Measurement of chaperone-mediated effects on polyglutamine protein aggregation by the filter trap assay

The formation of aggregates by polyglutamine-containing (polyQ) proteins in neurons is a key to the pathogenesis of several progressive neurodegenerative diseases such as Huntington’s disease (HD) spinocerebellar ataxias (SCAs), and spinal and bulbar muscular atrophy (SBMA). In order to study whether the members of the heat shock protein (HSP) families, by virtue of their molecular chaperone activity, can inhibit the formation of polyQ aggregates, we developed a cell culture model expressing the GFP tagged fragment of exon1 of the huntingtin gene with an expanded polyQ chain and tetracycline inducible chaperones. Expression of mutated Huntington’s protein leads to the formation of 2% SDS insoluble high molecular weight polyQ aggregates that are retarded on a cellulose acetate membrane in the so-called filter trap assay (FTA). This chapter explains in detail the protocols of the FTA and how it can be a useful tool to study the effect of HSPs or their functional mutants on aggregation of polyglutamine proteins. Moreover, the assay is useful to investigate how externally added polyQ peptides can act as nucleation seeds for internally expressed polyQ proteins

P-31-NMR studies on acid-base balance and energy metabolism of acid-exposed fish

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Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species

HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones

A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro. Whether they also act protectively in cells against polyQ aggregation has so far only been studied for few of them (e. g. HSPB1, HSPB5 and HSPB8). Here, we compared the 10 members of the human HSPB family for their ability to prevent aggregation of disease-associated proteins with an expanded polyQ stretch. HSPB7 was identified as the most active member within the HSPB family. It not only suppressed polyQ aggregation but also prevented polyQ-induced toxicity in cells and its expression reduces eye degeneration in a Drosophila polyQ model. Upon overexpression in cells, HSPB7 was not found in larger oligomeric species when expressed in cells and-unlike HSPB1-it did not improve the refolding of heat-denatured luciferase. The action of HSPB7 was also not dependent on the Hsp70 machine or on proteasomal activity, and HSPB7 overexpression alone did not increase autophagy. However, in ATG5-/- cells that are defective in macroautophagy, the anti-aggregation activity of HSPB7 was substantially reduced. Hence, HSPB7 prevents toxicity of polyQ proteins at an early stage of aggregate formation by a non-canonical mechanism that requires an active autophagy machinery

HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones

A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro. Whether they also act protectively in cells against polyQ aggregation has so far only been studied for few of them (e. g. HSPB1, HSPB5 and HSPB8). Here, we compared the 10 members of the human HSPB family for their ability to prevent aggregation of disease-associated proteins with an expanded polyQ stretch. HSPB7 was identified as the most active member within the HSPB family. It not only suppressed polyQ aggregation but also prevented polyQ-induced toxicity in cells and its expression reduces eye degeneration in a Drosophila polyQ model. Upon overexpression in cells, HSPB7 was not found in larger oligomeric species when expressed in cells and-unlike HSPB1-it did not improve the refolding of heat-denatured luciferase. The action of HSPB7 was also not dependent on the Hsp70 machine or on proteasomal activity, and HSPB7 overexpression alone did not increase autophagy. However, in ATG5-/- cells that are defective in macroautophagy, the anti-aggregation activity of HSPB7 was substantially reduced. Hence, HSPB7 prevents toxicity of polyQ proteins at an early stage of aggregate formation by a non-canonical mechanism that requires an active autophagy machinery

Vestnik statistiki : ezemesjacnyj teoreticeskij naucno-prakticeskij zurnal Gosudarstvennogo Komiteta SSSR po Statistike

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species