SCIENTIFIC REVIEW
Endoplasmic Reticulum Quality Control and Pharmacological Chaperones
Incilay (SİNİCİ) LAY*°
Endoplasmic Reticulum Quality Control and Pharmacological Chaperones
Summary
In cells, the quality of newly synthesized proteins is monitored with endoplasmic reticulum quality control (ERQC) in regard to proper folding and correct assembly in the early secretory pathway. Sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion. Recently, ERQC has been mathematically modeled by breaking it into three subpathways; termed the Endoplasmic Reticulum Associated (ERA) Degradation (ERAD), ERA-Folding (ERAF) and ERA-Transport pathways. The major aim of ERQC is to assure that only properly folded and assembled proteins in the endoplasmic reticulum (ER) are transported for furher maturation and secretion. Disturbances in the ERQC have been linked with the pathophysiology of many human diseases; diabetes mellitus, alzheimer, parkinson, lysosomal storage diseases etc.
Enzyme enhancement therapy, which uses pharmacological chaperones, is an emerging therapeutic approach that has the potential to treat many genetic diseases, associated with a mutant protein having difficulty in folding and/or assembling into active oligomers in the ER. This review focuses on the ERQC and its pathways, unfolded protein response in the ER, pharmacological chaperones and their action mechanisms.
Key Words: Endoplasmic reticulum quality control (ERQC), unfolded protein response (UPR), pharmacological chaperones (PC), misfolding protein diseases, endoplasmic reticulum associated degradation (ERAD).
Received: 23.03.2010 Revised: 26.05.2010 Accepted: 02.06.2010
Endoplazmik Retikulum Kalite Kontrolü ve Farmakolojik Şaperonlar
ÖzetHücrelerde yeni sentezlenen proteinler, erken sekretuvar yolakta uygun katlanma ve altbirimlerin doğru birleşmesine ilişkin olarak endoplazmik retikulum kalite kontrolü (ERQC) ile kontrol edilirler. Ardışık kontrol noktaları, protein sekresyonunun verimine ve doğruluğuna izin verecek şekilde erken sekretuvar yolakta uygun şekilde dağılmışlardır. Yakın zamanda, ERQC üç altyolağa ayrılacak şekilde matematiksel olarak modellenmiştir; Endoplazmik Retikulum İlişkili (ERA) Yıkım, ERA- Katlanma (ERAF) ve ERA- Transport yolakları olarak adlandırılmışlardır. ERQC’nun asıl amacı, endoplazmik retikulum’da (ER) sadece uygun katlanmış ve birleşmiş proteinlerin ileri olgunlaşma ve sekresyon basamaklarına ilerleyebilmesini garanti etmesidir. ERQC’deki bozukluklar diabetes mellitus, alzheimer, parkinson, lizozomal depo hastalıkları gibi birçok hastalıkların patofizyolojisiyle ilişkilidir. Enzim artırma tedavisi, ER’da katlanma ve/veya aktif oligomerlere birleşmede zorluk yaşayan bir mutant protein ile ilişkili birçok genetik hastalıkları tedavi etmek üzere farmakolojik şaperonları kullanan yeni bir tedavi yaklaşımıdır.
Bu derleme, ERQC ve yolakları, ER’da katlanmamış protein cevabı, farmakolojik şaperonlar ve çalışma mekanizmalarına odaklanmıştır.
Anahtar kelimeler: Endoplazmik retikulum kalite kontrol (ERQC), katlanmamış protein cevabı (UPR), farmakolojik şaperonlar (PC), yanlış katlanmış protein hastalıkları, endoplazmik retickulum ilişkili yıkım (ERAD).
* Hacettepe University, Faculty of Medicine, Department of Biochemistry, 06100 Ankara, Turkey
° Corresponding author E-mail: isinici@hacettepe.edu.tr INTRODUCTION
Majority of the protein synthesis in the cell occurs on the cytosolic surface of the endoplasmic reticulum (ER). These soluble proteins pass cotranslationally
into the ER lumen (1,2). Over one third of the newly synthesized proteins are destined for secretion or for transfer to the lumen of other organelles. Proteins
fold, oligomerize, disulfide bonds are formed and N-linked oligosaccharides are added in the ER. The high flux of proteins through the ER, efficient folding, selection of misfolded and correctly folded proteins require a tight regulation. This is achieved by Endoplasmic Reticulum Quality Control (ERQC) (3).
The term ERQC was first announced by Hurtley and Helenius, indicating the processes of conformation- dependent molecular sorting of secretory proteins (4). It includes a high concentration of molecular chaperones that maintain polypeptide solubility, enzymes that posttranslationally modify proteins, and factors that directly assist in the folding of newly synthesized polypeptides.
ERQC system has been mathematically modeled by breaking it into three subpathways; termed the
– ERA-Folding (ERAF)
– Endoplasmic Reticulum Associated (ERA) Degradation (ERAD),
– ERA-Transport pathways (5).
Export efficiency of a particular cargo protein depends on the activity of the ERAF, ERAD and export systems, which in turn are influenced by the proteome expressed by the cell. ERAF and ERAD rely on chaperones and devoted resident enzymes.
ERQC serves different roles (Table 1). The major aim of ERQC is to assure that only properly folded and assembled proteins in the ER are transported for further maturation.
ERA- Folding (ERAF)
Upon cotranslational translocation, proteins enter the crowded environment of the ER lumen and
soon begin folding into more stable, lower energy, conformation(s) (6). In the folding process some proteins function as catalysts which help many proteins that are translocated into the ER to fold and assemble correctly. These proteins are called molecular chaperones. Unfolded proteins are recognized by ER associated-molecular chaperones with hydrophobic sites, presence of immature glycans, exposure of reactive thiols. ER contains heat shock proteins (Hsp) 60 and Hsp 70s (a special Hsp 70 called BiP exist in ER), calnexin/calreticulin system (in folding of glycoproteins), Hsp 90 and Hsp 100 as molecular chaperones (Table 2). Mitochondria contain their own Hsp 60s and Hsp 70s that are distinct from those that function in cytosol or ER. Each Hsp 60 and Hsp 70 proteins work with their own small set of associated proteins in helping other proteins to fold, such as Hsp 70 machinery requires Hsp 40 (2). The Hsp 70 machinery acts early in the life of many proteins before the proteins leave the ribosome. It also prevents soluble substrate aggregation prior to degradation (7).
In contrast, Hsp 60 machinery, which forms a large barrel-shaped structure, acts later in a protein’s life, after it has been fully synthesized. They form an
‘isolation chamber’ into which misfolded proteins are fed, preventing their aggregation and providing them with a favorable environment in which they can attempt to refold (2). Action mechanisms of these molecular chaperones have widely been studied (8- 12). They bind and release polypeptides in an ATP- dependent cycle (13). Activity of Hsp70s is greatly enhanced by the members of the other chaperone families, GrpE and DnaJ (Hsp40) (14-20). Hsp70s stabilize the extending proteins with polyglutamins and have roles in the translocation of proteins from membranes. Another chaperone family, the Hsp90, is abundant and composed of essential proteins that have been most intensely studied for their role in
Table 1. Functions of ERQC
Prepares an environment suitable for protein maturation Maintains homeostasis in the early secretory pathway Prevents aberrant, unfolded protein conformers
Inhibits aggregation of the terminally misfolded proteins
Provides the delivery of misfolded proteins to degradative pathways Reduces the proteotoxicity of misfolded proteins
Important in storing proteins for regulated secretion
kinase and steroid hormone receptor maturation (21).
Hsp 100s maintain the solubility of the aggregated proteins. Lectin-like chaperone proteins, calnexin and calreticulin play roles in glycoprotein folding, help unfolded glycoproteins for entering refolding processes. Calnexin is an ER membrane-bound chaperone protein. Calreticulin is a soluble ER resident protein. ERp57 is another chaperone that collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER (22).
Most of the proteins destined to the Golgi, lysosomes, plasma membrane or extracellular space are glycosylated in the ER. During protein translocation, a special lipid dolichol immediately transfers oligosaccharide chain (NAc-Gln2-Man9- Glc3) to NH2 group of asparagines in the protein.
The transfer is catalyzed in a single enzymatic step with oligosaccharyl transferase and called N-linked glycosylation. The diversity of oligosaccharides on mature glycoproteins results from the later modifications of the original precursor oligosaccharide. Three glucoses and one mannose are quickly removed from the oligosaccharides.
Fortunately, these oligosaccharides are used as tags to mark the state of protein folding. Calnexin and calreticulin bind to oligosaccharides on incompletely folded proteins and retain them in the ER, prevent them from irreversible aggregation.
They also promote the association of incompletely folded proteins with another ER chaperone. They recognize oligosaccharides that contain a single terminal glucose. Therefore, they bind proteins only after two of the three glucoses have been removed by ER glucosidases I and II. When the third glucose has been removed, the protein dissociates from its chaperone and can leave the ER to their final destinations. If the substrate is still misfolded, it is recognized by the glucosyl
transferase (GT), which preferentially recognizes unfolded or molten globule (23-25) species and reglucosylates the polypeptide. This permits lectin-like chaperones, calnexin and calreticulin rebinding and prevents the misfolded protein from leaving the ER (Figure 1).
How do calnexin and calreticulin distinguish properly folded proteins from incompletely folded ones? GT keeps adding a glucose to those oligosaccharides that have lost their last glucose. However, it adds glucose only to oligosaccharides that are attached to unfolded proteins. Thus, an unfolded protein undergoes continuous cycles of glucose trimming (by glucosidase) and glucose addition (by GT), maintaining an affinity for calnexin and calreticulin until it achieves its fully folded state.
Thus, molecular chaperones play an important role in the correct folding of newly synthesized proteins and protein quality control is intimately linked to the processes of folding.
ERA Degradation (ERAD)
Selecting proteins from the ER for degradation is a challenging process. Misfolded proteins or unassembled subunit proteins should be degraded but folding intermediates of newly made proteins and correctly folded proteins should not. Help in making this distinction comes from N-linked oligosaccharides, which serve as timers that measure how long a protein has spent its stay in the ER (22).
The pattern of N-linked glycosylation is used to indicate the extent time of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that fail to acquire their native conformation should be translocated back to the cytosol, where they are deglycosylated, ubiquitylated and degraded in proteosomes or removed from the Table 2. Molecular chaperones in ER
Hsp 60 (GroEL-GroES) Hsp 70 (DnaK) Hsp 40 (DnaJ) Hsp 90 (HtpG)
Hsp 100 (Clp ailesi) (unfoldase) Calnexin and calreticulin
ER through the macroautophagy-lysosome system (MALS) (26). ERAD was first described in the mid 1990s with studies on CFTR and MHC class I (27-29) proteins that the proteins fail to fold or assemble are eventually retrotranslocated across the ER membrane for degradation by cytosolic proteasomes. Another mechanism utilized by the cell to destroy misfolded proteins, especially those that aggregate in the secretory pathway or in the cytoplasm is MALS (30).
Misfolded proteins in the ER cannot cycle between calnexin/calreticulin and the GT perpetually, and thus a folding ‘timer’ aborts this cycle and induces the destruction of dead-end products. The timer is an ER mannosidase that trims mannose residues from the core glycan (31-33). This reduces the efficiency of calnexin/calreticulin rebinding and diverts substrates to a second, putative lectin known as ER degradation, enhancing α-mannosidase-like protein (EDEM) (34-36). Three EDEM homologues have been identified in mammals (37,38), and one homologue resides in the yeast ER (39,40). Although the protein was originally thought to simply recognize polypeptides with trimmed mannose residues, recent data suggest that EDEM may be a
mannosidase and exhibits chaperone-like activity (41,42). It was also originally thought that EDEM might recognize only modestly mannose-trimmed species, but again more recent data suggest that ERAD substrates may be trimmed more extensively (43). Terminally misfolded proteins become substrates for ER α-mannosidases and subsequently ligands for the EDEM, which then induces the retro translocation and degradation process of ERAD. Removal of a mannose residue from Man9 N-linked oligosaccharides by ER α-mannosidase I is a critical step for preventing misfolded proteins from reentering the ER folding pathway and serves as a signal for retrotranslocation and proteasomal degradation. Although an N-glycan seems to be a critical element for the ERAD of misfolded glycoproteins, it is less clear how nonglycosylated misfolded proteins are recognized and targeted for degradation. The importance of this pathway is underscored by the fact that treatment of cells with tunicamycin, an inhibitor for N-glycosylation, results in the accumulation of misfolded proteins in the ER and induction of unfolded protein response (UPR).
Once the decision of the degradation for soluble Figure 1. Folding of glycoproteins with calnexin in the ER (92)
but misfolded proteins in the ER is being made, they are retrotranslocated to the cytosol with the help of EDEM, ubiquitylated and degraded in the proteasomes. Nearly, all ERAD substrates are ubiquitinated prior to their degradation, a process that requires the sequential action of an E1 ubiquitin- activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases, each of whose catalytic sites are located in the cytoplasm (44). An undiscovered protein-conducting channel probably facilitates the retrotranslocation of substrates to the cytoplasm. Short-lived (functional) proteins in the cytosol are also degraded by the cytosolic ubiquitin-proteosome system. On the other hand, misfolded aggregated proteins from both the cytosol [e.g. Huntingtin (poly-Gln expansion mutants), α-Synuclein (Parkinson’s)] and the ER [e.g. α1- antitrypsin (Z mutation, Glu342Lys), Vasopressin (Cys67X mutation)]; intracellular organelles (mitochondria and peroxisomes); long lived cytosolic proteins (general/ structural) and other macromolecules endocytosed from the extracellular environment are degraded through the MALS, with which lysosomes are also associated (45,46). The two degradation systems interact and both systems can be up-regulated in response to ER-stress resulting from the UPR in the ER. MALS can be activated via ER-activated autophagy. Wei et al also found that chemical disruption of lysosomal homeostasis induces ER-stress in normal fibroblasts, suggesting the existence of a cross-talk between the lysosomes and the ER such as an abnormality in one organelle can adversely affect the other. Fusion between macroautophagic vesicles (autophagosomes) and lysosomes produces the autolysosome, and then the process of cargo-degradation and flux begins. The inhibition of autolysosome formation results in an increase in the number of autophagosomes; but this is due to a decrease in autophagic flux, not an increase in autophagy. Decreased flux results in a build up of undegraded, defective, polyubiquitinated proteins and most importantly, dysfunctional mitochondria, which in turn can initiate apoptosis (47,48). Mutant misfolded proteins in the ER are sufficient to inhibit autophagic flux and initiate apoptosis (49-51).
Blocking ERAD stimulates autophagy via signalling
pathways that involve elements of the UPR and Ire1-dependent JNK phosphorylation (52-54).
On the other hand, since EDEM is constitutively degraded by autophagic pathways, inhibition of the latter stimulates glycoprotein dislocation and degradation. An intriguing is that EDEM, targets some glycoproteins to autophagic degradation (55).
Wu et al show that ER Man I is rapidly degraded by nonproteasomal pathways in hepatoma cells (56).
Further studies are needed to determine the interac- tions between the two ERAD systems; ubiquitin-pro- teasome system and MALS.
ERA-Transport pathways
Proteins that fold and exit from the ER faster than the action of the mannosidase would, therefore, escape degradation and get targeted to their sites where their functions begin. The plasma membrane and membranes of golgi, lysosomes, endosomes are all form part of a membrane system that communicates with the ER by means of transport vesicles that transfer proteins. The pathway from the ER to the cell surface involves many sorting steps, which continually select membrane and soluble luminal proteins for packaging and transport in vesicles or organelle fragments that bud from the ER and Golgi (57-59). Golgi is a sorting and dispatching station for the products of ER. A large proportion of carbohydrates that Golgi makes are attached as oligosaccharide side chains to the proteins which come from the ER. These oligosaccharide groups serve as tags to direct proteins into vesicles, and then transport them to lysosomes or other destinations.
In the specialized ER exit sites, proteins are first packaged into COPII-coated transport vesicles with a selective process and bud to destine for Golgi.
Cargo proteins display exit (transport) signals on their surface that are recognized by complementary receptor proteins. Proteins without such exit signals can also get packaged in vesicles so that even proteins that normally function in the ER (ER resident proteins) can slowly leak out of the ER. Secretory proteins that are made in high concentrations may also leave the ER without the help of sorting receptors. The ERGIC53 protein seems to serve as a
receptor for packaging some secretory proteins into COPII-coated vesicles. After transport vesicles have budded from ER exit site, they begin to fuse with one another. Vesicular tubular clusters are formed with homotypic fusion and with the interactions between v-SNAREs, t-SNAREs and Rabs (60,61).
There are tethering factors which are a diverse group of peripherally associated membrane proteins that bridge newly formed transport vesicles to ensure correct docking and fusion (62). Evidence suggests the existence of three distinct functional classes: (1) oligomeric complexes that bind to SNAREs and typically act as Rab effectors (the DCGE group that includes Dsl1 complex, conserved oligomeric Golgi (COG) complex, Golgi-associated retrograde protein (GARP) complex, and exocyst), (2) oligomeric complexes that function as guanine nucleotide exchange factors (GEFs) for Rab proteins (transport protein particle TRAPP I and TRAPP II complexes and HOPS (HOPS is both, a GEF and a Rab effector));
and (3) coiled-coil tethers. Distinct tethers are localized to different compartments of the secretory and endocytic pathways. ER–Golgi traffic appears to require the activity of Dsl1 and COG (both are members of the DCGE class), TRAPP I (a GEF tether) and p115 (a coiled-coil tether). Together with SNAREs, Rabs, coats and GEFs, tethers ensure correct docking of vesicular membranes prior to vesicle fusion (62).
Membrane-bound Rabs are activated through a GDP/GTP exchange and like all GTPases, Rabs cycle between an inactive GDP-bound state and an active GTP-bound form that interacts with effector proteins (63). The reaction is facilitated by Rab GEFs (64). Three complexes (TRAPPI, TRAPPII and HOPS) have been shown to function as Rab GEFs. Their GEF activity results in membranes of vesicles and acceptor membranes that are loaded with specific GTP- bound Rab molecules, thus allowing the subsequent recruitment of other oligomeric complexes and coiled-coil tethers. Thus, the activity of GEF tethers precedes the function of other oligomeric and coiled- coil tethers which are Rab effectors. This implies that the positioning of GEF tethers on vesicular membranes is the most upstream event in tethering.
Mammalian Rab1 localizes to COPII vesicles and is
required for their fusion (65).
For SNAREs, only the correct vesicle containing the appropriate SNARE zip code will tether to and fuse with a given acceptor membrane. Many tethers have been shown to interact with SNAREs. Among them Dsl1, COG and GARP complexes, which contain evolutionary conserved sequence MUN domains, have been shown to actively interact with step specific SNARE complexes and are implicated in regulation of their assembly and/or stability (66,67).There are other tethers that have been shown to interact with coat components (Table 1). The GEF, TRAPPI and coiled-coil tethers bind Sec23/Sec24 components of the COPII coat and promote homotypic COPII vesicle tethering (68). Dsl1 and Cog complexes bind to COPI coat and are likely to promote the tethering of COPI vesicles back to the ER or Golgi, respectively.
Tethers might first interact with coats to ensure that a vesicle of either the anterograde or retrograde type is coming, followed by tether interactions with the SNARE machinery to provide a more stringent level of recognition. They bind coats as part of the recognition process and also trigger or facilitate uncoating of vesicles to ensure unhampered SNARE pairing prior to fusion.
Thus, binding and working together of the Rabs, SNAREs, other tethers and/or additional yet un- known membrane receptors mediate the destination of a vesicle.
The vesicular tubular clusters are short lived and generated continually and function as transport packages that bring proteins from the ER to the Golgi. While COPII-coated vesicles carry proteins to the Golgi, COPI-coated vesicles carry back the ER resident proteins that have escaped as well as proteins that participated in the ER budding reaction and are being returned to the ER. Resident ER proteins contain signals that bind directly to COPI coats. This signal (KKXX sequence) consists of two lysines, followed by any two other amino acids, at the extreme C-terminal end of the ER membrane protein. Soluble ER resident protein Hsp70 (BiP) has a different short retrieval signal at their C-terminal end that consists of a KDEL sequence. The affinity of KDEL
receptor for the KDEL sequence is different in the ER and Golgi. Receptors have high affinity for KDEL sequence in vesicular tubular clusters and Golgi for capturing ER resident proteins that are present there at low concentrations. They have a low affinity for KDEL sequence in the ER to unload its cargo protein in spite of high concentration of KDEL containing resident proteins in the ER: The affinity of KDEL receptor change depending on the compartment in which it resides is related to the different pH values established in different compartments. There is another suggested mechanism for retention of ER resident proteins independent of their KDEL signals that ER resident proteins bind to one another, thus forming complexes that are too big to enter transport vesicles. Because of high concentration of ER resident proteins in the ER, low affinity interactions would be sufficient to have the proteins tied up in such complexes (22).
Unfolded protein response (UPR)
Accumulation of misfolded proteins in the ER triggers an UPR. The ER has evolved the UPR pathways to cope with stress. The UPR induces profound changes in cellular metabolism including generalized translation attenuation. It includes transcription of genes encoding ER chaperones, proteins involved in retrotranslocation and protein degradation in the cytosol (ERAD) and many other proteins that help to increase the protein folding capacity of the ER.
UPR activates several pathways to handle the in- crease of unfolded proteins (69-71):
1. Misfolded proteins in the ER signal to nucleus to increase the transcription of genes enhancing ERAF (ER chaperones and folding enzymes) and ERAD: Misfolded proteins in the ER activate a transmembrane protein kinase in the ER, which causes the kinase to oligomerize and phosphorylate itself. The oligomerization and autophosphorylation activates an endoribonuclease domain in the cytosolic portion of the same molecule, which cleaves a specific, cytosolic RNA molecule at two positions, excising an intron. The separated exons joined by an RNA ligase, generating a spliced mRNA, which is
translated to produce an active gene regulatory protein. This protein activates the transcription of the genes encoding the proteins that mediate the UPR. Gene regulatory protein enters nucleus and activates genes encoding ER chaperones and ERAD pathway components. Thus, chaperones are made in ER, where they facilitate the folding of proteins.
2. UPR signal to decrease the protein translation and entry of proteins into the ER: Misfolded proteins also activate a second transmembrane kinase in the ER, which inhibits a translation initiation factor by phosphorylating it, and thereby reduces the production of new proteins through the cell.
One consequence of the reduction in protein translation is lowering the flux of proteins into the ER, thereby limiting the load of proteins that need to be folded there.
3. Transcription activation of genes encoding the proteins involved in UPR: Gene regulatory protein is initially synthesized as an integral ER membrane protein. Because it is covalently tethered to the membrane, it can not activate the transcription of genes in the nucleus. When misfolded proteins accumulate in the ER, the transmembrane protein is transported to the Golgi, where it encounters proteases that cleave off its cytosolic domain, which can now migrate to the nucleus and help activate the transcription of the genes encoding proteins involved in the UPR. ,
4. Selective degradation of certain mRNAs enco- ding secretory proteins (72).
The relative importance of each of these pathways differs in different cell types enabling each cell type to tailor the UPR to its particular needs. If these measures are not sufficient for eliminating misfolded proteins from the ER, apoptotic pathways are activated (73). The UPR serves a key role in ER stress.
Pharmacological Chaperones
Disturbances in the ERQC have been linked with the pathophysiology of many human diseases (diabetes mellitus, alzheimer, parkinson, lysosomal storage diseases etc.). Diseases can arise due to mutations
in cargo proteins as well as in folding, transport or signaling molecules. Clearly, different therapeutic strategies have to be envisaged in each of these (5,74). One approach is an enzyme enhancement therapy, which uses pharmacological chaperones (PC). Candidate diseases are those associated with a mutant protein that has difficulty in folding and⁄or assembling into active oligomers in the ER. Aim of the PC therapy is to use a small molecule to stabilize the native conformation of a mutant enzyme in the ER, allowing it to pass ERQC, avoiding ERAD, and be transported to the target site where it functions (74,75). They act like physiological chaperones in the ER as to assist correct folding of the mutant proteins, and thus increase the mutant enzyme activities at the target sites. Many lysosomal storage diseases are candidates for this therapeutic approach and have the additional advantage of requiring only 5–10%
of normal enzyme levels to reduce and⁄or prevent substrate accumulation, and thus the disease. To date successful PCs have also been competitive inhibitors of their target enzymes. A seeming paradox arises:
‘how can an inhibitor of an enzyme result in its increased activity?’ It is known that for most proteins, there is only a small thermodynamic difference favoring the native fold over some inactive folding intermediate, and many missense mutations decrease that difference by only a small amount (74,76).
Nevertheless, they can result in a dramatic reduction in the number of protein molecules, able to reach and⁄or retain their native fold, and pass ERQC (77).
Thus, the effect of destabilizing missense mutation is often reflected in a decrease in thermostability (78). Enzymologists have long known that enzymes in the presence of a substrate or inhibitor are protected from thermodenaturation. Putting these facts together suggests that the destabilizing effects of some mutations may be offset by the stabilizing effects of a bound substrate or inhibitor. Small molecule compounds known as PCs function either as agonists or competitive inhibitors and have been used to rescue several misfolded proteins from ERAD (3,79,80). However, the severity of the mutation’s effect on the translated protein’s ability to fold and exit the ER is important in identifying candidates for this emerging therapeutic approach (79). The missense mutations and juvenile, chronic types of
lysosomal storage diseases are more suitable for the treatment with PCs as they have some residual enzyme activities (79,81). Although the infantile/
acute type mutations are severe, only a small increase in enzyme activity is needed for a well improved life, as based on the critical threshold hypothesis of Sandhoff et al which indicates that, in vivo, as little as 10% of normal Hex A levels are needed to turn over all its substrates (79,82,83). How PCs act? First, the in vivo substrates of the mutant enzyme are not present in the ER (84) where the inhibitors function as PC.
PCs bind to mutant enzymes in the ER to assist their correct conformation. Second, the in vivo substrates of the mutant enzymes are highly concentrated, well above the Km of the mutant enzymes, in patients’
lysosomes (83). This large difference in endogenous substrate versus inhibitor concentrations in the two compartments (ER and lysosomes), would favor inhibitor binding by the enzyme in the ER (locking the mutant enzyme into a conformation compatible with exit from the ER), but favor inhibitor release once the enzyme-inhibitor complex reache the substrate-rich lysosome (the target site where it functions). Ideally, a PC should bind tighter at the neutral pH of the ER than at the acidic pH of the lysosome. PCs should be displaced by the high levels of stored substrates once the enzyme-PC complex reaches the lysosome (85).
PCs represent a very tractable therapeutic approach for a large proportion of genetic diseases where the point mutation does not totally prevent the formation of some functional enzyme⁄receptor. PC therapy has showed promising preclinical results in at least four enzyme deficiencies in lysosomal storage diseases¸
β-glucosidase, β-galactosidase, α-galactosidase A and β-hexosaminidase A and several Phase I and Phase II clinical trials are underway, e.g., http://
www.amicustherapeutics.com/pipeline/overview.
asp (81,86-88).
An advantage of this approach is that it can readily be implemented using the existing drug production infrastructure, unlike gene therapy approaches where no such infrastructure exists. Authors have screened the Maybridge library of 50 000 compounds using a real-time assay for non-carbohydrate-based β-hexosaminidase inhibitors in lysosomal storage
diseases and identified several that functioned as PCs in patient cells. Two of these inhibitors had derivatives that had been tested in humans for other purposes. These observations lead them to screen the NINDS library of 1040 Food and Drug Administration approved compounds for PCs.
Pyrimethamine, an antimalarial drug with well documented pharmacokinetics, was confirmed as a β-hexosaminidase PC and compared favorably with their best carbohydrate-based PC in patient cells with various mutant genotypes (74). Another example is phenyketonuria. Patients with mild phenylketonuria respond to high doses of the cofactor BH4, in an allele dependent fashion, resulting in increased activity of the mutated enzyme phenylalanine hydroxylase and concomitant decrease (> 30%) in serum phenylalanine levels (89). Although the effect was initially attributed to decreased binding affinity of the cofactor, BH4 has been shown to stabilize and increase the half-life of
mutated phenylalanine hydroxylase in a variety of expression systems (90,91). Thus, the response of phenylketonuria patients to BH4 supplementation is in fact, a bona fide example of a PC that was used to treat a genetic disease successfully. Chaperones are capable of crossing the BBB and may, therefore, have therapeutic potential for the CNS. Table 3 documents the advantages and disadvantages of the PC therapy, which is called as enzyme enhancement therapy.
CONCLUSION
As a conclusion, more than 30% of the newly synthesized proteins are normally degraded without ever reaching the target sites where they function. Thus, the ERQC, that composed of ERAD, ERAF and ERA-transport pathways, is essential for further secretion and functions of the proteins.
Table 3. Advantages and disadvantages of PCs therapy
Advantages Disadvantages
Small, hydrophobic molecules; easily accessible through membranes (important for blood brain barrier, central nervous system)
Mutation specific therapy (Severity of the mutation should be known)
Mostly, reversible competitive inhibitors of the enzymes (specific binding to their misfolded enzymes in the ER and are easily displaced with high concentrated substrates at the target sites)
Mostly, a little residual mutant enzyme activity is needed
At low concentrations they function as chaperones
(sufficient to use at small amounts) For clinical phase studies, a transgenic animal model with a little residual enzyme activity is needed (knockout animals have no enzyme activities and are not suitable) Usage of small amounts provide no or little side effects
At low concentrations no inhibitor effects is observed Oral intake
Act additively or even synergistically with the other therapeutic approaches (recombinant enzyme replacement therapy, substrate deprivation therapy, etc.)
Cost-effective and economic
Can be readily implemented using the existing drug production infrastructure (NINDS and Maybridge library)
The importance of each of these pathways differs in different cell types, enabling each cell type to tailor the UPR to its particular needs. The UPR has a key role in the ER stress in many diseases and it is important for recovery to overcome the ER stress by determining ERQC in those diseases. For defining the new therapeutic strategies and maybe the potential biomarkers in diseases, the ERQC pathways should be cleared with further studies in more details.
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