Reconciling theories of chaperonin accelerated folding with experimental evidence

For the last 20 years, a large volume of experimental and theoretical work has been undertaken to understand how chaperones like GroEL can assist protein folding in the cell. The most accepted explanation appears to be the simplest: GroEL, like most other chaperones, helps proteins fold by preventing aggregation. However, evidence suggests that, under some conditions, GroEL can play a more active role by accelerating protein folding. A large number of models have been proposed to explain how this could occur. Focused experiments have been designed and carried out using different protein substrates with conclusions that support many different mechanisms. In the current article, we attempt to see the forest through the trees. We review all suggested mechanisms for chaperonin-mediated folding and weigh the plausibility of each in light of what we now know about the most stringent, essential, GroEL-dependent protein substrates.     

Molecular mechanisms of nitrosative stress-mediated protein misfolding in neurodegenerative diseases

Nitrosative and oxidative stress, associated with the generation of excessive reactive oxygen or nitrogen species, are thought to contribute to neurodegenerative disorders. Many such diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Accumulating evidence implies that at least two pathways affect protein folding: the ubiquitin-proteasome system (UPS) and molecular chaperones. Normal protein degradation by the UPS can prevent accumulation of aberrantly folded proteins. Molecular chaperones – such as protein-disulfide isomerase, glucose-regulated protein 78, and heat shock proteins – can provide neuroprotection from aberrant proteins by facilitating proper folding and thus preventing their aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Here, we present evidence for the hypothesis that nitric oxide contributes to degenerative conditions by S-nitrosylating specific chaperones or UPS proteins that would otherwise prevent accumulation of misfolded proteins. Received 5 December 2006; received after revision 7 February 2007; accepted 15 March 2007     

Scaffolding proteins and their role in viral assembly

Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.     

Hsp70 chaperones: Cellular functions and molecular mechanism

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.Received 21 October 2004; received after revision 24 November 2004; accepted 6 December 2004     

Immunophilins: for the love of proteins

Immunophilins are chaperones that may also exhibit peptidylprolyl isomerase (PPIase) activity. This review summarizes our knowledge of the two largest families of immunophilins, namely cyclophilin and FK506-binding protein, and a novel chimeric dual-family immunophilin, named FK506- and cyclosporin-binding protein (FCBP). The larger members of each family are modular in nature, consisting of multiple PPIase and/or protein-protein interaction domains. Despite the apparent difference in their sequence and three-dimensional structure, the three families encode similar enzymatic and biological functions. Recent studies have revealed that many immunophilins possess a chaperone function independent of PPIase activity. Knockout animal studies have confirmed multiple essential roles of immunophilins in physiology and development. An immunophilin is indeed a natural ‘protein-philin’ (Greek ‘philin’ = friend) that interacts with proteins to guide their proper folding and assembly. Received: 7 May 2006; received after revision 3 July 2006; accepted 24 August 2006     

Protein folding and degradation in bacteria: to degrade or not to degrade? That is the question

In Escherichia coli protein quality control is carried out by a protein network, comprising chaperones and proteases. Central to this network are two protein families, the AAA+ and the Hsp70 family. The major Hsp70 chaperone, DnaK, efficiently prevents protein aggregation and supports the refolding of damaged proteins. In a special case, DnaK, together with the assistance of the AAA+ protein ClpB, can also refold aggregated proteins. Other Hsp70 systems have more specialized functions in the cell, for instance HscA appears to be involved in the assembly of Fe/S proteins. In contrast to ClpB, many AAA+ proteins associate with a peptidase to form proteolytic machines which remove irreversibly damaged proteins from the cellular pool. The AAA+ component of these proteolytic machines drives protein degradation. They are required not only for recognition of the substrate but also for substrate unfolding and translocation into the proteolytic chamber. In many cases, specific adaptor proteins modify the substrate binding properties of AAA+ proteins. While chaperones and proteases do not appear to directly cooperate with each other, both systems appear to be necessary for proper functioning of the cell and can, at least in part, substitute for one another. RID="*" ID="*"Corresponding author.     

Crystallin proteins and amyloid fibrils

Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, α-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer’s and Parkinson’s). In this review, the literature on the interaction of αB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials. Received 13 June 2008; received after revision 29 July 2008; accepted 05 August 2008     

The folding process of hen lysozyme: a perspective from the ‘new view’

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the ‘new view’ of protein folding based on energy surfaces and land scapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together.     

sHsps and their role in the chaperone network

Small Hsps (sHsps) encompass a widespread but diverse class of proteins. These low molecular mass proteins (15—42 kDa) form dynamic oligomeric structures ranging from 9 to 50 subunits. sHsps display chaperone function in vitro, and in addition they have been suggested to be involved in the inhibition of apoptosis, organisation of the cytoskeleton and establishing the refractive properties of the eye lens in the case of α-crystallin. How these different functions can be explained by a common mechanism is unclear at present. However, as most of the observed phenomena involve nonnative protein, the repeatedly reported chaperone properties of sHsps seem to be of key importance for understanding their function. In contrast to other chaperone families, sHsps bind several nonnative proteins per oligomeric complex, thus representing the most efficient chaperone family in terms of the quantity of substrate binding. In some cases, the release of substrate proteins from the sHsp complex is achieved in cooperation with Hsp70 in an ATP-dependent reaction, suggesting that the role of sHsps in the network of chaperones is to create a reservoir of nonnative refoldable protein.     

Hsp70 in mitochondrial biogenesis: From chaperoning nascent polypeptide chains to facilitation of protein degradation

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix.     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

SecB,one small chaperone in the complex milieu of the cell

SecB is only one of a plethora of cytosolic chaperones in E. coli whose common property is that they bind nonnative proteins. It plays a crucial role during protein export via the general secretory pathway by modulating the partitioning of precursors between folding or aggregation and delivery to the membrane-bound translocation apparatus. In this latter role SecB demonstrates specific binding to a unique partner, SecA. SecB has the potential to participate in functions outside of export acting as a general nonspecific chaperone to provide buffering capacity of the nonnative state of proteins in the cytosolic pool. We discuss the interactions of SecB with its many binding partners in light of its recently determined structure, emphasizing both kinetic and thermodynamic parameters. RID="*" ID="*"Corresponding author.     

To be, or not to be — molecular chaperones in protein degradation

To be, or not to be--that is the question not only for Hamlet in Shakespeare's drama but also for a protein associated with molecular chaperones. While long viewed exclusively as cellular folding factors, molecular chaperones recently emerged as active participants in protein degradation. This places chaperones at the center of a life or death decision during protein triage. Here we highlight molecular mechanisms that underlie chaperone action at the folding/degradation interface in mammalian cells. We discuss the importance of chaperone-assisted degradation for the regulation of cellular processes and its emerging role as a target for therapeutic intervention in cancer and amyloid diseases.     

Cellular maintenance of nuclear protein homeostasis

The accumulation and aggregation of misfolded proteins is the primary hallmark for more than 45 human degenerative diseases. These devastating disorders include Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. Over 15 degenerative diseases are associated with the aggregation of misfolded proteins specifically in the nucleus of cells. However, how the cell safeguards the nucleus from misfolded proteins is not entirely clear. In this review, we discuss what is currently known about the cellular mechanisms that maintain protein homeostasis in the nucleus and protect the nucleus from misfolded protein accumulation and aggregation. In particular, we focus on the chaperones found to localize to the nucleus during stress, the ubiquitin–proteasome components enriched in the nucleus, the signaling systems that might be present in the nucleus to coordinate folding and degradation, and the sites of misfolded protein deposition associated with the nucleus.     

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

In the hepatitis delta virus, ribozymes are encoded in both the genomic strand RNA and its complement, the antigenomic strand. The two ribozymes are similar in sequence and structure, are most active in the presence of divalent cation and catalyze RNA cleavage reactions which generate a 5′-hydroxyl group and a 2′,3′-cyclic phosphate group. Recent progress has been made in understanding the catalytic mechanism. One key was a crystal structure of the genomic ribozyme that revealed a specific cytosine positioned to act as a general acid-base catalyst. The folding of the ribozyme in the context of the longer viral RNA is another area of interest. The biology requires that each ribozyme act only once, and mechanisms proposed for regulation of ribozyme activity sometimes invoke alternative RNA structures. Likewise, interference of ribozyme function by polyadenylation of the antigenomic RNA strand could be controlled through alternative structures, and a model for such control is proposed. Received 21 June 2001; received after revision 18 July 2001; accepted 20 July 2001     

Heat-shock protein 90, a chaperone for folding and regulation

Heat-shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. Hsp90 fulfills a housekeeping function in contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. A remarkable proportion of its substrates are proteins involved in cell cycle control and signal transduction. Hsp90 acts with a cohort of Hsp90 co-chaperones that modulate its substrate recognition, ATPase cycle and chaperone function. The large conformational flexibility of Hsp90 and a multitude of dynamic co-chaperone complexes contribute to generating functional diversity, and allow Hsp90 to assist a wide range of substrates.     

Helix insertion into bilayers and the evolution of membrane proteins

Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed.     

The perspectives of studying multi-domain protein folding

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding. Received 3 December 2008; accepted 23 December 2008     

BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum

BiP is a constitutively-expressed resident protein of the endoplasmic, reticulum (ER) of all eucaryotic cells, and belongs to the highly conserved hsp70 protein family. In the ER, BiP is involved in polypeptide translocation, protein folding and presumably protein degradation as well. These functions are essential to cell viability, as has been shown for yeast. In this review, I will summarize the structural features of hsp70 proteins and focus on those experiments which revealed the biological function of BiP.