Abstract
Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Multicellular organisms contain a large number of different sHsps, raising questions as to whether they function redundantly or are specialized in terms of substrates and mechanism. To gain insight into this issue, we undertook a comparative analysis of the eight major human sHsps on the aggregation of both model proteins and cytosolic lysates under standardized conditions. We discovered that sHsps, which form large oligomers (HspB1/Hsp27, HspB3, HspB4/αA-crystallin, and HspB5/αB-crystallin) are promiscuous chaperones, whereas the chaperone activity of the other sHsps is more substrate-dependent. However, all human sHsps analyzed except HspB7 suppressed the aggregation of cytosolic proteins of HEK293 cells. We identified ∼1100 heat-sensitive HEK293 proteins, 12% of which could be isolated in complexes with sHsps. Analysis of their biochemical properties revealed that most of the sHsp substrates have a molecular mass from 50 to 100 kDa and a slightly acidic pI (5.4-6.8). The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, but also B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, although these were also efficient in preventing the aggregation of cytosolic proteins.
Highlights
Small heat shock proteins are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins
The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, and B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, these were efficient in preventing the aggregation of cytosolic proteins
We found that PBS is more suitable as a standard buffer because the aggregation of the model substrates was significantly faster in this buffer compared with HEPES buffer
Summary
Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Among others, insulin [19], yeast alcohol dehydrogenase (ADH) [20], citrate synthase (CS) [21], and malate dehydrogenase (MDH) [22] These assays are excellent tools to determine whether a given protein has chaperone properties and to analyze specific features of the chaperone mechanism. In this context it is an important open question of how well these assays mimic the in vivo situation and reflect the cellular functions of sHsp and which (if any) of the model substrate assays represents a good surrogate for the in vivo situation are yet unclear. There is indirect evidence for at least some sHsps that their
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