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The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

Heat stress can lead to protein misfolding and aggregation, potentially causing cell death due to the loss of essential proteins. Bacteria, being particularly exposed to environmental stress, are equipped with disaggregases that rescue these aggregated proteins. The bacterial Hsp70 chaperone DnaK and the ATPase associated with diverse cellular activities protein ClpB form the canonical disaggregase in bacteria. While this combination operates effectively during physiological heat stress, it is ineffective against massive aggregation caused by temperature‐based sterilization protocols used in the food industry and clinics. This leaves bacteria unprotected against these thermal processes. However, bacteria that can withstand extreme, man‐made stress conditions have emerged. These bacteria possess novel ATPase associated with diverse cellular activities disaggregases, ClpG and ClpL, which are key players in extreme heat resistance. These disaggregases, present in selected Gram‐negative or Gram‐positive bacteria, respectively, function superiorly by exhibiting increased thermal stability and enhanced threading power compared to DnaK/ClpB. This enables ClpG and ClpL to operate at extreme temperatures and process large and tight protein aggregates, thereby contributing to heat resistance. The genes for ClpG and ClpL are often encoded on mobile genomic islands or conjugative plasmids, allowing for their rapid spread among bacteria via horizontal gene transfer. This threatens the efficiency of sterilization protocols. In this review, we describe the various bacterial disaggregases identified to date, characterizing their commonalities and the specific features that enable these novel disaggregases to provide stress protection against extreme stress conditions. [ABSTRACT FROM AUTHOR]

The ability to reverse protein aggregation is vital to cells.sup.1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore.sup.3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp70.sup.4,5. However, the model is also controversial, as the necessary motor activity has not been identified.sup.6-8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting.sup.9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore.sup.11,12. Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97).sup.13 and degradation by the 26S proteasome.sup.14. A combination of optical tweezers and fluorescent-particle tracking is used to dissect the dynamics of the Hsp100 disaggregase ClpB, and show that the processive extrusion of polypeptide loops is the mechanistic basis of its activity., Author(s): Mario J. Avellaneda [sup.1] , Kamila B. Franke [sup.2] , Vanda Sunderlikova [sup.1] , Bernd Bukau [sup.2] , Axel Mogk [sup.2] , Sander J. Tans [sup.1] [sup.3] Author Affiliations: [...]

The oligomeric AAA+ chaperones ClpB/Hsp104 mediate the reactivation of aggregated proteins, an activity that is crucial for the survival of cells during severe stress. Hsp104 is also essential for the propagation of yeast prions by severing prion fibres. Protein disaggregation depends on the cooperation of ClpB/Hsp104 with a cognate Hsp70 chaperone system. While Hsp70 chaperones are also involved in prion propagation, their precise role is much less well defined compared with its function in aggregate solubilization. Therefore, it remained unclear whether both ClpB/Hsp104 activities are based on common or different mechanisms. Novel data show that ClpB/Hsp104 uses a motor threading activity to remodel both protein aggregates and prion fibrils. Moreover, transfer of both types of substrates to the ClpB/Hsp104 processing pore site requires initial substrate interaction of Hsp70. Together these data emphasize the similarity of thermotolerance and prion propagation pathways and point to a shared mechanistic principle of Hsp70-ClpB/Hsp104-mediated solubilization of amorphous and ordered aggregates. Key words: AAA+ protein, ClpB, Hsp104, protein disaggregation, prion propagation. Les chaperons oligomeriques ClpB/Hsp104 membres de la famille AAA+ permettent la reactivation de proteines agregees, une activite cruciale a la survie cellulaire lors d'un stress severe. Hsp104 est aussi essentielle a la propagation des prions chez la levure en sectionnant les fibres du prion. La desagregation des proteines depend aussi de la cooperation entre ClpB/Hsp104 et le systeme de chaperon apparente; Hsp70. Alors que les chaperons Hsp70 sont aussi impliques dans la propagation des prions, leur role precis est beaucoup moins bien defini comparativement a leur fonction dans la solubilization des agregats. Ainsi, on ne sait pas clairement si les deux activites de ClpB/Hsp104 sont basees sur des mecanismes communs ou distincts. De nouvelles donnees montrent que ClpB/Hsp104 utilise une activite; motrice d'enfilage pour remodeler les agregats de proteines et les fibrilles de prions. De plus, le transfert des deux types de substrats vers le pore de traitement de ClpB/Hsp104 requiert une interaction initiale du substrat avec Hsp70. Ensemble, ces donnees soulignent les similarites qui unissent le mecanisme de tolerance a la chaleur et la voie de propagation des prions et pointent vers un principe mecanistique commun de solubilization des proteines amorphes et des agregats ordonnes qui depend de Hsp70 et ClpB/Hsp104. Mots-cles: proteines AAA+, ClpB, Hsp104, desagregation des proteines, propagation de prions. [Traduit par la Redaction], Introduction An evolutionary conserved network of molecular chaperones and proteases, termed the protein quality control system, ensures cell survival during various stress situations by either refolding or degrading misfolded proteins. [...]

Molecular chaperones help in the folding of newly synthesized proteins in the cytosol. It is shown that Deltatig::kan mutants lacking trigger factor have not defects in growth or protein folding, but combined Deltatig::kan and DeltadnaK mutations lead to synthetic lethality. DnaK depletion in the Deltatig::kan mutant leads to major aggregation of cytosolic proteins.