Abstract
Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1.
Highlights
Small heat shock proteins form a large family of proteins ubiquitously expressed in practically all kingdoms that play an important role in cellular homeostasis [1,2,3,4]
This paper deals with the investigation of some properties of G34R, P39L and E41K mutants of heat shock protein B1 (HspB1) associated with type II Charcot-Marie-Tooth diseases [32] or distal hereditary motor neuropathy [33]
The recombinantly expressed wild type (WT) HspB1 and the three N-terminal mutants could all be readily purified from the soluble fraction of the E. coli lysate, where none constructs yielded inclusion bodies
Summary
Small heat shock proteins (sHsp) form a large family of proteins ubiquitously expressed in practically all kingdoms that play an important role in cellular homeostasis [1,2,3,4]. Certain members of the sHsp family possess antiapoptotic activity [20, 21], regulate proliferation [22], stabilize the cytoskeleton [23, 24] and seem to be involved in the regulation of many other vital cellular processes Since these proteins participate in many different cellular processes, it is not surprising that their mutations have been associated with different congenital diseases such as cataract, desmin-related myopathy, cardiomyopathy, distal hereditary motor neuropathy and CharcotMarie-Tooth disease [25,26,27]. This paper deals with the investigation of some properties of G34R, P39L and E41K mutants of HspB1 associated with type II Charcot-Marie-Tooth diseases [32] or distal hereditary motor neuropathy [33] These mutations result in different manifestations of disease. In order to begin to understand the molecular mechanisms underlying these diseases we investigated the physico-chemical properties of these three HspB1 mutants
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