hsp110 Protects Heat-denatured Proteins and Confers Cellular Thermoresistance

Abstract

The 110-kDa heat shock protein (hsp110) has long been recognized as one of the primary heat shock proteins in mammalian cells. It belongs to a recently described protein family that is a significantly diverged subgroup of the hsp70 family and has been found in organisms as diverse as yeast and mammals. We describe here the first analysis of the ability of hsp110 to protect cellular and molecular targets from heat damage. It was observed that the overexpression in vivo of hsp110 conferred substantial heat resistance to both Rat-1 and HeLa cells. In vitro heat denaturation and refolding assays demonstrate that hsp110 is highly efficient in selectively recognizing denatured proteins and maintaining them in a soluble, folding-competent state and is significantly more efficient in performing this function than is hsc70. hsp110-bound proteins can then be refolded by the addition of rabbit reticulocyte lysate or hsc70 and Hdj-1, whereas Hdj-1 does not itself function as a co-chaperone in folding with hsp110. hsp110 is one of the principal molecular chaperones of mammalian cells and represents a newly identified component of the primary protection/repair pathway for denatured proteins and thermotolerance expression in vivo.It has been long recognized that the major heat shock proteins (hsps) 1 of mammalian cells are observed at 28, 70, 90, and 110 kDa (1-3) and other hsp families, e.g. hsp60 and hsp40, have been subsequently identified. All of these stress protein groups have been intensively studied, excluding the hsp110 species. The cloning of hsp110 from hamster, mouse, yeast, arabadopsis, and a variety of other species has been recently described (4 -11, 29, 30). Moreover, as is the case with the hsp70 family, multiple members of the hsp110 family have also been found in individual organisms (8 -11). These studies indicate that hsp110 is a significantly enlarged and diverged relative of the hsp70 family of proteins but also includes unique sequence components. The notable constitutive expression and stress inducibility of hsp110 is highly suggestive of a major role in unstressed cells as well as in the heat shock response and the expression of thermotolerance (1, 2). A description of the heat shock response in eucaryotes is not possible without an understanding of the roles played by this major stress protein.We describe here an analysis of the characteristics of hsp110, both in vivo and in vitro. EXPERIMENTAL PROCEDURESPurification of Recombinant His-tagged HSP110 -cDNA for hsp110 was cloned into pRSET vector (Invitrogen), resulting in introduction of a His 6 -(enterokinase recognition sequence)-Arg-Ser tag to the amino terminus of hsp110 (pRSET-hsp110). pRSET-hsp110 was transformed into Escherichia coli strain JM109(DE3) cells. The transformant containing pRSET-hsp110 was grown at 37°C in LB medium with ampicillin until the OD reached 0.6, when the expression of His-hsp110 was induced by the addition of 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside during further incubation at 30°C for 5 h. Cells were lysed in 20 ...

Characterization of Heat Shock Protein 110 and Glucose-Regulated Protein 170 as Cancer Vaccines and the Effect of Fever-Range Hyperthermia on Vaccine Activity

“…They appear to possess many of the secondary structural features of hsp70 and are peptide chain-binding proteins. Although little is known about the cellular functions of hsp110, deletion mutational studies have defined its basic domains and indicate that it has a peptide-binding domain generally analogous to that of hsp70, while it also exhibits major functional differences from those of hsp70 (15,18). Less is understood at the molecular level of grp170 structure and function; however, cellular studies have shown that it binds to Ig chain in the ER, may be the ATPase responsible for protein import into the ER, and actively binds peptides from TAP (i.e., the transporter associated with Ag processing; Refs.…”

“…The existence of hsp110 in parallel with hsp70 in the cytoplasm and of grp170 in parallel with grp78 in the ER of (apparently) all eukaryotic cells argues for important differential functions for these distantly related protein families. Indeed, present data indicate important functional differences between these large and small stress protein groups; e.g., hsp110 appears to be significantly more efficient than hsp70 in binding peptide chain but does not bind to ATP agarose, as does hsp70 (15,18); grp170 binds peptide from TAP, whereas grp78 does not (19,20).…”

“…Although individual stress proteins have been studied for several years (in some cases intensively studied, e.g., hsp70), the largest of the above hsp and grp groups, hsp110 and grp170, have been almost entirely ignored. These stress proteins have only been cloned within the last few years, and their characterization remains at a very preliminary level (12)(13)(14)(15)(16). Curiously, they have both been found by sequence analysis to represent large and highly "diverged" relatives of the hsp70 family.…”

The yeast Hsp110, Sse1p, exhibits high‐affinity peptide binding

“…Like Hsp70s, Hsp110s exhibit ATPase activity [3,4]. Unlike Hsp70s, however, Hsp110s do not actively fold proteins, but rather act as holdases to maintain the solubility of denatured model protein substrates [3,[5][6][7]. Hsp110s also function as NEFs for Hsp70s [3,8,9].…”

Unique Peptide Substrate Binding Properties of 110-kDa Heat-shock Protein (Hsp110) Determine Its Distinct Chaperone Activity

“…They also show chaperone activity of their own. Unlike Hsp70s, Hsp110s lack the hallmark activity to assist in protein folding; however, they exhibit high activity in preventing aggregation of denatured proteins for which they are even more efficient than Hsp70s (40,41). For this reason, Hsp110s are called "holdases" in comparison with "foldases" for Hsp70s.…”