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Beyond the Signal Sequence: Protein Routing in Health and Disease

Oregon National Primate Research Center (P.M.C., C.C-F., G.M-N.) and Departments of Physiology and Pharmacology and Cell and Developmental Biology (P.M.C.), Oregon Health and Science University, Beaverton, Oregon 97006; and Research Units in Developmental Biology (C.C-F., G.M-N.), and Reproductive Medicine (P.M.C.), Instituto Mexicano del Seguro Social, Mexico Distrito Federal 06725, Mexico

Correspondence: Address all correspondence and requests for reprints to: P. Michael Conn, Oregon National Primate Research Center/Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu

	Abstract

Top Abstract I. Introduction II. Protein Processing and... III. Diseases Caused by... IV. Diseases Caused by... V. Rescue of Defective... VI. Conclusions References

 
Receptors, hormones, enzymes, ion channels, and structural components of the cell are created by the act of protein synthesis. Synthesis alone is insufficient for proper function, of course; for a cell to operate effectively, its components must be correctly compartmentalized. The mechanism by which proteins maintain the fidelity of localization warrants attention in light of the large number of different molecules that must be routed to distinct subcellular loci, the potential for error, and resultant disease. This review summarizes diseases known to have etiologies based on defective protein folding or failure of the cell’s quality control apparatus and presents approaches for therapeutic intervention.

I. Introduction

II. Protein Processing and the Role of Chaperones

A. ER processing and quality control (QC)

B. Golgi apparatus

C. Mitochondria

D. Chaperones: the role of folding in QC and routing

III. Diseases Caused by Defective Routing

A. G Protein-Coupled Receptors (GPCRs) in disease states

B. Wild-type protein degradation and oligomerization in protein processing

C. Other proteins

IV. Diseases Caused by Conformational Errors

A. AD and neuropathies

B. Parkinson’s disease (PD)

C. Huntington’s disease (HD)

D. Viral prions

E. Cataracts

F. 1-AT deficiency

V. Rescue of Defective Proteins

A. Temperature

B. Chemical chaperones

C. Nonspecific agents

VI. Conclusions

	I. Introduction

Top Abstract I. Introduction II. Protein Processing and... III. Diseases Caused by... IV. Diseases Caused by... V. Rescue of Defective... VI. Conclusions References

 
IN 1971 GÜNTER BLOBEL presented a first version of the "signal hypothesis." He postulated that, in the case of secretory proteins, there is an intrinsic signal that governs protein movement to and across membranes. Blobel’s laboratory and others subsequently showed that other distinct "address tags" direct proteins to intracellular organelles. These principles are universal, operating similarly in yeast, plant, and animal cells; errors in these tags can result in hereditable disease. In 1999, the Nobel Prize recognized Blobel’s work.

Recent evidence, coming largely from mutations that result in human disease, led to another inescapable conclusion: the successful intracellular routing of many proteins is also governed by a sensitive quality control (QC) system that recognizes particular structural motifs, then retains and degrades defective molecules (1). Abnormal proteins are extremely dangerous to cells because they interfere with normal functions and eventually may result in cell death. The cell limits the accumulation of aberrant conformations with a chaperone system to assist the overall folding process (2, 3). A picture is now emerging that, for some wild-type (wt) proteins, such as the opiate receptor and human (but not rodent) GnRH receptor (GnRHR), only a modest percentage of the newly synthesized protein is routed correctly; the balance is destroyed. The reason for this apparent inefficiency is unclear but suggests the possibility of an unappreciated level of regulation that may provide receptors for up-regulation, enzymes for feedback adjustment, or ion channels to adapt to rapidly changing conditions.

In some cases, protein oligomerization is requisite for export of the nascent protein from the endoplasmic reticulum (ER); mutant or defective proteins may oligomerize with and retain wt proteins, leading to a dominant-negative phenotype.

Diseases caused by abnormal proteins include circumstances in which a specific protein, or protein complex, fails to fold correctly [e.g., cystic fibrosis (CF)], or is not sufficiently stable to perform its normal function (e.g., some forms of cancer). They also include conditions in which abnormal folding results in the failure of protein to be correctly positioned within the cell [e.g., hypogonadotrophic hypogonadism (HH), ₁-antitrypsin (₁-AT) deficiency, and some forms of retinitis pigmentosa (RP)]. Other misfolding diseases, amyloidoses, are caused by conformational changes coupled to the aggregation and cytotoxicity of misfolded proteins outside the cell (Table 1). This review addresses the molecular mechanisms of protein misfolding and protein aggregation and the newly appreciated role of this event in health and disease.

	II. Protein Processing and the Role of Chaperones

Top Abstract I. Introduction II. Protein Processing and... III. Diseases Caused by... IV. Diseases Caused by... V. Rescue of Defective... VI. Conclusions References

View larger version (45K): [in this window] [in a new window]   FIG. 1. The QC process in the secretory pathway. The ER is a lamellar organelle associated with ribosomal protein synthesis, co- and posttranslational modification, and protein folding. Proteins enter the ER in an unfolded state via the Sec61 translocon to receive posttranslational modifications; if the protein is not properly folded/matured, it will remain in the ER and is eventually degraded without reaching its normal cellular site of action. The discrimination between properly folded proteins and misfolded ones takes place in the ER by the QC machinery; sometimes aberrant forms acquire their correct conformation after several passages through the folding chaperone system. Terminally misfolded proteins are retrotranslocated across the ER membrane to be degraded by proteasomes in a process known as ER-associated degradation. Correctly folded proteins are allowed to exit the ER and enter the secretory pathway, through exit sites located at the ER membrane. Proteins are packed into COPII-coated vesicles and leave the ER to be transported to the Golgi apparatus, where they fuse with the target membrane elements in the ERGIC, near the Golgi. Once the proteins reach the Golgi complex, they are sorted to peripheral compartments such as vacuoles, plasma membrane, and secretory vesicles. Retrieval of misfolded proteins from the Golgi complex to the ER occurs; also defective proteins can be transported to the endosomal system for lysosomal or vacuolar degradation. SV, Secretory vesicle.

The effectiveness of the QC system depends, in large part, on the ability of functional proteins to avoid capture because their hydrophobic regions are generally buried. However, when these hydrophobic surfaces are exposed, ER resident chaperones interact with them, retaining them in the ER (8). It appears that the glucosyl transferases recognize misfolded proteins when hydrophobic residues situated on the carboxyl side of the glycosylation site are present. The QC mechanism, which involves a remarkable series of glycosylation and deglycosylation reactions, enables correctly folded proteins to be distinguished from misfolded ones (15) (Fig. 2). However, if the glycoprotein is misfolded it results in retention in the ER where it can undergo a new cycle of reglycosylation by a uridine diphosphate-glucose:glycoprotein glycosyltransferase (UGT) (3, 16). Another protein, ER degradation-enhancing 1,2-mannosidase I (a recently discovered lectin), binds misfolded proteins that are released from calnexin and promotes their degradation, rather than allowing entry into another UGT-calnexin cycle (13). It was initially believed that ER-resident proteinases and peptidases degraded misfolded and orphan proteins (17). However, terminally misfolded molecules are "retrotranslocated" or "dislocated" across the ER membrane to be degraded by cytosolic proteasomes (Fig. 3). The misfolded or unassembled proteins are retained; proteins that normally form associations with other proteins cannot progress through the secretory pathway until they are bound to their partners. Instead, the misfolded proteins are recognized by ER chaperones or by other factors; they are then again translocated to the cytosol through the translocon Sec61 (18). This process, known as ER-associated degradation (ERAD), selects the QC substrates to be degraded, targets them to the translocon, and transports them back to the cytosol, where the proteolytic components of the protein degradation pathway such as ubiquitin, ubiquitin-conjugating enzymes, and the cytosolic 26S proteasome are used (6). After retrotranslocation from the ER, misfolded proteins are polyubiquitylated before degradation. Lack of polyubiquitylation prevents proteasomal degradation and leads to failure in transport of the protein from the ER lumen or membrane out to the cytoplasm, causing the protein to remain in the ER. Ubiquitination may facilitate the recruitment of factors that actively extract the polypeptide from the ER membrane.

View larger version (27K): [in this window] [in a new window]   FIG. 2. The calnexin/calreticulin cycle. The QC mechanism involves a series of glycosylation and deglycosylation reactions that enable correctly folded proteins to be distinguished from misfolded ones. It appears that the glucosyl transferases recognize misfolded proteins when hydrophobic residues situated on the carboxyl side of the glycosylation site are present. When proteins translocate the ER membrane, a core of oligosaccharides (Glc3Man9-GlcNAc2) is attached to asparagine residues in the Asn-X-Ser/Thr consensus sequence (step 1). After the folding process, two of the outermost glucose residues are eliminated by the glucosidases I and II (step 2). Disulfide bonds are transiently formed by the Erp57 (a thiol oxidoreductase chaperone), which interacts specifically with calnexin and calreticulin, and the monoglucosylated protein (step 3). After the last glucose residue is eliminated by glucosidase II, the complex dissociates and releases a protein with a carbohydrate structure of Man9GlcNAc2 (step 4). Correctly folded proteins are then transported out of the ER to the secretory pathway. When the glycoprotein is not correctly folded, the N-glycan is reglycosylated by the UGT (steps 5 and 6), which induces a new round of calnexin/calreticulin binding preventing the exit of the protein from the ER.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Ubiquitin proteasome pathway. Terminally misfolded proteins are retrotranslocated across the ER membrane to be degraded by proteasomes. The misfolded protein is targeted to the Sec61 translocon and then is transported back to the cytosol (step 1). After retrotranslocation from the ER, misfolded proteins are polyubiquitylated before degradation. The lack of polyubiquitylation prevents proteasomal degradation and leads to failure in transport of the protein from the ER lumen or membrane out to the cytoplasm, causing the protein to remain in the ER. The ubiquitin is attached to a cysteine residue of an ubiquitin-activating enzyme (E1), and then transferred to a cysteine of an ubiquitin-conjugating enzyme (E2), and finally to the substrate by the ubiquitin-protein ligase (E3, step 2). The proteins that undergo proteasomal degradation by the proteolytic complex 26S proteasome are marked by the binding of a ubiquitin chain to a lysine residue. The 26S proteasome is composed of a core 20S particle and one or two 19S regulatory particles. The entry channel is quite narrow and, even in its open state, it allows entry of only unfolded proteins in a complex ATP-dependent mechanisms to recognize, unfold, and linearize substrates and to inject them into the 20S proteasome (step 3). These proteins are digested to small peptides, which in turn are rapidly hydrolyzed to amino acids by peptidases (step 4). After proteolysis, the polyubiquitin chain is released and hydrolyzed into single ubiquitin moieties that go through a new cycle of protein degradation.

When aberrant proteins accumulate in the ER due to ineffective ER degradation machinery, there is a different response that occurs. This "unfolded protein response" (UPR) leads to synthesis of ER-resident chaperones and enzymes. Drugs that block N-linked glycosylation or disulfide bond formation or alter calcium homeostasis produce this response. If the response fails to clear the ER, apoptosis is induced (8). The UPR and ERAD are partially overlapping responses that lead to elimination of misfolded secretory proteins. Induction of the UPR up-regulates multiple genes involved in ERAD/proteasomal protein degradation. Defects in ERAD constitutively activate the UPR, and simultaneous deficiency of both pathways decreases cell viability.

In contrast, correctly folded proteins are transported out of the ER and do not go through this QC system, which only retains and degrades misfolded proteins (7, 24). Defective components can cause the QC system to lead to destruction of recoverable proteins at a great cost to energy input. A disturbed QC leads to disease and eventually to cell death.

Proteins are allowed to exit the ER and enter the secretory pathway when they are properly folded, through exit sites located at the ER membrane (25, 26, 27, 28). Misfolded proteins and ER chaperones do not usually exit the ER; however, it has been observed that some misfolded proteins that are exported from the ER shuttle between the ER and the Golgi apparatus during their maturation. The UPR also induces the expression of genes required for ER export (28, 29). Proper function of the QC machinery in the ER is essential for a healthy cellular metabolism.

B. Golgi apparatus
Coated vesicles mediate intracellular transport of proteins. Correctly folded proteins are packed into coat protein complex II (COPII)-coated vesicles and leave the ER at the exit sites and are transported to the Golgi apparatus, where they fuse with the target membrane elements in the ER-Golgi intermediate compartment (ERGIC), near the Golgi (25, 26, 27, 28). ER exit can be by bulk flow or it can be facilitated by transport receptors, such as ERGIC-53, a transmembrane lectin that cycles between ER, ERGIC, and Golgi. ERGIC-53 possesses an ER exit determinant in its cytosolic domain that mediates binding to COPII-coated vesicles. Once in the Golgi, the proteins are sorted to peripheral compartments of the cell-like vacuoles, plasma membrane, and secretory vesicles (28, 30).

Cells also have a back-up QC system, which prevents transport of misfolded proteins beyond the ER. The Golgi complex also makes conformation-based discrimination of abnormal proteins, which are targeted for degradation. In certain cases, retrieval of misfolded proteins from the Golgi complex to the ER occurs (31). The recycling of misfolding proteins might be required by ERAD, assisting the ER QC machinery (7, 28, 32, 33, 34). It appears that misfolded soluble proteins require a modification obtained in the Golgi, presumably in the cis-Golgi, to be recognized as ERAD substrates. It is possible that retrotranslocation occurs from a specialized compartment that soluble proteins can reach only via the Golgi apparatus.

It is not yet clear how the QC system operates in the Golgi complex, because the chemical environment of the Golgi differs from that in the ER. The abundant chaperones and folding enzymes that are found in the ER are lacking in the Golgi; the Golgi has a different ionic milieu and less soluble protein content and is more acidic than the ER. However, the intermediate compartment has calreticulin, glucosyltransferase, and glucosidase II, indicating a potential for glycoprotein folding to occur (35, 36). A Golgi-specific endo--mannosidase ("endomannosidase") has been demonstrated to function as a trimming endoglycosidase that has been found to work in conjunction with calreticulin. It has been suggested that this endomannosidase has a role in the QC of N-glycosylation. However, it is not yet clear whether the endomannosidase will dissociate the calreticulin-glycoprotein complex of properly folded glycoproteins and/or of improperly folded ones that have escaped the ER (37). There is a QC mechanism that resides in the Golgi complex, which routes defective proteins to the endosomal system to be degraded (38). When the quantity of misfolded proteins increases in the ER, some move to the intermediate compartment and to the cis-Golgi (30). Proteins that are targets for lysosomal or vacuolar degradation can either be targeted directly to the endosomal system or can be first routed to the plasma membrane before they are delivered to the endosomal system (12). It is possible that the QC mechanism for some misfolded or aggregated proteins is initiated in the endosomes, thereby precluding these proteins from reaching the cell surface (39, 40, 41).

Golgi complex can be a reservoir for misfolded membrane proteins, but whether the proteins are degraded or remain in the Golgi for a long period of time is unknown (42). Misfolded membrane proteins tend to expose polar residues, which are recognized as defects by the QC. Retrieval of membrane proteins from Golgi to the ER can be triggered by the presence of these polar residues in the transmembrane domains. These polar residues can also cause entry from the Golgi or cell surface into the endosomal system. A multispanning membrane protein of the Golgi, Tul1, which is a putative ubiquitin ligase, is an enzyme that recognizes polar residues and might separate membrane proteins that are incorrectly folded in the transmembrane region or not completely oligomerized for vacuolar targeting (7).

Evidence based on lysosomal enzyme inhibitors suggests that the turnover of secretory pathway membrane proteins may happen by targeting from the trans-Golgi network to the endosome/lysosome system (43).

The QC mechanisms in the Golgi complex became particularly important for exportable proteins that achieved their posttranslational modification in the Golgi apparatus. Native or mutant proteins can be directed to degradation to maintain the local environment of the secretory pathway.

C. Mitochondria
The mitochondrion is an organelle that consists of an outer and inner membrane that separates the matrix from the intermembrane space. Mitochondria provide the majority of the ATP to the cell by coupling the oxidative phosphorylation with respiration. The mitochondrial respiratory chain is composed of five enzyme complexes: reduced nicotinamide adenine dinucleotide-Coenzyme Q reductase (complex I), succinate Coenzyme Q reductase (complex II), ubiquinol-cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F₁F₀-ATPase (complex V). In the respiratory enzyme complexes these components transfer electrons to each other and ultimately to molecular oxygen; they also translocate protons across the inner mitochondrial membrane. This proton gradient activates the ATP synthase that catalyzes the conversion of ADP to ATP, completing then the process of oxidative phosphorylation (44, 45).

Studies in yeast suggest that a protein QC system exists in mitochondria similar to that in the ER (46). Proteins that cross the mitochondrial membranes do so in an extended conformation, being coupled to the processes of protein unfolding in the cytosol and protein refolding in the matrix. Precursor proteins destined for the mitochondrion are escorted to their proper cellular location and are maintained in a conformation compatible with import by molecular chaperones within the cytosol, such as the heat shock protein 70 (Hsp70) and mitochondrial import stimulation factor. Both of these chaperones exploit the inherent instability of the precursor in unfolding the protein so that it can properly interact with the import machinery. Once the precursor polypeptide translocation into the matrix is complete, the precursor must adopt its mature conformation to be able to function in the organelle. Molecular chaperones in the mitochondria are involved in translocation, refolding, and assembly of imported proteins and proteins encoded in the mitochondria, and protein degradation (47, 48).

After completion of translocation, folding routes may differ for individual proteins. Some proteins can fold directly after release from the membrane-associated form of Hsp70, or they can be transferred from there directly to Hsp60, chaperonins that mediate the folding of proteins to their native state, or into monomers that subsequently undergo oligomeric assembly (49). There are several other matrix proteins, in addition to the molecular chaperones, that are also involved in protein maturation and folding, such as peptidases and peptidyl prolylisomerases. However, the peptidases have no access to the precursors while they are attached to chaperones.

When there is an unfavorable condition that provokes proteins to become unfolded and denatured, the molecular chaperones, in addition to binding proteins that are folding de novo, bind to (preexistent) proteins that have become unfolded and maintain them in a soluble state before the favorable conditions are restored. This situation is also observed when proteins are not able to fold correctly because of a chemical modification, lack of assembly partners, or because of mutation. These misfolded proteins will be targeted for degradation. The chaperones help to maintain these misfolded proteins in a soluble state, to prevent them from aggregation. If there is a lack of correct chaperone function, then these proteins aggregate and cannot be degraded. The ATP-dependent degradation of misfolded proteins in the mitochondrial matrix is accomplished by the proteolysis in mitochondria 1 protease (47).

D. Chaperones: the role of folding in QC and routing
Molecular chaperones are proteins that interact transiently with unfolded or partially folded intermediates, stabilizing exposed residues with particular physicochemical characteristics and preventing them from forming inappropriate intra- or intermolecular contacts (50). Chaperones increase the efficiency of the overall folding process by reducing the probability of aggregation and by protecting proteins as they fold and by rescuing misfolded and even aggregated proteins and enable them to have a second chance to fold correctly (1, 51, 52). The concentrations of many chaperones are substantially increased during cellular stress. Chaperones frequently utilize a cycle of ATP-driven conformational changes to fold or refold their targets. Protein folding typically takes place in the cytoplasm, for cytoplasmic proteins, or in the ER, for transmembrane and secretory proteins (53).

The Hsps have been divided into groups according to their mol wt. They are classified in two main groups: high mol wt ATP-dependent Hsp that requires cochaperones to modulate their conformation and ATP binding. This group includes four major families (Hsp70, Hsp60, Hsp90, and Hsp100). The second group of chaperones includes the small ATP-independent Hsp, such as Hsp27, that trap denatured proteins on their surfaces. The basic reactivity of all chaperones is similar, but their structural characteristics differ; they participate in very diverse cellular processes, and they can be targeted to different subcellular compartments (53, 54). Misfolded proteins can interact with one or more of the Hsp70 class of chaperones, which protects them against aggregation (55, 56). Hsp70 molecules form multichaperone complexes with cochaperones such as the Hsp40, and they can function in an ATP-dependent process to catalyze the refolding of denatured or partially denatured molecules into enzymatically active forms (51, 57, 58). Certain combinations of chaperones, e.g., Hsp70, Hsp104, and Hsp40, are capable of disassembly of intracellular protein aggregates and acceleration of the refolding of the insoluble molecules into soluble, native species (21, 59, 60, 61).

The molecular chaperones that are used in QC are abundant in the ER. Retention-based, chaperone-mediated QC in the ER is well characterized in the case of glycoproteins. The two homologous lectins, calnexin, which is a transmembrane protein, and calreticulin, which is a soluble luminal protein, operate as chaperones in the QC of newly synthesized glycoproteins, ensuring that correctly folded glycoproteins leave the ER, and they can also mediate glycoprotein degradation of the misfolded protein (15, 62, 63, 64, 65, 66). The lectin-assisted glycoprotein folding involves cycling of de- and reglycosylation until correct folding is achieved. If the protein is correctly folded, it will leave the ER. Misfolded it will be recognized by the ER enzyme UGT and reglycosylated, allowing it to reassociate with the QC machinery. The protein will be able to get completely folded as long as it goes through the calnexin-calreticulin cycle repeatedly. Both calnexin and calreticulin suppress aggregation of misfolded proteins via a polypeptide-binding site in their globular domains. Calnexin and calreticulin form complexes with ERp57, a member of the PDI family, which form transient disulfide bonds with calnexin- and calreticulin-bound glycoproteins, facilitating correct disulfide bond formation. PDI is one of the chaperones transcriptionally up-regulated when unfolded proteins accumulate in the cell (67).

One of the most abundant ER molecular chaperones is BiP, the main protein of the Hsp70 family. It stabilizes newly synthesized polypeptides during folding, prevents aggregation by binding to and stabilizing exposed hydrophobic regions of unfolded proteins, mediates retention in the ER, suppresses formation of nonnative disulfide bonds, and has a role in the translocation of newly synthesized proteins across the ER membrane and in degradation of misfolded proteins. BiP is also up-regulated when misfolded proteins accumulate in the ER (7).

Chaperone ERGIC-53 is a lectin that serves as an escort protein carrying high-mannose N-linked glycans, which cycles between the ER and the Golgi complex; this molecule also helps to prevent proteins from interacting with ligands during the early secretory pathway.

When there is an excess of misfolded proteins due to physical or chemical stresses or to mutations, there is an increase in chaperone and proteolytic activities, to reduce the presence of damaging nonnative proteins. Several chaperones can stabilize proteins undergoing denaturation and allow their refolding when the conditions are back to normal. However, when the chaperone’s function on protein stabilization is not optimal, it results in protein aggregation.

	III. Diseases Caused by Defective Routing

Top Abstract I. Introduction II. Protein Processing and... III. Diseases Caused by... IV. Diseases Caused by... V. Rescue of Defective... VI. Conclusions References

 
A. G protein-coupled receptors (GPCRs) in disease states
1. HH.
HH is characterized by absent or decreased gonadal function and manifests as delayed or disrupted sexual development in affected individuals; it constitutes one of the most common causes of hereditary hypogonadism. Mutations in the GnRHR gene are one etiology of HH. Heterologous expression of these GnRHR alleles reveals that the majority encode misfolded receptors with an increased propensity for targeted degradation or intracellular retention. Mutations are predominantly in transmembrane segments, yet (somewhat surprisingly) are widely dispersed across the entire sequence of the GnRHR (68, 69, 70).

In HH due to GnRH resistance, affected individuals are either compound heterozygous or homozygous for the mutation. To date, 16 inactivating mutations in the human (h)GnRHR gene and a splice junction mutation at the intron 1-exon 2 boundary have been described as causes of HH and were isolated from patients with phenotypes from partial to complete hypogonadism. The majority of these misfolded translation products do not route correctly to the cell membrane, where the mutant protein is otherwise fully functional (71). The expression of 13 naturally occurring hGnRHR point mutations shows that 11 of these mutants become nonfunctional misrouted proteins that are rescuable by genetic or pharmacological approaches (Fig. 4; Refs.68 , 72 , and 73).

View larger version (26K): [in this window] [in a new window]   FIG. 4. hGnRHR mutant rescue with a complex indole and peptidomimetics antagonist of the GnRHR "IN3" (264 ). HH is characterized by absent or decreased gonadal function and manifests as delayed or disrupted sexual development in affected individuals and constitutes one of the most common causes of HH. Mutations in the GnRHR gene are one etiology of HH. Heterologous expression of these GnRHR alleles reveals that the majority encode misfolded receptors with an increased propensity for targeted degradation or intracellular retention. Mutations are predominantly in transmembrane segments, yet (somewhat surprisingly) widely dispersed across the entire sequence of the GnRHR. The majority of these misfolded translation products do not route correctly to the cell membrane, where the mutant protein is otherwise fully functional. The expression of 13 naturally occurring hGnRHR point mutations, shows that 11 of these mutants become nonfunctional misrouted proteins that are rescuable by a cell membrane-permeant antagonist of GnRH, IN3. Most of misfolded hGnRHR mutants are rescued by IN3, which allows them to escape the ER and avoid a degradation pathway.

Pharmacological chaperone treatment approximately doubles the expression of wt hGnRHR (but not rat GnRHR) at the plasma membrane. Based on this observation, it has been suggested that more than half of the synthesized hGnRHR never arrives at the plasma membrane. The disparity between the human and rat sequences appears related to specific structural differences in the human sequence that result in instability of the human sequence (77).

One mutant that results in HH is the E90K mutation of the hGnRHR gene; this mutant may be rescued by deleting the amino acid K191, an "extra" amino acid in primate GnRHR (not found in rodents) that appears to destabilize the hGnRHR, thus precluding movement of much of the translation product to the plasma membrane (72). Patients bearing the E90K defect express a severe phenotype of hypogonadotrophic hypogonadism (78). Peptidomimetic, cell membrane-permeant GnRH antagonists from three different chemical classes have been shown to serve as templates (pharmacological chaperones or "pharmacoperones") for naturally occurring and "designer" (misfolded) GnRHR mutants and thereby effect rescue by stabilizing the GnRHR (68, 69, 71). Unexpectedly, even the use of green fluorescent protein or "hemagglutin-tags" on receptor mutations, has a dramatic effect on the wt receptor and resulted in altered routing (79), a finding that, presumably, is not unique to this receptor.

2. RP.
Rhodopsin (Rh) is the visual pigment protein of vertebrate rod cells and is an integral membrane glycoprotein. Rh synthesis requires membrane insertion of the polypeptide opsin, and it has a covalently bound reverse agonist, the chromophore 11-cis-retinal, which interacts with amino acid residues located in transmembrane -helices (80, 81).

To date, more than 150 opsin mutants have been identified that cause RP, a heterogeneous group of inherited disorders that cause progressive retinal degeneration and that lead to photoreceptor death and subsequent severe loss of peripheral and night vision. In humans, more than 25% of all RP cases are caused by dominant mutations in the gene encoding Rh, producing misfolded Rh, which, presumably, interferes with the maturation of wt molecules in the ER (82).

The opsin mutants are divided into three different classes depending on their distinct biochemical features. Class I mutants that accumulate at the cell surface; Class II mutants retained in the ER; and Class III mutants mainly retained in the ER but also found on the cell surface (80, 81). The most common mutation linked to RP is the P23H mutation, which is a Class III type mutant, located in the N-terminal tail within one of the ß-strands in close proximity to residues that form the retinal plug, which keeps the chromophore in its proper position. P23H-opsin is retained intracellularly, predominantly in a perinuclear region. The P23H mutant leads to formation of conjugated hydrogen bonding that exposes the chromophore-binding site. Mutations located within this region result in improper folding of opsin and poor binding of the chromophore. The P23H mutant has a structural alteration that decreases the stability of the protein, leading to aberrant folding and subsequent aggregation. However, there is a pharmacological chaperone, an 11-cis-retinal analog that facilitates proper folding and stabilization of P23H-opsin (80, 81).

3. Nephrogenic diabetes insipidus (NDI).
In nephrogenic diabetes insipidus (NDI), a disease that can be inherited or acquired, the kidney is unable to concentrate urine in response to the antidiuretic hormone arginine vasopressin, despite normal or elevated plasma concentrations of such hormone. NDI describes only those conditions in which arginine vasopressin release fails to induce the expected increase in the permeability of the cortical and medullary collecting ducts to water. Vasopressin regulates body water, resulting in water reabsorption from urine, by fusion of vesicles containing aquaporin-2 (AQP2) water channels with the apical membrane of renal collecting ducts, this being the final step in the antidiuresis-signaling pathway that leads to increased water permeability in the duct. Mutations in either the vasopressin type 2 receptor (V2R) gene or the AQP2 gene can cause congenital NDI. Mutations in the V2R gene cause the X-linked form of inheritance of NDI, and mutations in the AQP2 gene cause a rare non-X-linked form of inheritance of NDI (83, 84, 85).

There are 155 known mutations within the V2R and, as in the case of the GnRHR, the majority are in the transmembrane segments of the V2R, but are broadly distributed in the sequence. More than 90% of these mutations failed to properly fold and therefore have defective intracellular transport, being retained in intracellular compartments instead of being transported to the cell membrane. In some cases, accumulation of V2R mutants away from the plasma membrane, possibly in the ER, occurs because there is little maturation (sugar trimming) of the glycosyl-groups (86).

In the AQP2 gene there have been several point mutations identified that are associated with NDI. Autosomal-recessive NDI-causing AQP2 mutations have a significant defective protein trafficking that results in retention of the mutant protein in the ER, although they do not present major differences in intrinsic water permeability or protein stability. A mutation in the AQP2 gene, in which there was a substitution of a lysine for a glutamic acid at position 258 (AQP2-E258K), has been shown to result in an autosomal-dominant form of inherited NDI. This mutant is localized mainly in the Golgi complex region in Xenopus oocytes, and it is stable as the wt AQP2 (85).

The AQP2-E258K mutant forms mixed oligomers with the wt AQP2 and has a dominant-negative effect on the function of the wt form, which might explain the dominant inheritance of NDI, whereas in the recessive inheritance of NDI there appears to be a lack of oligomerization of the mutant AQP2 protein with the wt AQP2 (84, 85). The AQP2-E258K mutant exits the ER, after which it heterotetramerizes with wt AQP2. AQP2 mutants seem to have an impaired routing and thereby differ in their retention site. For example, the T126M AQP2 mutant is retained in the ER and rapidly degraded by the proteasome; in contrast, the E258K AQP2 mutant is retained in the Golgi and then degraded by proteasomes and lysosomes (87).

B. Wild-type protein degradation and oligomerization in protein processing
There is evidence showing that in many cells, approximately 30–60% of the newly synthesized proteins never achieve their completely folded native structure and are thereby targeted for degradation. This is documented in the case of the GnRH and the opioid receptors, where exit from the ER is the limiting step in processing of the newly synthesized receptors. Frequently, receptor folding intermediates are retained in the ER until the correct conformation is attained. In the case of the opioid receptors, there is an apparent inefficiency of the maturation process because only 40% of the precursor receptor was converted to the mature form and reached the cell surface, whereas the other 60% is retained in the ER and degraded (88).

For the human GnRHR, about half of the synthesized receptor is expressed at the plasma membrane. This is not the case for the rodent GnRHR, which is more stable and is almost completely routed to the cell surface. It has been shown that in the presence of pharmacological chaperones, there is a higher expression of hGnRHR at the plasma membrane (75).

For many years the GPCRs have been believed to be monomeric structures, each one being independently activated by the binding of a single ligand. These receptors then activate G proteins. However, this classical model may be oversimplified because an increasing number of studies have demonstrated that many GPCRs exist (in the plasma membrane) as oligomers or heterooligomers (89).

The existence of a dimeric GPCR was suggested by using bivalent antibodies directed against a peptide antagonist of the GnRHR; this antibody converted the antagonist into an agonist and promoted receptor activation (90). It has been demonstrated that several GPCRs form constitutive dimers during biosynthesis. For example, the dimerization of the metabotropic glutamate receptor (mGluR1a) takes place in the ER in the HEK293 cell line (91). Also it has been shown that oxytocin, vasopressin V1a, and V2 receptors are present as dimers in an ER-enriched fraction (92). Several studies also suggested that some GPCRs form dimers in the presence of agonists and that sometimes the formation of a constitutive dimer is dependent on receptor density (93, 94, 95).

It has been assumed that GPCR mutants do not have an effect on the function of the wt receptor. However, it has been demonstrated that heterodimerization between the wt and the mutant receptors contributes, at least in part, to the dominant-negative phenotypes associated with heterozygous mutations. A loss-of-function CCR2(Y139F) mutant acts as a dominant-negative agent, blocking signaling through the wt CCR2 (93). Other examples include the V2R (96) and D2 receptors (97). In the case of GnRHR and the fly Frizzled family of Wnt receptors, the mutant forms heterooligomers between the wt and the dominant-negative receptors, trapping them both in the ER (76, 98).

Sometimes, the simultaneous presence of two different GPCR moieties is necessary to constitute a functional receptor. For example, neither type 1 nor type 2 -aminobutyric acid B receptor (GABA_BR1 nor GABA_BR2) when expressed individually, activates GIRK-type potassium channels. However, the combined expression of GABA_BR1 and GABA_BR2 produces a oligomer that is capable of stimulation of channel activity upon binding agonist by the complex (99). The heterodimerization of the GABA_BR1 and GABA_BR2 in the ER is necessary for exporting GABA_BR1 to the cell surface (100, 101). In the heterodimeric complex GABA_BR2 is required to activate G proteins, and GABA_BR1 is responsible for the binding of its ligand (100).

The retention of the wt receptor proteins as well as the formation of oligomers seems to modify the overall processing of the newly synthesized proteins. The retention of the wt and mutant receptors by oligomerization in the ER could be associated with different diseases. Further analysis of such mechanisms will likely lead to a better understanding of the regulation of GPCRs.

C. Other proteins
1. Cancer (p53).
The tumor suppressor p53 activates numerous genes and leads to cell cycle arrest or apoptosis, protecting the cell against tumor growth. Because of its growth-inhibitory activity, p53 must be maintained at low levels to ensure cell survival and proper organism development; this is regulated by MDM2, which inhibits the function of p53 by targeting it for ubiquitin-dependent proteasomal degradation (102). The mutant form of p53 loses the ability to induce cell cycle arrest or apoptosis. Approximately 50% of the human cancers lose p53 function mainly by point mutations in the core domain, which results in loss of sequence-specific DNA binding and transcription function. These mutations mainly affect residues that are directly involved in contacting DNA and that are important for maintaining the structural stability of the highly conserved DNA-binding domain by thermodynamically destabilizing p53, which results in its unfolding and inactivation (Fig. 5; Ref.103). The core domain is relatively unstable, because the majority of residues in the p53 core domain are targets of mutation in tumors.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Mutant p53 pathways that result in destabilization, unfolding, and inactivation. The p53 point mutations mainly affect residues that are localized in the core domain. A, Unfolded p53 loses sequence-specific DNA binding and transcriptional function by blocking interaction with target DNA. B, Misfolded mutants have a dominant-negative effect by aggregating with wt p53, thus forming functionally inactive oligomers that are incapable of entering the nucleus. C, External small synthetic compounds can bind to p53, stabilizing the DNA-binding conformation, allowing nuclear transport and normal function.

Missense single-point mutations are the most common alterations in the p53 gene; however, many cancers lose p53 activity by inhibition of the wt protein rather than by mutation. Tumor growth suppression can be achieved by reintroduction of wt p53, and intermittent threshold levels of p53 activity may be sufficient, functioning as an important target in the development of cancer therapies. Various strategies have been designed to restore function to mutant p53. The restoration of p53 function, based upon molecular modeling, introduced additional DNA contacts or enhanced the stability of the folded state. One strategy to reactivate destabilized p53 mutants leading to thermodynamic stabilization and activity restoration is by using a small molecule that binds the native, but not the denatured, state and drives the conformational equilibrium toward the native state. Furthermore, the introduction of second-site suppressor mutations, by site-directed mutagenesis of single amino acid substitutions can restore, at least partially, the conformation for DNA binding or the stability of the protein (103, 106). Moreover, it has been shown that specific DNA binding of mutant p53 as well as p53-dependent apoptosis in tumor cells can be restored by synthetic peptides that have been derived from the p53 C terminus.

2. Cystinuria.
The membrane glycoprotein rat liver bile acid coenzyme A: amino acid N-acetyltransferase (rBAT) is localized at kidney and small intestine membranes, which are sites for high-affinity L-cystine absorption. This glycoprotein produces a large amount of amino acid transport. Mutations of the membrane glycoprotein rBAT have been found to cause the inheritable disorder cystinuria type I, which is an autosomal-recessive failure of dibasic amino acid transport across the luminal membrane of kidney cells and also affects intestinal absorption of cysteine and provokes a low solubility of cysteine, leading to a severe development of cysteine "calculi," which are abnormal stony masses in the kidney commonly known as kidney stones (107). So far, 32 cystinuria-specific mutations of rBAT have been described in patients with cystinuria, which include missense and nonsense mutations, deletions, and insertions. The most common point mutation is M467T, localized in the third transmembrane domain and found in 26% of cystinuria type I genes (108).

There is a diminution of membrane-expressed proteins M467T compared with wt rBAT, and the intracellular amounts of the mutant form are higher, which suggests that there might be a defect in the delivery of the mutants to the plasma membrane because of their retention in the ER, probably due to improper folding. rBAT is assembled with other proteins to constitute the complete amino acid carrier complex, and this assembly has slower or impaired activity if rBAT is improperly folded. The transport out of the ER is accomplished after assembly of proteins into native homo- or heterooligomers (108).

Another mutant of rBAT, R365W, is located in the extracellular domain of rBAT and was found in some cystinuria patients. This mutant behaves as a temperature-sensitive folding mutant. It is probable that the cystinuria phenotype related to this mutant form of rBAT is a consequence of a severe trafficking defect. Most cystinuria-specific rBAT mutations are trafficking mutants, supporting the proposed role of rBAT in the routing of the holotransporter, formed by two different subunits (the rBAT heavy chain and a rBAT light chain), to the plasma membrane (109).

3. CF.
CF is an autosomal-recessive disorder affecting the apical membrane of epithelial cells of the pancreas, bronchial glands, biliary tree, intestinal glands, reproductive organs, and sweat glands. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, which functions as a Cl^– channel and regulator of other ion channels, resulting in Cl^–-impermeable epithelial cells in the affected organs (Fig. 6; Refs.110 and 111).

View larger version (42K): [in this window] [in a new window]   FIG. 6. Model of the lung epithelium in CF. In CF, the apical membrane of epithelial cells of the respiratory tract, along with other organs, lose plasma membrane CFTR channel expression. Mutations in the CFTR channel cause the disease, the most common being the 508 CFTR mutation. A, In the normal healthy state of the lung, the CFTR is localized to the apical membrane of the lung epithelium, and Cl–, and thus Na+ and water, pass freely from the bloodstream into the lungs. B, In a CF patient, the mutant channel remains largely trapped in the cell, thereby impeding the normal flux of Cl–, and thus Na+ and water, across the epithelium; as a result the airway surface dries out and the mucus accumulates and becomes condensed and thick, impeding the passage of air. C, In the presence of a chemical chaperone or a nonspecific agent such as glycerol, the mutant CFTR is relocalized to the cell surface, recovering the Cl–, and thus Na+ and water, flux, rehydrating the airway membrane and allowing mucus clearing.

Unlike the hGnRHR and V2R, most patients share a common CFTR mutation, this being a deletion of phenylalanine at position 508 (508). This site is normally in the cytoplasmic domain of CFTR; the mutant remains in the ER and is ultimately degraded by the ubiquitin-proteasome system. The disease in the homozygous patient is quite severe, even though it has been demonstrated that F508 CFTR retains some function as a Cl^– channel, although less than the wt protein. The CF phenotype associated with F508 CFTR appears to originate from mislocalization and premature degradation, rather that the altered functional properties or decreased stability of mutant CFTR (110). Misfolding CFTR causes this protein to be targeted to the degradative apparatus before it can be translocated to the plasma membrane. There is evidence for a temperature sensitivity of F508 CFTR maturation, although there may be no apparent decrease in thermal lability of the mutant protein (114). Calnexin binds to the newly synthesized CFTR and dissociates from it when it reaches its native conformation. In contrast, the F508 CFTR remains associated to calnexin therefore, after a prolonged interaction leading the F508 CFTR to ERAD (115).

The F508 CFTR mutant is functional once it reaches the native state. Growth conditions that favor proper folding of the mutant CFTR allow increased recovery of function. Identification of a compound that corrects the F508 CFTR folding defect would be of great potential therapeutic benefit, and some studies that utilize small molecules identified from screening techniques are underway. CFTR activators may also correct the impaired chloride transport in cells expressing mutant CFTRs in CF patients (116).

4. Gitelman’s syndrome.
Gitelman’s syndrome is an autosomal-recessive disease characterized by renal salt wasting with hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria (117). The first clinical presentation usually occurs at 6 yr of age or older and may include muscular pain and cramping with exercise and fatigue and weakness. The disease is generally mild, and some patients even remain without symptoms (118, 119). This disease is caused by mutations in the SLC12A3 gene, which encodes the thiazide-sensitive NaCl cotransporter (NCCT; Fig. 7). The mutation in the NCCT gene results in salt wasting from the distal convoluted tubule, increasing aldosterone secretion, which augments Na⁺ reabsorption. Because of the indirect coupling of Na⁺ reabsorption to K⁺ and H⁺ secretion in the distal nephron, the augmented Na⁺ reabsorption activity occurs at the expense of increased K⁺ and H⁺ secretion, accounting for the observed hypokalemia and metabolic alkalosis (120, 121). The tertiary structure of NCCT contains 12 transmembrane domains and intracellular amino- and carboxy-terminal regions (122). The mutations identified in Gitelman’s syndrome are located throughout the entire coding sequence of the protein and include missense, frameshift, nonsense, and splice-site mutations (123).

View larger version (21K): [in this window] [in a new window]   FIG. 7. Model of the NCCT. The tertiary structure of NCCT contains 12 transmembrane domains and intracellular amino- and carboxy-terminal regions. The mutations identified in Gitelman’s syndrome are located throughout the entire coding sequence of the protein. Thirteen human NCCT mutations have been functionally tested in Xenopus laevis oocytes. The missense mutations G439S, T649R, and G741R were partially glycosylated and impaired in their route to the plasma membrane, and instead they are retained in the ER/pre-Golgi complex. In contrast, the rest of the mutations impair their insertions of a functional protein into the plasma membrane.

5. Hemochromatosis (HC).
Hereditary HC is an autosomal-recessive disorder characterized by an abnormal increase in the absorption of iron in the gastrointestinal tract. The organs in which iron deposits primarily are liver, pancreas, heart, joints, and endocrine organs (125). Clinical consequences of iron accumulation in these organs include cirrhosis of the liver, hepatocellular carcinoma, diabetes, heart failure, arthritis, and hypogonadism (126). The majority of North American patients with HC have a mutation in cysteine 282 (to tyrosine) in a major histocompatibility complex (MHC) class I-like gene, called hemochromatosis gene (HFE) (127, 128). A second mutation (H63D) was reported to be also present in HC patients who are heterozygous for the C282Y mutation (129).

The human HFE protein is a membrane protein homologous to MHC class I molecules, which contain an extracellular peptide-binding region (1 and 2 loops), an Ig-like domain (3), a transmembrane region, and a short cytoplasmic tail. HFE contains intramolecular disulfide bridges in the 2- and 3-domains that stabilize its tertiary structure (127). The disulfide bridge in the 3-domain of MHC class I molecules is required for their association with ß₂-microglobulin (ß₂M), leading to efficient intracellular processing and transport to the plasma membrane (130, 131). The C282Y mutation prevents the interaction between HFE with ß₂M and eliminates cell surface presentation. The role of ß₂M/heavy chain interaction is to facilitate and stabilize the folding of the heavy chain during biosynthesis (130, 132).

The process by which HFE may affect iron uptake is poorly understood. Recent evidence demonstrated that HFE diminishes the binding affinity of iron-loaded transferrin to its receptor (133, 134). In fact, the binding of HFE and transferrin receptor is required for HFE transport to endosomes (125). However, the physiological relevance of this finding remains unclear.

The association of the heavy chain with the ß₂M occurs in the ER and is then transported through the cis-Golgi network, to the middle and trans-Golgi cisternae where the glycosyl side chain is modified to a more complex form en route to the plasma membrane (135, 136). Class I molecules that fail to assemble properly are recycled between the ER and Golgi, rather than being retained exclusively in the ER (137).

6. Hypercholesterolemia.
Plasma low-density lipoproteins (LDL) bind to the LDL receptor (LDLR), a transmembrane glycoprotein that mediates LDL cellular uptake, internalization, and its delivery to lysosomes. Loss of LDLR function leads to decreased LDL catabolism, and its cholesterol is released, resulting in elevated levels of plasma cholesterol. Mutations in the LDLR gene results in protein misfolding and ER retention, causing familial hypercholesterolemia, which is a common autosomal-dominant inherited disorder (138, 139). To date, there are more than 600 mutations that have been characterized in the LDLR; however, 50% of these mutations in familial hypercholesterolemia patients lead to a class 2 mutation, which are proteins that are partially or totally retained in the ER or have an ER-Golgi-defective transport (140). The BiP chaperone has been shown to bind to LDLR by coimmunoprecipitation studies, where an interaction of Grp78 with either the wt or the mutant LDLR was observed, and there was no interaction with other proteins. Moreover, the interaction of Grp78 with mutant LDLR was shown to be a prolonged one compared with that of the wt LDLR, demonstrating that Grp78 is the chaperone responsible for retention of the misfolded receptors in the ER. The mechanisms responsible for this LDLR retention in the ER are not yet clear (138).

A prolonged retention of misfolded or incompletely folded proteins in the ER leads to their degradation. The proteasomal degradation pathway mediates the LDLR mutant degradation, but the precise mechanism for the LDLR is still unknown. The receptor-associated protein (RAP) has been identified to function as a specialized chaperone in the folding and trafficking of LDLR along the early secretory pathway. RAP, which binds only to members of the LDLR family, promotes the receptor folding and maturation by preventing the formation of nonproductive intermolecular disulfide bonds and premature ligand binding within the early secretory pathway (141). It is still not known whether RAP is also involved in ER retention and/or the proteasomal degradation of the LDLR mutants (139).

	IV. Diseases Caused by Conformational Errors

Top Abstract I. Introduction II. Protein Processing and... III. Diseases Caused by... IV. Diseases Caused by... V. Rescue of Defective... VI. Conclusions References

 
The so-called conformational diseases are disorders in which the constituent protein involves change in size or fluctuations in shape with resultant self-association and tissue deposition as amyloid fibrils (142). This diverse group of diseases includes disorders such as Alzheimer’s and Parkinson’s diseases, amyloidoses, ₁-antitrypsin deficiency, and the prion encephalopathies. These diseases result when a specific protein is subjected to a conformational rearrangement that results in aggregation and deposition within tissues or cellular compartments (143, 144). These disorders have common features: they have either sporadic or inherited patterns, they appear later in life (generally after the fourth or fifth decade), and all are characterized by neuronal loss and synaptic abnormalities. In contrast, the clinical symptoms and disease progression are very different among such disorders (145, 146).

Amyloid structures can be recognized because of Congo red and thioflavin S binding and a core structure based on the presence of highly organized ß-sheets. This characteristic makes them very resistant to proteolytic degradation (147, 148). When these aggregates are formed, they are stable for long periods, forming a bed for the progressive accumulation of more deposits in tissue and eventual conversion into amyloid fibrils (1). The deposits in amyloidoses are extracellular but also include some intracellular aggregates. These can often be observed as thin fibrillar structures, sometimes assembled further into larger aggregates or plaques (Fig. 8; Refs.146 and 148). The capacity of polypeptide chains to form amyloid structures seems to be a generic feature of polypeptide chains, and it is not restricted to the relatively small number of proteins associated with recognized clinical disorders (149, 150). The polypeptides involved in amyloid diseases include full-length proteins (e.g., lysozyme or Ig light chains), biological peptides (amylin, atrial natriuretic factor), and fragments of larger proteins produced as a result of specific processing (e.g., the Alzheimer ß-peptide). These disorders may also result from fragments due to general degradation [e.g., polyglutamine stretches cleaved from proteins with polyglutamine extensions such as huntingtin, ataxins, and the androgen receptor (148)].

View larger version (31K): [in this window] [in a new window]   FIG. 8. Schematic representation of the possible mechanism of formation of amyloid fibrils. The so-called conformational diseases are disorders in which the constituent protein involves change in size or fluctuations in shape with resultant self-association and tissue deposition as amyloid fibrils. When a specific protein is subjected to a conformational rearrangement that promotes aggregation, it induces the deposition within tissues or cellular compartments resulting in these disorders. The core structure of amyloid fibrils contains highly organized ß-sheets, which makes them very resistant to proteolytic degradation. When these aggregates are formed, they are stable for long periods, forming a bed for the progressive accumulation of more deposits in tissue and eventual conversion into amyloid fibrils. These can often be observed as thin fibrillar structures, sometimes assembled further into larger aggregates or plaques. The impairment of cellular function is the result of direct interaction between the aggregated proteins with some cellular components. However, it has been demonstrated that, in many cases, the prefibrillar aggregates are more toxic to the cells than mature fibrils.

The molecular chaperones and other QC mechanisms are remarkably efficient in ensuring that prefibrillar aggregates are neutralized before they can do any damage. If the efficiency of the QC mechanisms is impaired, the probability of pathogenic behavior is increased. Most of the amyloid diseases are associated with old age, and the explanation could be a decrease in the efficiency of these protective mechanisms, when there is an increased tendency for protein to become misfolded or damaged, coupled with a decreased efficiency of the molecular chaperone and UPRs (154). The increase in human life expectancy is associated with augmentation in the prevalence of amyloid diseases, in particular in highly developed countries; this can be explained by the inefficiency of the QC mechanisms to prevent the protein aggregation (148, 155).

A. AD and neuropathies
AD is, at present, an incurable neurodegenerative disease and the most common form of dementia (156). The clinical manifestations in AD are the gradual loss of memory, followed by a progressive deterioration of higher cognitive functions and behavior (157), caused by a massive loss of neurons (158). Prevalence is estimated to affect 5–8% of persons over 65 yr of age, and the incidence of inherited forms ranges from 1–30% (159).

Primarily, extracellular plaques and intracellular neurofibrillary tangles characterize the pathology of AD. Plaques are composed mainly of the amyloid-ß-peptide (Aß), whereas tangles are composed of the cytoskeletal protein (142). The formation of tangles is considered as a secondary event, i.e., a consequence of the formation of the Aß-plaques (160, 161). The diseases called "tautopathies" are different from AD, and the brain lesions in these patients are the intracellular aggregation of -proteins, without accumulation of Aß-plaques (162).

To date, mutant forms of three genes, ß-amyloid precursor protein (APP) gene (located in chromosome 21), presenilin 1 (PS1, on chromosome 14), and presenilin 2 (PS2, chromosome 1), have been shown to cause early-onset familial AD (158). In addition, polymorphisms in four genes, i.e., apolipoprotein E (apo E), -2 microglobulin, very low density lipoprotein receptor, and low density lipoprotein receptor-related protein, are shown to be risk factors for AD pathogenesis (158, 163).

Amyloid formation by the Alzheimer’s Aß-protein is the most extensively studied. The Aß-peptides are degradation products of a transmembrane cell surface glycoprotein APP (Fig. 9). Three different proteases called -, ß-, and -secretases can cleave APP. When APP is concomitantly hydrolyzed by ß-secretase at the N terminus and by the -secretase within the membrane, the two main products, Aß(1–40) and Aß(1–42), migrate outside the cell and produce fibrils (164). Peptides containing the 40- or 42-residue form of Aß, and shorter derivatives, form amyloid-like fibrils in vitro, with characteristics similar to fibrillar aggregates extracted from AD amyloid plaques (146, 165, 166). The amyloid cascade hypothesis predicts that the overproduction of Aß, or the failure to eliminate this peptide, induces AD primarily through amyloid deposition, which is postulated to be involved in neurofibrillary tangle formation (167, 168, 169). These lesions are then associated with cell death and consequent memory impairment (142, 170, 171). The precise involvement of amyloid in sporadic cases of AD and the formation of neurofibrillary tangles should be explained.

View larger version (26K): [in this window] [in a new window]   FIG. 9. Hypothetical mechanism of conformational change for Aß-peptides. Three different proteases called -, ß-, and -secretases can cleave APP. When APP is concomitantly hydrolyzed by ß-secretase at the N terminus and by the -secretase within the membrane, the two main products, Aß(1–40) and Aß(1–42), migrate outside the cell and produce amyloid fibrils.

B. Parkinson’s disease (PD)
PD is the second most common, late-onset neurodegenerative disorder. The degeneration of dopaminergic neurons in the substantia nigra resulted in the typical characteristics of PD, which include muscular rigidity, postural instability, and resting tremor. The clinical hallmark of PD is the deposition in brain cells of intracytoplasmic inclusion bodies called Lewy bodies (LBs) (158, 159, 175).

Inherited forms of PD are caused by mutations in three genes, -synuclein, parkin, and ubiquitin carboxyl-terminal hydrolase L1(158, 175, 176). Autosomal-dominant PD results in mutations in the -synuclein gene, and autosomal-recessive PD is due to mutations in the parkin gene. Among other factors, apolipoprotein E 2/4 genotype and exposure to pesticides might be risk factors for sporadic PD (177, 178).

The -synuclein is a highly conserved 140-amino acid protein very abundant in the central nervous system, particularly in the presynaptic terminals (179, 180), but its physiological functions are largely unknown. This protein was originally identified as a precursor of the nonamyloid component of the senile plaques in AD (181) and is a major constituent of LBs and other disease-specific lesions such as the abnormally shaped neurites known as Lewy neurites (182, 183, 184). The -synuclein is also found in the intracellular inclusions of other neurodegenerative disorders, such as dementia with LB and the LB variant of AD commonly referred to as a "-synucleinopathies," and also in the filamentous glial and neuronal inclusions of multiple-system atrophy (175, 185).

It has been reported th