AAA+ protein unfoldases-the Moirai of the proteome

AAA+ ATPases safeguard proteostasis by harnessing ATP hydrolysis to unfold misfolded or damaged proteins. Depending on their specific structural motifs and partners, they either channel substrates to peptidases for degradation or disentangle aggregates for refolding. This conserved mechanism across all life domains relies on the conversion of chemical energy into mechanical force. Although structural, biochemical and single-molecule studies have provided valuable insights into ATP-driven unfolding, the precise threading mechanism remains unresolved. This review synthesises current knowledge on three classes of AAA+ unfoldases: (a) self-contained proteases with both ATPase and peptidase domains, (b) ATPases cooperating with separate peptidases and (c) ATPases dedicated to disaggregation and refolding without proteolysis. We discuss existing models, underline technological limitations and outline experimental approaches to address these challenges. Gaining a clearer picture of substrate threading will enhance our grasp of basic protein quality control and also enable modulation of proteostasis in eukaryotic cells, relevant to neurodegenerative diseases and antibiotic targeting of bacterial unfoldases. Like the mythological Moirai, AAA+ enzymes govern the fate of proteins, though their exact decision-making and handling of their protein substrates remains elusive.