Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.

The biological functions of proteins are governed by their three-dimensional fold. Protein folding, maintenance of proteome integrity, and protein homeostasis (proteostasis) critically depend on a complex network of molecular chaperones. Disruption of proteostasis is implicated in aging and the pathogenesis of numerous degenerative diseases. In the cytosol, different classes of molecular chaperones cooperate in evolutionarily conserved folding pathways. Nascent polypeptides interact cotranslationally with a first set of chaperones, including trigger factor and the Hsp70 system, which prevent premature (mis)folding. Folding occurs upon controlled release of newly synthesized proteins from these factors or after transfer to downstream chaperones such as the chaperonins. Chaperonins are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. This review focuses on recent advances in understanding the mechanisms of chaperone action in promoting and regulating protein folding and on the pathological consequences of protein misfolding and aggregation.

The identification of proximate amino acids by chemical cross-linking and mass spectrometry (XL-MS) facilitates the structural analysis of homogeneous protein complexes. We gained distance restraints on a modular interaction network of protein complexes affinity-purified from human cells by applying an adapted XL-MS protocol. Systematic analysis of human protein phosphatase 2A (PP2A) complexes identified 176 interprotein and 570 intraprotein cross-links that link specific trimeric PP2A complexes to a multitude of adaptor proteins that control their cellular functions. Spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and PP2A and revealed the topology of TCP1 ring complex (TRiC) chaperonin interacting with the PP2A regulatory subunit 2ABG. This study establishes XL-MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes.

Hsp70 molecular chaperones function in protein folding in a manner dependent on regulation by co-chaperones. Hsp40s increase the low intrinsic ATPase activity of Hsp70, and nucleotide exchange factors (NEFs) remove ADP after ATP hydrolysis, enabling a new Hsp70 interaction cycle with non-native protein substrate. Here, we show that members of the Hsp70-related Hsp110 family cooperate with Hsp70 in protein folding in the eukaryotic cytosol. Mammalian Hsp110 and the yeast homologues Sse1p/2p catalyze efficient nucleotide exchange on Hsp70 and its orthologue in Saccharomyces cerevisiae, Ssa1p, respectively. Moreover, Sse1p has the same effect on Ssb1p, a ribosome-associated isoform of Hsp70 in yeast. Mutational analysis revealed that the N-terminal ATPase domain and the ultimate C-terminus of Sse1p are required for nucleotide exchange activity. The Hsp110 homologues significantly increase the rate and yield of Hsp70-mediated re-folding of thermally denatured firefly luciferase in vitro. Similarly, deletion of SSE1 causes a firefly luciferase folding defect in yeast cells under heat stress in vivo. Our data indicate that Hsp110 proteins are important components of the eukaryotic Hsp70 machinery of protein folding.

No abstract