Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

A protein's function is determined by the wide range of motions exhibited by its 3D structure. However, current experimental techniques are not able to reliably provide the level of detail required for elucidating the exact mechanisms of protein motion essential for effective drug screening and design. Computational tools are instrumental in the study of the underlying structure-function relationship. We focus on a special type of proteins called "hinge proteins" which exhibit a motion that can be interpreted as a rotation of one domain relative to another.

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that function as “holdases” and prevent protein aggregation due to changes in temperature, pH, or oxidation state. sHsps have a conserved α-crystallin domain (ACD), which forms the dimer building block, flanked by variable N- and C-terminal regions. sHsps populate various oligomeric states as a function of their sequestrase activity, and these dynamic structural features allow the proteins to interact with a plethora of cellular substrates. However, the molecular mechanisms of their dynamic conformational assembly and the interactions with various substrates remains unclear. Therefore, it is important to gain insight into the underlying physicochemical properties that influence sHsp structure in an effort to understand their mechanism(s) of action. We evaluated several disease-relevant mutations, D109A, F113Y, R116C, R120G, and R120C, in the ACD of HspB5 for changes to in vitro chaperone activity relative to that of wildtype. Structural characteristics were also evaluated by ANS fluorescence and CD spectroscopy. Our results indicated that mutation Y113F is an efficient holdase, while D109A and R120G, which are found in patients with myofibrillar myopathy and cataracts, respectively, exhibit a large reduction in holdase activity in a chaperone-like light-scattering assay, which indicated alterations in substrate–sHsp interactions. The extent of the reductions in chaperone activities are different among the mutants and specific to the substrate protein, suggesting that while sHsps are able to interact with many substrates, specific interactions provide selectivity for some substrates compared to others. This work is consistent with a model for chaperone activity where key electrostatic interactions in the sHsp dimer provide structural stability and influence both higher-order sHsp interactions and facilitate interactions with substrate proteins that define chaperone holdase activity.

Heat shock proteins (Hsps) are central components of the cellular stress response and serve as the first line of defense against protein misfolding and aggregation. Disruption of this proteostasis network is a hallmark of neurodegenerative diseases, including tauopathies, a class of neurodegenerative diseases characterized by intracellular tau accumulation in neuronal and glial cells. Although specific Hsps are enriched in glial cells, and some have been shown to directly bind tau and influence its aggregation, the broader interplay between Hsps and tau remains poorly understood. In particular, it is unclear whether tau expression affects the heat shock response and whether this interaction is modulated in a sex-specific fashion. Here, we used a Drosophila model of tauopathy to examine both inducible and constitutive Hsp expression in response to heat stress in the context of glial tau expression. We found that Hsp expression displays sexually dimorphic expression patterns at basal levels and in response to heat stress. Moreover, tau expression in glia disrupts the normal induction of specific heat shock proteins following heat stress. This work provides new insight into how tau interacts with the cellular stress response and highlights sex-specific differences in Hsp regulation. Understanding these molecular connections is crucial to understanding how the presence of tau in glial cells influences the stress response and potentially contributes to tauopathy pathogenesis.

Heat shock proteins (Hsps) are central components of the cellular stress response and serve as the first line of defense against protein misfolding and aggregation. Disruption of this proteostasis network is a hallmark of neurodegenerative diseases, including tauopathies – a class of neurodegenerative diseases characterized by intracellular tau accumulation in neuronal and glial cells. Although specific Hsps are enriched in glial cells, and some have been shown to directly bind tau and influence its aggregation, the broader interplay between Hsps and tau remains poorly understood. In particular, it is unclear whether tau expression affects the heat shock response, and whether this interaction is modulated in a sex-specific fashion. Here, we used a Drosophila model of tauopathy to examine both inducible and constitutive Hsp expression in response to heat stress in the context of glial tau expression. We found that Hsp expression displays sexually dimorphic expression patterns at basal levels and in response to heat stress. Moreover, tau expression in glia disrupts the normal induction of specific heat shock proteins following heat stress. This work provides new insight into how tau interacts with the cellular stress response, and highlights sex-specific differences in Hsp regulation. Understanding these molecular connections is crucial to understanding how the presence of tau in glial cells influences the stress response, and potentially contributes to tauopathy pathogenesis.