News Physiol Sci 16: 123-126, 2001;
1548-9213/01 $5.00
News in Physiological Sciences, Vol. 16, No. 3, 123-126,
June 2001
© 2001 Int. Union Physiol. Sci./Am. Physiol. Soc.
A Nonconventional Role of Molecular Chaperones: Involvement in the Cytoarchitecture
Peter Csermely
P. Csermely is in the Department of Medical Chemistry, Semmelweis University, H-1444 Budapest, and the Biorex Corporation, H-8200 Veszprém, Hungary.
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Abstract
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A hallmark of chaperone action is assistance in protein folding. Indeed, folding of nascent prokaryotic proteins proceeds mostly as a chaperone-assisted, posttranslational event. On the contrary, in nonstressed eukaryotic cells folding-related tasks of eukaryotic chaperones are restricted to a subset of proteins, and "jobless" chaperones may form an extension of the cytoarchitecture, facilitating intracellular traffic of proteins and other macromolecules.
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Introduction
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Protein folding is characterized by two major steps in vitro (Fig. 1
; Ref. 4). In the first steps, most of the secondary structure is already formed. Folding usually starts with the formation of 
-helices, since ß-sheet formation requires hydrogen bonds between amino acids, which are far from each other in the primary sequence. In this step, the unfolded protein is collapsed and a (more or less) stable intermediary, the molten globule, is formed. The partially folded state of molten globules can be characterized by a developed secondary structure that is mostly unorganized, showing almost no tertiary structure. Molten globules still have large hydrophobic surfaces and therefore are subjects of extensive aggregation. The volume of molten globules, however, is almost as small as that of the native, folded protein.
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FIGURE 1. Major steps of protein folding in vitro. The unfolded protein first undergoes fast hydrophobic collapse, during which most of its hydrophobic surfaces become buried. The folding intermediate develops its final, native structure in a slower process. Although folding of small proteins (in the range of 10–30 kDa) may be a rather straightforward process, larger proteins are often trapped in various misfolded states. These trapped intermediates usually have hydrophobic surfaces and are prone to aggregation. Figure is adapted from Ref. 2.
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The last steps of protein folding are the slow, rate-limiting steps. In these steps the inner, hydrophobic core of the protein becomes tightly packed (2) and unique, high-energy bonds are formed, such as disulfide bridges or ion pairs. The free energy gain of these latter processes enables the formation of local, unstable protein structures, which are stabilized by the favorable conformation of the rest of the protein. These unstable protein segments can stabilize themselves by forming complexes with another molecule. Thus they often serve as active centers of enzymes or as contact surfaces between various proteins involved, e.g., in signal transduction.
For larger proteins, folding is not a straightforward process. Their unordered, hesitating, zigzag pathways need a lot of help. Besides this, aggregation of partially unfolded or misfolded proteins is a great danger. Molecular chaperones serve to prevent aggregation and to rescue misfolded proteins from their folding traps (1, 5). In the case of chaperone machines that surround their targets, rearrangement of the hydrophobic core of the target protein is aided by periodic pulling and water percolation (Fig. 2; Ref. 2), whereas other molecular chaperones grab a hydrophobic peptide segment of their client proteins.
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FIGURE 2. A possible mechanism of chaperone action. Anfinsen-cage type molecular chaperones, which surround their target proteins, exert a periodic multidirectional pulling of the target by using their periodic conformational changes governed by the hydrolysis of ATP. In the pulling process, the hydrophilic exterior of the target protein becomes immobilized, whereas its hydrophobic core becomes mobilized and gets another chance to rearrange itself. During the chaperone-mediated expansion of the target, water molecules enter its hydrophobic core and facilitate its rearrangement further. This (partially hypothetical) view is supported by fluorescence anisotropy, electron spin resonance measurements, and hydrogen/deuterium exchange studies. Figure is adapted from Ref. 2.
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Protein folding in crowded cells: conventional roles of chaperones
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In vivo protein folding first occurs when a protein is born. Prokaryotic proteins are synthesized quickly. Most of their folding occurs after translation and needs the help of chaperones. In eukaryotes, protein synthesis is a slower process: proteins fold during their emergence from the ribosomes, i.e., cotranslationally (8). Folding of these proteins may occur sequentially. Different domains of the protein fold one after the other, and the process is helped by the ribosomal machinery itself. After synthesis, chaperones help the translocation of proteins through membranes. Pores of most cellular membranes (with the notable exception of the nuclear pores) are too small to accommodate fully folded, globular proteins. Proteins have to unfold to get through and refold in the lumen of the organelle. These processes are facilitated by molecular chaperones.
The cellular environment is much more crowded than usual in vitro experimental conditions in protein folding studies. Estimated protein concentrations reach 200–300 mg/ml (20–30% wt/vol), which is close to the theoretical "overlap" concentration for a typical 50-kDa protein (7). Molecular crowding promotes aggregation, which makes the chaperone-mediated protection of folding proteins even more desirable. Crowding also stabilizes chaperone-target complexes, which increases the efficiency and fidelity of chaperone action.
Chaperones also help refolding of damaged proteins. After environmental stress, protein damage becomes abundant; therefore, an increased capacity of the "chaperone machines" is highly advantageous. Indeed, many stressors (such as alcohol, other poisons, sunburn, anxiety, etc.) induce the synthesis of these chaperones (called heat-shock or stress proteins), and in case of bacterial and viral infections the developing fever also helps this process (13). In stressed cells, chaperones not only help proteins to survive but also help their destruction by presenting ultimately damaged proteins to the lysosomal protein degradation or to the proteasome. Chaperones may also play an important role in helping RNA folding and association of RNA-protein complexes.
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Are eukaryotic chaperones jobless?
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Eukaryotes accommodated mitochondria, which enabled them to produce a vast amount of chemical energy in the form of ATP. This energy richness may be one reason why eukaryotes discard rather than repair a lot of misfolded proteins (10, 12). Similarly, as much as 97% of RNAs never leave the nucleus but become almost instantly degraded (6). In resting eukaryotic cells, chaperones have a smaller role in folding than in prokaryotes. Most cytoplasmic chaperone machineries are specialized to help the folding of a small subset of proteins, such as nuclear hormone receptors, protein kinases, actin, or tubulin. On the other hand, many of the eukaryotic chaperones, such as the 90-kDa heat shock protein (Hsp90) are expressed constitutively and form 1–5% of cellular proteins. Moreover, Hsp90 and other chaperones are mandatory for the life of eukaryotic cells; their deletion is lethal (3, 9). Why do we need so much of these chaperones, if their specific targets are at least a hundred times less abundant than the chaperones themselves? Are they just waiting for a stress to occur? Recent data from Pratt et al. (9), and from our own lab (unpublished observations) indicate that eukaryotic chaperones may also participate in the cytoplasmic organization and traffic. This eukaryote-specific role fits the increased need for compartmentalization and organization in eukaryotic cells.
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A nonconventional role: chaperones and the cytoarchitecture
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In the late 1970s, Porter and co-workers (14) found a cytoplasmic meshwork of various filaments called the "microtrabecular lattice." Although a rather energetic debate has developed about the validity of the electronmicroscopical evidence for the microtrabeculae, several independent findings support the existence of a cytoplasmic mesh-like structure (7). However, the identity of the constituents of this lattice, besides the regular microtubular, microfilamental, and intermediate filamental network, remained rather elusive. The extensions of the regular cytoarchitecture obviously must bind to the microtubular, microfilamental, and intermediate filamental network, must be abundant proteins by themselves, and their association must be a low affinity, highly dynamic association, making them difficult to isolate and study by conventional biochemical techniques.
Besides other proteins, such as members of the glycolytic pathway (7), molecular chaperones are excellent candidates for this purpose (Fig. 3; Ref. 3). They are abundant and all bind to filamentous actin, to tubulin, and most probably to the intermediate filaments. They form low affinity and highly dynamic complexes with each other, with the filaments, and with their target proteins. Besides their structural and perhaps protective role, chaperones also participate in the direction of cytoplasmic protein (9) and maybe RNA traffic. Disruption of Hsp90-organized chaperone complexes (often called the foldosome) leads to a slower translocation of various signaling molecules, including steroid receptors and several protein kinases. Fast translocation of these signaling components is linked to the cytoskeleton and directed by the foldosome-component immunophilins or p50cdc37 chaperone (9).
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FIGURE 3. Chaperones as putative constituents of the cytoarchitecture. The figure summarizes the current view about the complex cytoplasmic meshwork attached to the microfilamental, microtubular, and intermediate filamental structures. Recent data indicate that molecular chaperone complexes may be an important parts of this meshwork. Figure is adapted from Ref. 14.
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Summary and perspectives
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Besides the well established role of molecular chaperones in aiding protein folding, recent data raised the possibility of their participation in the eukaryotic cytoarchitecture, facilitating intracellular traffic of proteins and other macromolecules. Further studies are needed to explore whether these roles may be performed in parallel or whether they, at least in part, compete with each other. The early observations that cellular stress provokes an increased Brownian motion of endogenous intracellular particles (7) as well as the reduced chaperone efficiency of filamentous archebacterial 60-kDa heat shock proteins (11) indicate that chaperone-assisted folding and participation in the cytoarchitecture may be competing processes. Cellular stress induces the buildup of massive amounts of misfolded proteins; chaperones help to rescue and refold them. This may impair their anchorage to the cytoarchitecture, and an increased diffusion may develop. Similarly, an accelerated protein synthesis, such as that of malignant or virally infected cells, may also impair cellular rigidity by shifting the chaperone aid toward protein folding instead of the regular support of the cytoarchitecture (Fig. 4).
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FIGURE 4. Possible interplay between chaperone-assisted protein folding and participation in the cytoarchitecture. Indirect evidence suggests that chaperone-assisted protein folding and chaperone participation in the cytoarchitecture may be competitive processes. This raises the possibility that cellular conditions requiring a more intensive assistance in protein folding (environmental stress, malignant transformation, viral infection, etc.) may disrupt the cytoplasmic meshwork and vice versa; a more developed cytoplasmic meshwork (e.g., in differentiated or senescent cells) may impair chaperone-assisted protein folding.
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Acknowledgments
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Work in the author's laboratory was supported by research grants from International Centre for Genetic Engineering and Biotechnology, the Hungarian Science Foundation (OTKA-T25206), the Hungarian Ministry of Social Welfare (ETT-21/00), and the Volkswagen Foundation (I/73612).
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Introduction
Protein folding in crowded...
