No abstract

Alzheimer's disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-beta precursor protein (APP), the amyloid-beta peptide (Abeta). CAA is also the major pathological lesion in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), caused by a mutation in the gene coding for the Abeta peptide. Several members of the small heat shock protein (sHsp) family, such as alphaB-crystallin, Hsp27, Hsp20 and HspB2, are associated with the pathological lesions of AD, and the direct interaction between sHsps and Abeta has been demonstrated in vitro. HspB8, also named Hsp22 of H11, is a recently discovered member of the sHsp family, which has chaperone activity and is observed in neuronal tissue. Furthermore, HspB8 affects protein aggregation, which has been shown by its ability to prevent formation of mutant huntingtin aggregates. The aim of this study was to investigate whether HspB8 is associated with the pathological lesions of AD and HCHWA-D and whether there are effects of HspB8 on Abeta aggregation and Abeta-mediated cytotoxicity. We observed the expression of HspB8 in classic SPs in AD brains. In addition, HspB8 was found in CAA in HCHWA-D brains, but not in AD brains. Direct interaction of HspB8 with Abeta(1-42), Abeta(1-40) and Abeta(1-40) with the Dutch mutation was demonstrated by surface plasmon resonance. Furthermore, co-incubation of HspB8 with D-Abeta(1-40) resulted in the complete inhibition of D-Abeta(1-40)-mediated death of cerebrovascular cells, likely mediated by a reduction in both the beta-sheet formation of D-Abeta(1-40) and its accumulation at the cell surface. In contrast, however, with Abeta(1-42), HspB8 neither affected beta-sheet formation nor Abeta-mediated cell death. We conclude that HspB8 might play an important role in regulating Abeta aggregation and, therefore, the development of classic SPs in AD and CAA in HCHWA-D.

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4,8,12,16,20 and 24 subunits, each maintaining a strict 3:1 HSPB2:HSPB3 subunit ratio. These complexes are thermally stable up to 40 °C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or αB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could efficiently associate with HSP20. Taken altogether, this study brings evidence that despite the high sequence homology within the sHSP family, the biochemical properties of the HSPB2/B3 complex are distinctly different from other sHSPs, indicating that the HSPB2/B3 assembly likely possesses other cellular functions than its family members. Various mammalian small heat shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintains a strict 3:1 HSPB2:HSPB3 subunit ratio. Analyzing the thermal stability of the HSPB2/B3 assembly by far-UV circular dichroism spectroscopy revealed subtle structural changes, occurring slightly above 40 °C, and an unfolding curve with an inflection point at approximately 56 °C. Furthermore, HSPB2/B3 exerted poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Finally, coimmunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer does not interact with HSP20, HSP27 or αB-crystallin. However, HSPB2 that ...

Alzheimer's disease (AD) is associated with plaque deposition in the brain of AD patients. The major component of the aggregate is a 39-42 long peptide termed b-amyloid (Ab). Except for Ab, plaques contain several other components which co-precipitate together with Ab. One such component is the small heat shock protein (sHSP) aB-crystallin. Instead of preventing the cell from the neurotoxicity of Ab, aB-crystallin induces an increased neurotoxicity.We find -using solution state NMR spectroscopy -that aBcrystallin competes efficiently for Ab monomer-monomer interactions. Interactions between Ab and aB-crystallin involve the hydrophobic core residues 17-21 as well as residues 31-32 of Ab, and thus the same chemical groups which are important for Ab aggregation. In the presence of aB-crystallin, Met35 in Ab becomes efficiently oxidized. In order to quantify the redox properties of the different complexes consisting of Ab/aB-crystallin/copper, we suggest an NMR assay which allows to estimate the electrochemical properties indirectly by monitoring the rate of glutathion (GSH) auto-oxidation. The oxidation of the side chain Met35 in Ab might account for the increased neurotoxicity and the inability of Ab to form fibrillar structures, which has been observed previously in the presence of aB-crystallin [Stege, G.J. et al. (1999) The molecular chaperone aB-crystallin enhances amyloid-beta neurotoxicity. Biochem. Biophys. Res. Commun. 262,[152][153][154][155][156].

Crosslinking of proteins by tissue transglutaminase (tTG) is enhanced in amyloid (Ab) deposits characteristic of Alzheimer's disease and sporadic inclusion body myositis. Small heat shock proteins (sHsps) also occur in amyloid deposits. We here report the substrate characteristics for tTG of six sHsps. Hsp27, Hsp20 and HspB8 are both lysine-and glutaminedonors, aB-crystallin only is a lysine-donor, HspB2 a glutaminedonor, and HspB3 no substrate at all. Close interaction of proteins stimulates crosslinking efficiency as crosslinking between different sHsps only takes place within the same heteromeric complex. We also observed that aB-crystallin, Hsp27 and Hsp20 associate with Ab in vitro, and can be readily crosslinked by tTG.