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Highlights • Acid stress induces dissociation of Hsp20.1 and Hsp14.1 oligomers. • Dissociated Hsp20.1 and Hsp14.1 show chaperone activities in acid stress. • Hsp20.1 and Hsp14.1 maintain oligomers and show chaperone activities at 50 °C. • sHsps might function in different modes in different stressed conditions., A great number of studies have proven that sHsps protect cells by inhibiting protein aggregation under heat stress, while little is known about their function to protect cells under acid stress. In this work, we show that Hsp20.1 and Hsp14.1 oligomers dissociated to smaller oligomeric species or even dimer/monomer at low pH (pH 4.0 and pH 2.0), whereas no prominent quaternary structural changes were seen at 50 °C. Both oligomers and smaller oligomeric species exhibited abilities to suppress client aggregation at low pH and at 50 °C. These results suggest that sHsps may function in different modes in different stressed conditions.

Highlights ► Utrophin actin binding domain crystallises as an open dimer but is monomeric in solution. ► We introduced cysteines in the CH domains to lock the monomer closed. ► Utrophin bound to F-actin with reduced affinity in the closed conformation. ► Differential scanning calorimetry confirmed the open conformation of utrophin on actin., Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin.

Highlights • Mithramycin (MTR) binds to core histones but not to linker histone H1. • Unlike MTR–DNA interaction, MTR–histone association is metal independent. • MTR alters H3K18 acetylation in vitro and ex vivo. • MTR is a dual binder (binds to both DNA and histones) in the chromatin context., Mithramycin (MTR) is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin – histone proteins – as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode). The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR–DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg2+. As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo. Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.