Disaggregating chaperones: an unfolding story.

Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Here, we review the role of molecular chaperones as a network of cellular defenses against the formation of cytotoxic protein aggregates. Chaperones favour the native folding of proteins either as "holdases", sequestering hydrophobic regions in misfolding polypeptides, and/or as "unfoldases", forcibly unfolding and disentangling misfolded polypeptides from aggregates. Whereas in bacteria, plants and fungi Hsp70/40 acts in concert with the Hsp100 (ClpB) unfoldase, Hsp70/40 is the only known chaperone in the cytoplasm of mammalian cells that can forcibly unfold and neutralize cytotoxic protein conformers. Owing to its particular spatial configuration, the bulky 70 kDa Hsp70 molecule, when distally bound through a very tight molecular clamp onto a 50-fold smaller hydrophobic peptide loop extruding from an aggregate, can locally exert on the misfolded segment an unfolding force of entropic origin, thus destroying the misfolded structures that stabilize aggregates. ADP/ATP exchange triggers Hsp70 dissociation from the ensuing enlarged unfolded peptide loop, which is then allowed to spontaneously refold into a closer-to-native conformation devoid of affinity for the chaperone. Driven by ATP, the cooperative action of Hsp70 and its co-chaperone Hsp40 may thus gradually convert toxic misfolded protein substrates with high affinity for the chaperone, into non-toxic, natively refolded, low-affinity products. Stress- and mutation-induced protein damages in the cell, causing degenerative diseases and aging, may thus be effectively counteracted by a powerful network of molecular chaperones and of chaperone-related proteases.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.