F. U. Hartl

Pathways of chaperone-mediated protein folding in the cytosol

Cells are faced with the task of folding thousands of different polypeptides into a wide range of conformations. For many proteins, the folding process requires the action of molecular chaperones. In the cytosol of prokaryotic and eukaryotic cells, molecular chaperones of different structural classes form a network of pathways that can handle substrate polypeptides from the point of initial synthesis on ribosomes to the final stages of folding.

Converging concepts of protein folding in vitro and in vivo

Most proteins must fold into precise three-dimensional conformations to fulfill their biological functions. Here we review recent concepts emerging from studies of protein folding in vitro and in vivo, with a focus on how proteins navigate the complex folding energy landscape inside cells with the aid of molecular chaperones. Understanding these reactions is also of considerable medical relevance, as the aggregation of misfolding proteins that escape the cellular quality-control machinery underlies a range of debilitating diseases, including many age-onset neurodegenerative disorders.

Identification of in vivo substrates of the chaperonin GroEL.

The chaperonin GroEL has an essential role in mediating protein folding in the cytosol of Escherichia coli. Here we show that GroEL interacts strongly with a well-defined set of approximately 300 newly translated polypeptides, including essential components of the transcription/translation machinery and metabolic enzymes. About one third of these proteins are structurally unstable and repeatedly return to GroEL for conformational maintenance. GroEL substrates consist preferentially of two or more domains with αβ-folds, which contain α-helices and buried β-sheets with extensive hydrophobic surfaces. These proteins are expected to fold slowly and be prone to aggregation. The hydrophobic binding regions of GroEL may be well adapted to interact with the non-native states of αβ-domain proteins.

Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle.

The Hsc70-interacting protein Hip, a tetratricopeptide repeat protein, participates in the regulation of the eukaryotic 70 kDa heat shock cognate Hsc70. One Hip oligomer binds the ATPase domains of at least two Hsc70 molecules dependent on activation of the Hsc70 ATPase by Hsp40. While hydrolysis remains the rate-limiting step in the ATPase cycle, Hip stabilizes the ADP state of Hsc70 that has a high affinity for substrate protein. Through its own chaperone activity, Hip may contribute to the interaction of Hsc70 with various target proteins. We propose a mechanism for the regulation of eukaryotic Hsc70 that is distinct from that of bacterial Hsp70. The Hsc70/Hsp40/Hip system is apparently independent of a GrpE-like nucleotide exchange factor.

Chaperones increase association of tau protein with microtubules

Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative diseases, including Parkinsons disease and Huntingtons disease, which are characterized by accumulation of protein aggregates (e.g., α-synuclein and huntingtin, respectively). These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimers disease, a neurodegenerative disorder characterized by β-amyloid and tau protein aggregates. Here, we report an inverse relationship between aggregated tau and the levels of heat shock protein (Hsp)70/90 in tau transgenic mouse and Alzheimers disease brains. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. We have also demonstrated a direct association of the chaperones with tau proteins. Our results suggest that up-regulation of molecular chaperones may suppress formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation.

Molecular chaperones in protein folding: the art of avoiding sticky situations

Molecular chaperones are a class of proteins that interact with the non-native conformations of other proteins. The major role of chaperones of the Hsp70 and Hsp60 families is to prevent aggregation of newly synthesized polypeptides and then to mediate their folding to the native state. As a result of functional studies of these proteins, there has been a revision of the long-held view that protein folding in the cell is a spontaneous process.

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T‐complex polypeptide 1 (TCP‐1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP‐1 to be part of a hetero‐oligomeric 970 kDa complex containing several structurally related subunits of 52–65 kDa. These members of a new protein family are assembled into a TCP‐1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP‐dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co‐chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes

Tumor and viral antigens elicit a potent immune response by heat shock protein–dependent uptake of antigenic peptide with subsequent presentation by MHC I. Receptors on antigen-presenting cells that specifically bind and internalize a heat shock protein–peptide complex have not yet been identified. Here, we show that cells expressing CD40, a cell surface protein crucial for B cell function and autoimmunity, specifically bind and internalize human Hsp70 with bound peptide. Binding of Hsp70–peptide complex to the exoplasmic domain of CD40 is mediated by the NH2-terminal nucleotide–binding domain of Hsp70 in its ADP state. The Hsp70 cochaperone Hip, but not the bacterial Hsp70 homologue DnaK, competes formation of the Hsp70–CD40 complex. Binding of Hsp70-ADP to CD40 is strongly increased in the presence of Hsp70 peptide substrate, and induces signaling via p38. We suggest that CD40 is a cochaperone-like receptor mediating the uptake of exogenous Hsp70–peptide complexes by macrophages and dendritic cells.

PRINCIPLES OF PROTEIN FOLDING IN THE CELLULAR ENVIRONMENT

The ability of newly synthesised protein chains to fold into their functional conformations has evolved within the complex intracellular environment. Until recently, however, this ability has been studied largely as the refolding of denatured mature proteins in dilute simple solutions. Recent work aimed at understanding how proteins fold in vivo has allowed some general statements to be postulated.

Regulation of the Heat-shock Protein 70 Reaction Cycle by the Mammalian DnaJ Homolog, Hsp40

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a ~7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.