Clerico, E. M. | Search bioRxiv

bioRxiv2026

Survey of the human proteostasis network: the ubiquitin-proteasome system

Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1430 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3150 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

bioinformatics↗

bioRxiv2025

The nucleotide exchange factor, GrpE, modulates substrate affinity by interaction of its N-terminal tails with the DnaK substrate-binding domain.

The 70-kDa heat shock proteins (Hsp70s) assist in protein folding through allosteric communication between their nucleotide-binding domains (NBDs) and substrate-binding domains (SBDs), which are connected by an interdomain linker. Their nucleotide-dependent allosteric cycle is modulated by ligand binding and co-chaperones, including nucleotide exchange factors (NEFs). GrpE, the NEF for the E. coli Hsp70, DnaK, has been proposed to have a dual effect on the chaperone, facilitating the exchange of ADP for ATP in the NBD in a temperature-dependent fashion and promoting substrate release from the SBD. We recently reported NMR-based evidence that GrpE binding to DnaK has a direct structural effect on the SBD. Here, we expanded on these findings and obtained new evidence for a model in which the disordered N-terminal tails of GrpE facilitate peptide dissociation from the nucleotide-free DnaK/GrpE complex by transiently binding to the canonical substrate-binding site in the SBD. This GrpE/SBD interaction, while weak, is favored by the high local concentration of the tails around the SBD after complex formation. Moreover, we identified the DnaK binding motif in GrpEs N-terminal disordered tails as 17IIM19, which is conserved across many bacterial species. Excitingly, our data further suggest a mechanism for the temperature-dependence of GrpEs modulation of DnaKs refolding activity: as the temperature increases, unfolding of GrpEs coiled-coil weakens its contacts with the SBD, reducing N-terminal tail binding, and thus increasing DnaK affinity to substrates.

biochemistry↗