions inside the nucleus. Despite the fact that both proteins are normally part of the same complex, their behavior in neonatal cardiac myocytes appears to be very different. The sarcomeric association of αB-crystallin occurs under milder conditions and persists for a longer period of time in comparison with hsp25. Our findings suggest that αB-crystallin and hsp25 are both involved in protection of the cytoskeleton during stress situations in the heart, although in different manners. In addition, hsp25 also plays a role inside the nucleus.
In the present study, we have analyzed the response of human smooth muscle cell (SMC)s to oxidative stress, in terms of recruitment of key elements of the stress-activated protein kinase (SAPK) ...pathway, such as Rac(1), p38, and the small heat shock protein (HSP)27. The level of expression of three small HSPs, alphaB-crystallin, HSP20, HSP27, as well as the phosphorylation levels of HSP27 and p38, were higher in cultured, asynchronously growing SMCs originating from left interior mammary artery (LIMA) than those originating from aorta, saphenous vein, and umbilical vein, validating the choice of SMCs from LIMA as a model system in our study. In synchronized, quiescent SMCs from LIMA, oxidative stress (H(2)O(2) stimulation)-induced membrane translocation of Rac(1), p38 phosphorylation, membrane translocation, and phosphorylation of HSP27. In these cells, simvastatin (S), an HMG-CoA reductase inhibitor, blocked, in a mevalonate-dependent way, oxidative stress-induced membrane translocation of Rac(1). However, S pretreatment prior to oxidative stress increased the levels of p38 phosphorylation, HSP27 membrane translocation/phosphorylation, actin polymerization, and apoptosis in these cells, in a mevalonate-dependent way. These results establish that S pretreatment has a stimulatory effect on the stress-activated p38/HSP27 pathway, despite its blocking effect on Rac(1) activation.
The amine‐donor substrate specificity of tissue‐type transglutaminase has been studied in a series of recombinant αA‐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 αA‐crystallin. A biotinylated amine‐acceptor hexapeptide was used as a probe for labelling the amine‐donor sites. Wild‐type bovine αA‐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 αA‐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.
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