Abstract
In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Folding and assembly of proteins and their function depend on each other. Like in industrial production lines, in cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm (Bennett et al. 2005; Bukau et al. 2006; Dobson 2003; Ellgaard and Helenius 2003; Park et al. 2007; Ravid et al. 2006; Roth 2002; Sitia and Braakman 2003; Zhang and Kaufman 2006). Protein quality control is a basic cellular phenomenon through which aberrant proteins become eliminated. Aberrant proteins can occur as waste products at a certain rate during de novo synthesis, or are caused by cellular stress, or due to disease-causing mutations (Aridor and Hannan 2002; Gregersen et al. 2006; Kim and Arvan 1998; Kopito 2000; Lukacs et al. 1994; Petäjä-Repo et al. 2000; Schubert et al. 2000; Turner and Varshavsky 2000; Ward and Kopito 1994). Once recognized as non-native or incompletely assembled, those proteins will be removed and degraded by a process generally termed ERAD, for ER-associated degradation (Hirsch et al. 2004; Meusser et al. 2005).
For secretory and membrane proteins, the molecular machinery involved in the recognition, retention and dislocation of aberrant proteins has been identified to a certain detail (Carvalho et al. 2006; Denic et al. 2006; Hirsch et al. 2003; Ismail and Ng 2006; Katiyar et al. 2005; Li et al. 2006; Lilley and Ploegh 2004; Schuberth and Buchberger 2005; Tsai et al. 2002; Ye et al. 2003, 2004). The ensuing final step consists of polyubiquitination of aberrant proteins condemning them for degradation by proteasomes (Eisele et al. 2006; Hochstrasser 1996; Jarosch et al. 2002; McCracken and Brodsky 2005; Wolf and Hilt 2004; Zwickl et al. 2002). In addition to various chaperones aiding proteins to achieve their proper conformation, various machinery proteins are involved in the recognition and retention of aberrant proteins. For glycoproteins, the importance of specific oligosaccharidic structures generated initially by trimming glucosidase II and UDP-glucose:glycoprotein glucosyltransferase and later on by ER-mannosidase I has been recognized (Helenius and Aebi 2004; Parodi 2000; Roth 2002; Roth et al. 2002). Glycoproteins bearing monoglucosylated oligosaccharides will be bound by calnexin or calreticulin. If the aberrant glycoproteins are considered, binding to calnexin or calreticulin will protect them temporarily from degradation. The complete deglucosylation by glucosidase II will result in their exit from the calnexin/calreticulin cycle. Subsequent trimming of mannose residue(s) of the oligosaccharide B branch by ER-mannosidase I opens the gate to dislocation and degradation of aberrant proteins. The link between the calnexin/calreticulin cycle and the dislocation process is apparently provided by two lectin-like proteins: EDEM1 (yeast ortholog Htm1p/Mnl1p) (Hosokawa et al. 2001; Jakob et al. 2001; Kanehara et al. 2007; Nakatsukasa et al. 2001; Oda et al. 2003) and Yos9p (mammalian orthologues OS-9 and XTP3-B) (Bhamidipati et al. 2005; Buschhorn et al. 2004; Gauss et al. 2006; Kanehara et al. 2007; Kostova and Wolf 2005; Szathmary et al. 2005).
Depending on the type of protein and the location of the lesion, different ERAD dislocation pathways have been identified (Carvalho et al. 2006; Denic et al. 2006; Ismail and Ng 2006; Schuberth and Buchberger 2005). Aberrant luminal proteins and membrane proteins with a defect in their luminal domain undergo the ERAD-L pathway, which is defined by the E3 ubiquitin ligase Hrd1p complex. The Hrd1p complex consists of several proteins including Hrd3p, an E2 complex (Ubc7p and its membrane-anchoring factor Cue1p), the Cdc48p complex (AAA-ATPase Cdc48p or p97, the Ufd1 and Npl4 cofactors, and the Ubx2p membrane anchor), Der1p, Yos9p, Kar2p (BiP) and Usa1p. It should be noted that the actual function of some of those proteins in the complex remains to be established. Aberrant membrane proteins with lesions in their cytosolic domain enter the ERAD-C pathway organized by the E3 ubiquitin ligase Doa10p complex. This complex is comparably simple and consists in addition to Doa10p only of the E2 complex and the Cdc48p complex. The ERAD-M pathway is followed by membrane proteins with a lesion in their transmembrane domain and involves only Hrd1p and Hrd3p. These dislocation pathways were established for yeast cells but most probably will apply to higher eukaryotes as well because of the evolutionary conservation of the ERAD pathways. The Doa10p complex also operates in the polyubiquitination of aberrant cytosolic and nuclear proteins, in addition to the ERAD-C pathway (Neuber et al. 2005; Ravid et al. 2006; Swanson et al. 2001).
This review will focus on how immunocytochemical labeling and electron microscopic analysis have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.
Machinery proteins of the protein quality control reside beyond the ER
For the quality control of glycoprotein folding, glucosidase II (Gls II) and UDP-glucose:glycoprotein glucosyltransferase (GT) in connection with the calnexin/calreticulin cycle are of eminent importance (Helenius and Aebi 2004; Parodi 2000; Roth 2002). Gls II is a luminal glycoprotein, which exists in two isoforms (Pelletier et al. 2000; Ziak et al. 2001) and does not contain known ER retention signals of the C-terminal KDEL type, nor any hydrophobic region characteristic of transmembrane proteins (Flura et al. 1997; Trombetta et al. 1996). As depicted in Fig. 1a, Gls II acts second to glucosidase I by removing the two inner α1,3-linked glucose residues (Brada and Dubach 1984; Burns and Touster 1982). The presence of three or two glucose residues on oligosaccharides can be considered to represent a trimming glyco-code whereas one glucose residue represents a trimming as well as folding glyco-code (Fig. 1b) (Jakob et al. 1998b). The involvement of Gls II and of mono-glucosylated oligosaccharides generated by the enzyme in the protein quality control is well documented (Hammond et al. 1994; Hebert et al. 1995; Jakob et al. 1998a, b). By confocal immunofluorescence, Gls II not unexpectedly exhibited a pattern typically observed for the ER as shown in Fig. 2b (Roth et al. 2003; Zuber et al. 2001). By high-resolution immunoelectron microscopy, ER localization of Gls II could be definitely established (Lucocq et al. 1986; Zuber et al. 2000, 2001). In addition to the rough ER including the nuclear envelope and the transitional ER, the smooth ER was also positive for Gls II. However, with the superior resolution of electron microscopic immunogold labeling, Gls II was additionally found in tubulovesicular clusters between transitional ER and the cis Golgi apparatus. They represent pre-Golgi intermediates involved in antero- and retrograde transport of cargo (Appenzeller-Herzog and Hauri 2006; Bannykh and Balch 1997, 1998; Hammond and Glick 2000; Palade 1975; Saraste et al. 1987; Schweizer et al. 1988).
Mono-glucosylated oligosaccharides of glycoproteins are targeted by the calnexin/calreticulin cycle and after being deglucosylated by Gls II, will be targeted by GT if not correctly folded (Fig. 1b). GT apparently senses exposed patches of charged amino acids and reglucosylates the aberrant glycoproteins, which is followed by their re-entry in the calnexin/calreticulin cycle (Parodi et al. 1983; Sousa and Parodi 1995; Trombetta and Parodi 2003). When the subcellular distribution of GT was studied by confocal immunofluorescence, its labeling pattern (Fig. 2a) was alike that observed for Gls II (Fig. 2b; Zuber et al. 2001). By immunoelectron microscopy, GT was detectable in the rough ER including the nuclear envelope and the transitional ER as well as the smooth ER (Fig. 2d). Unlike Gls II, for which the labeling intensity over rough and smooth ER was equal, labeling intensity for GT over smooth ER was only 11% that of the rough ER. GT immunolabeling was also discovered in the pre-Golgi intermediates (Fig. 2d). Notably, the pre-Golgi intermediate immunolabeling for GT was approximately twice that of rough ER (Zuber et al. 2001). Double immunogold labeling for GT combined with the pre-Golgi intermediate marker ERGIC-53 and the COPII component sec23p (Hughes and Stephens 2008) proved the identity of the GT-labeled structures. Interestingly, specific immunogold labeling for calreticulin was also observed in the pre-Golgi intermediates (Zuber et al. 2000, 2001).
Together, these results provided new insight into the in situ subcellular organization of some key elements of the protein quality control machinery. Gls II, GT and calreticulin were not only present in the rough ER, as expected, but also in the smooth ER and unequivocally present in pre-Golgi intermediates. This pattern was found in different rat cell lines and tissues as well as Drosophila tissue and cell lines. The presence of three functionally closely associated proteins is a strong evidence for the involvement of pre-Golgi intermediates in protein quality control. Of course, immunolocalization provides no direct evidence for the functionality of the detected protein at a certain location. However, there is no reason to assume that Gls II, GT and calreticulin would be only functional in the ER. Studies in yeast have provided strong evidence that multiple, sequentially acting quality control checkpoints exist along the secretory pathway extending as far as to the Golgi apparatus (Arvan et al. 2002; Caldwell et al. 2001; Sayeed and Ng 2005; Taxis et al. 2002; Vashist et al. 2001; Vashist and Ng 2004; Younger and Chen 2006). In mammalian and insect cells, protein quality control is apparently not restricted to the ER, and the pre-Golgi intermediates appear to be involved in this fundamental cellular process as well. As will be discussed later, pre-Golgi intermediates represent not only a quality control checkpoint, but are also sites of accumulation of aberrant proteins.
The ERAD factor EDEM1 defines a novel vesicular ER exit pathway
As mentioned in the “Introduction”, an impressive body of molecular and functional data exists concerning the macromolecular assemblies involved in the various ERAD pathways. The current well-founded basic conception of ERAD in yeast and higher eukaryotes is that the aberrant proteins after being removed from folding cycles are dislocated to the cytosol and eventually degraded by the 26S proteasome, and that this occurs in the ER. It is not clear whether this is a randomly occurring event or a more structured affair. Recent studies on EDEM1 in mammalian cells have provided preliminary evidence for a high level of subcellular organization.
The discovery that the Man8 B isomer oligosaccharide was actively involved in ERAD-L (Fig. 1c) in yeast (Jakob et al. 1998a) and mammalian cells (Liu et al. 1999) paved the way to the identification of a lectin-like protein with sequence similarity to class I α1,2-mannosidases in yeast -Htm1p/Mnl1p- and mammalian cells -EDEM1- (Hosokawa et al. 2001; Jakob et al. 2001; Nakatsukasa et al. 2001). EDEM1 of mammalian cells is a soluble glycoprotein (Olivari et al. 2005; Zuber et al. 2007), which is regulated by the unfolded protein response (Hosokawa et al. 2001) and seems to connect the calnexin/calreticulin cycle to the dislocation process (Molinari et al. 2003; Oda et al. 2003). EDEM1 appears to exist in complex with the dislocation proteins Derlin-2 and -3, and the AAA ATPase p97 (Oda et al. 2006). It is not fully understood how EDEM1 interacts with aberrant proteins. However, there is evidence for interaction with ER-mannosidase I-trimmed oligosaccharides such as depicted in Fig. 1c (Hosokawa et al. 2003). Overexpression of EDEM1 has been shown to prevent formation of dimers of misfolded Null Hong Kong variant of alpha1-antitrypsin (Hosokawa et al. 2006). Notably, EDEM1 and ER-mannosidase I do not exist in complexes, which can be immunoprecipitated (Hosokawa et al. 2003).
Recently, the subcellular distribution of endogenous EDEM1 in various mammalian cell types was established with a specific anti-peptide antibody (Zuber et al. 2007). Unexpectedly, its immunofluorescence pattern did not correlate with that of calnexin and other ER marker proteins. Rather, an unusual pattern of well distributed punctate structures along with some localized finger-like structures was revealed (Fig. 3a–c). The distribution patterns of endogenous EDEM1 and that of overexpressed tagged EDEM1 were dramatically different: instead of a punctate, non-ER pattern, a typical reticular ER pattern plus punctate staining was observed (Zuber et al. 2007). This striking difference in subcellular distribution between endogenous EDEM1 and overexpressed tagged EDEM1 was confirmed by Optiprep density gradients. Endogenous EDEM1 was restricted to the densest fractions, whereas tagged EDEM1 showed the same broad distribution as observed for calnexin, sec61β, and Derlin-1 and -2 (Zuber et al. 2007). In this context, it needs to be emphasized that previous biochemical analyses of EDEM1 interaction with quality control machinery proteins and ERAD substrates were performed with cells transiently overexpressing tagged EDEM1 (Hosokawa et al. 2003; Molinari et al. 2003; Oda et al. 2003). The nature of the EDEM1 immunofluorescence pattern was clarified by immunogold labeling and serial section analysis (Fig. 3d–h). It revealed the presence of EDEM1-reactive buds along rough ER cisternae which apparently gave raise to ∼150 nm vesicles. These buds and vesicles were devoid of a COPII coat, formed outside the canonical ER exit sites of the transitional ER and were not found in the tubulovesicular clusters of pre-Golgi intermediates (Fig. 3i). Occasionally, EDEM1 luminal immunolabeling in limited parts of distended ER cisternae was observed, which accounted for approximately 11% of the immunogold labeling for GT. Double confocal immunofluorescence for endogenous EDEM1 in rat hepatoma clone 9 cells stably expressing the Null Hong Kong variant alpha 1-antitrypsin showed co-distribution of the two proteins (Zuber et al. 2007). Together, these data revealed the existence of a vesicular transport pathway out of the rough ER through which the ERAD factor EDEM1 and an ERAD substrate became sequestered from the early secretory pathway. Through this pathway potentially harmful aberrant luminal proteins can be removed. These findings also indicate that the Gls II and GT containing pre-Golgi intermediates appear to be not involved in the dislocation of an ERAD-L substrate.
Endomannosidase assigns glucose trimming function to the Golgi apparatus
It is generally assumed the glucose trimming occurs exclusively by Gls I and II and, therefore, is limited to the ER and pre-Golgi intermediates. However, under conditions of inhibition of trimming glucosidases, formation of mature oligosaccharides has been observed. This apparent paradox could be explained by the existence of an alternate glucose-trimming pathway by neutral endo-alpha-mannosidase (Lubas and Spiro 1987, 1988; Moore and Spiro 1990, 1992; Spiro 2000). Endomannosidase is currently the only known endoglycosidase. In contrast to the trimming Gls I and II, it cleaves internally between the glucose-substituted mannose and the remaining oligosaccharide (Fig. 4a). Its substrate specificity (Fig. 4a) is basically that of Gls I and II (Moore and Spiro 1990, 1992; Rabouille and Spiro 1992). However, unlike Gls I and II, ER-mannosidase I trimmed mono-glucosylated oligosaccharides are a substrate of endomannosidase. The resulting Man8–5 GlcNAc isomer A is the specific product of endomannosidase. It should be noted that this oligosaccharide is no more a substrate for reglucosylation by GT. Biochemically, activity for endomannosidase was found to be enriched in Golgi membranes (Lubas and Spiro 1987). By immunofluorescence (Dong et al. 2000; Zuber et al. 2000), endomannosidase exhibited a crescent-shaped perinuclear staining and fine punctate staining throughout the cytoplasm which partially overlapped with immunofluorescence for Gls Golgi mannosidase II (Fig. 4b–d). High-resolution immunoelectron microscopy demonstrated endomannosidase in the peripheral and Golgi-associated pre-Golgi intermediates as well as cis and medial cisternae of the Golgi apparatus (Fig. 4e) (Zuber et al. 2000). Trans cisternae of the Golgi apparatus and the trans Golgi network were unreactive. Quantification revealed ∼85% of the immunogold labeling for endomannosidase in the Golgi apparatus and ∼15% in pre-Golgi intermediates. Although, both endomannosidase and Gls II could be detected in pre-Golgi intermediates by double immunogold labeling, they labeled different elements of the vesiculotubular clusters (Fig. 4e). Thus, endomannosidase and Gls I and II exhibited non-overlapping subcellular distributions (Roth et al. 2003; Zuber et al. 2000). Functionally, the presence of endomannosidase in the ER would interfere with the action of glucosyltransferase by preventing the reglucosylation of misfolded glycoproteins. Together, these findings demonstrating a predominantly Golgi apparatus localization of endomannosidase strongly indicated that glucose trimming of N-linked oligosaccharides is not limited to the ER.
Since glucose trimming is indispensable for the synthesis of mature oligosaccharide side chains, deglucosylation by endomannosidase in the Golgi apparatus ensures that this important process is not blocked. Further biochemical and morphological analyses demonstrated that Golgi apparatus localized endomannosidase-processed oligosaccharides of alpha 1-antitrypsin irrespective of their folding state (Torossi et al. 2006). From the literature, it is well known that disease-causing misfolded glycoproteins to a certain extent might escape the protein quality control and become secreted (Cox 2001; Desnick et al. 2001). As a case in point, in humans suffering from alpha1-antitrypsin deficiency, the Z-variant of alpha1-antitrypsin not only becomes partially secreted, but also is active as serine protease inhibitor (Cabral et al. 2002; Teckman and Perlmutter 1996). As experimentally shown for the Z-variant of alpha1-antitrypsin (Torossi et al. 2006), endomannosidase provided a back-up mechanism for its de-glucosylation en route through the Golgi apparatus. Processing of its oligosaccharides to mature ones is apparently important for their proper trafficking and correct functioning.
Organelle changes due to intracellular accumulation of misfolded proteins
As a general rule, misfolded proteins become targeted by the protein quality control and following polyubiquitination will be degraded by proteasomes (Eisele et al. 2006; Hochstrasser 1996; Jarosch et al. 2002; McCracken and Brodsky 2005; Wolf and Hilt 2004; Zwickl et al. 2002). Depending on various factors such as the efficiency of the ubiquitin–proteasome system, the intracellular amounts of misfolded glycoproteins and their biophysical properties as well as interactions with other proteins, a whole spectrum of organelle changes can be observed in protein folding diseases.
For some protein folding diseases, no significant structural aberrations of the early secretory pathways could be observed. An example is Fabry’s disease, an inherited deficiency of lysosomal alpha-galactosidase A (alpha-Gal A), which causes progressive lysosomal glycosphingolipid accumulation (mainly globotriosylceramide Gb3) (Desnick et al. 2001). Disease-causing mutant alpha-Gal A could be shown by immunofluorescence to be retained in the ER where it existed in complexes with the chaperone BiP (Yam et al. 2005, 2006). From this, we concluded that recognition and ER-retention of the mutant alpha-Gal A by the protein quality control machinery constituted the mechanism leading to lysosomal deficiency in alpha-Gal A. Electron microscopic analysis of cultured fibroblast from Fabry patients harboring different mutations did not reveal significant changes of the morphology of the ER and the pre-Golgi intermediates. As expected, the fibroblasts contained numerous large lysosomes with characteristic multilamellar inclusions. Thus, the intracellularly retained mutant alpha-Gal A apparently became dislocated and was efficiently degraded by the ubiquitin–proteasome system. A similar situation was observed for a polytope membrane protein, aquaporin-2, whose folding mutants can cause renal diabetes insipidus (Canfield et al. 1997; Morello and Bichet 2001). The T126M mutant aquaporin-2 was found to be retained in the ER and efficiently degraded by proteasomes without causing ER dilatation (Hirano et al. 2003). ER retention and rapid proteasomal degradation are also hallmarks of the pulmonary form of alpha 1-antitrypsin deficiency (Lomas and Parfrey 2004; Sifers et al. 1988). However, other types of protein folding diseases have been shown to result in the distention of the ER cisternae. One example is the congenital hypothyroid goiter in which the mutant thyroglobulin is misfolded (Kim et al. 1996; Kim and Arvan 1998; Kim et al. 1998, 2000; Medeiros Neto et al. 1996). In disorders of procollagen biosynthesis, distended ER cisternae were also observed (Bogaert et al. 1992). Other examples are represented by LDL receptor class 2 mutants (Lehrman et al. 1987; Pathak et al. 1988).
There are protein folding diseases associated with both distended ER cisternae and enlarged pre-Golgi intermediates. A misssense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupting one of the two interchain disulfide bonds is associated with intracellular accumulation of misfolded proinsulin (Wang et al. 1999). This resulted in a significant increase of the volume density of dilated ER profiles and of the pre-Golgi intermediates (Fig. 5a, b) (Fan et al. 2007; Zuber et al. 2004). For the latter, a significant increase of the tubular elements was observed. Although the mutant proinsulin was degraded through proteasomes (Wang et al. 1999), its accumulation in the early secretory pathway caused an activation of the unfolded protein response and induced apoptosis (Oyadomari et al. 2002a; Oyadomari et al. 2002b). Other mutant proteins such as the cystic fibrosis (Kopito 1999; Riordan 1999) causing delta F508 variant of the chloride channel (Gilbert et al. 1998) and misfolded major histocompatibility complex class I protein (Hsu et al. 1991; Raposo et al. 1995), have been shown to accumulate in the expanded pre-Golgi intermediates.
Certain other misfolded proteins are accompanied by the formation of insoluble aggregates in the lumen of the ER, which physically precludes dislocation to the cytosol and exposure to proteasomes. The stress-induced so-called intracisternal granules in the pancreas of starved guinea pigs (Palade 1956), which are composed of aggregated proenzymes (Fig. 6a) (Geuze and Slot 1980; Pavelka and Roth 2005), form a classical example. For the liver-disease-causing alpha 1-antitrypsin Z variant, about 15% of the non-secreted mutant protein is polymerogenic and thus forms insoluble aggregates in the ER lumen, which cannot be degraded (Lomas et al. 1992, 2004) The Glu342Lys substitution of the Z-variant results in a spontaneous loop-sheet polymerization of the protein. In contrast to the above-mentioned protein folding diseases, which all have in common a loss-of-function pathogenesis, the Z-variant-caused alpha 1-antitrypsin deficiency seems to involve a pathologic gain-of-function pathogenesis (Hidvegi et al. 2005). The ER inclusions described above belong to the category of Russell bodies. Russell bodies represent subregions of the rough ER in which insoluble proteins accumulate (Fig. 6b) (Alanen et al. 1985; Kopito and Sitia 2000; Valetti et al. 1991). They are typically found in cells synthesizing mutant immunoglobulins (Alanen et al. 1985; Kopito and Sitia 2000; Mattioli et al. 2006; Valetti et al. 1991) but also in cells synthesizing, for instance, mutant myocilin. Mutations of the myocilin gene are associated with primary open-angle glaucoma (Tamm 2002). Mutant myocilins are secretion-incompetent and have been shown biochemically to form intracellular detergent-insoluble complexes (Gobeil et al. 2004; Jacobson et al. 2001; Sohn et al. 2002). In cultured cells transfected to express both mutant and wild-type myocilin, heteromeric, detergent–insoluble protein complexes were formed which were segregated into typical Russell bodies (Yam et al. 2007c). Thus, myocilin-caused open-angle glaucoma represents a protein folding disease. Its pathogenesis involves a pathological gain-of-function mechanism because of the interaction and complex formation of mutant with wild-type myocilin (Gobeil et al. 2004; Joe et al. 2003; Sohn et al. 2002; Yam et al. 2007c). As a consequence, unfolded protein response factors and pro-apoptotic factors were up-regulated and cells underwent apoptosis (Yam et al. 2007c) as detected by the appearance of lobulated nuclei and the TUNEL assay (Taatjes et al. 2008).
All the mutant proteins discussed above are luminal or membrane proteins. What happens to aberrant cytosolic and nuclear proteins? Same like ER proteins, one extreme situation is that they become efficiently degraded by cytosolic and nuclear proteasomes subsequent to polyubiquitination (Schubert et al. 2000; Turner and Varshavsky 2000). Thus, cytosolic and nuclear quality control in normal cells suppresses the formation of aggregates of aberrant proteins by degrading them. The other extreme is represented by the formation of cytosolic and nuclear inclusion bodies due to inefficient degradation of aberrant proteins by the ubiquitin–proteasome system. Cytosolic, non-membrane bounded inclusion bodies are generally called aggresomes (Corboy et al. 2005; Kopito and Sitia 2000). They consist of pericentriolar protein aggregates surrounded by a cage of intermediate (vimentin) filaments that are the most consistent component of aggresomes in addition to ubiquitin, proteasomes and molecular chaperones. Aggresomes can be induced experimentally by forced overexpression of aggregation-prone mutant proteins or by experimentally inhibiting proteasomes (Fig. 7) (Anton et al. 1999; Fan et al. 2007; Johnston et al. 1998; Wigley et al. 1999). On the other hand, it has been shown that protein aggregates can directly impair the function of the ubiquitin–proteasome system (Bence et al. 2001). The formation of aggresomes is a multi-step process, which depends on the intact microtubules. Aggresomes are formed by the coalescence of small protein aggregates transported from the cell’s periphery along microtubules to centrioles (Garcia-Mata et al. 1999; Johnston et al. 1998; Kawaguchi et al. 2003; Vidair et al. 1966; Wigley et al. 1999; Wojcik et al. 1996). In the nucleus, the inclusion bodies can be found in association with the promyelocytic leukemia oncogenic domains (Anton et al. 1999).
Inclusion bodies have been observed in association with a number of chronic neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis (Johnston et al. 2000; Kabashi and Durham 2006; Rubinsztein 2006; Selkoe 2003; Shults 2006; Soto 2003). Inclusion bodies named Lewy bodies are a morphological hallmark of Parkinson’s disease and other neurodegenerative disorders (McNaught et al. 2002b; Olanow et al. 2004; Shults 2006). Lewy bodies in the dopaminergic neurons resemble aggresomes and represent spherical bodies commonly composed of a core of granular material and peripheral radiating filaments. They contain a variety of proteins such as alpha-synuclein, the alpha-synuclein-binding protein synphilin-1 torsin A, neurofilaments, ubiquitin, proteasomal subunits and various heat shock proteins as well as ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, ubiquitin ligase enzymes and proteasome activators. Furthermore, they contain centrosome-related gamma-tubulin and pericentrin. Thus, it has been proposed that the formation of Lewy bodies represents an aggresome-like response in dopaminergic neurons (McNaught et al. 2002c). Considering the observed impairment of the ubiquitin–proteasome system in patients with Parkinson’s disease (McNaught et al., 2001, 2002a, 2006), it is assumed that Lewy bodies, by segregating increasing levels of aberrant and potentially cytotoxic proteins, might protect the neurons (Olanow et al. 2004). It should be stressed, however, that the mechanism leading to selective neuronal death in Parkinson’s disease is not fully understood and the role Lewy bodies might be playing needs to be studied further studies (Lindholm et al. 2006).
Synthetic chaperones for treatment of protein folding disease
The various protein folding diseases mentioned above can be classified based on the pathogenetic mechanism. Efficient proteasomal degradation of the misfolded protein is characteristic of the loss-of-function pathogenesis. This is the case in protein folding diseases such as cystic fibrosis, the lung form of alpha 1-antitrypsin deficiency, aquaporin 2-caused renal diabetes insipidus, Gaucher’s disease and Fabry’s disease. Here, the missing function of the degraded protein alone can be the cause of the clinical symptoms, or secondary effects due to substrate accumulation like in lysosomal storage diseases. Intracellular accumulation due to inefficient proteasomal degradation of misfolded proteins is representative of a pathological gain-of-function mechanism, which is combined with a loss of function. Intracellular accumulation of misfolded proteins associated or not with protein aggregation can result in the activation of the unfolded protein response leading to ER stress and apoptosis. A pathological gain-of-function mechanism can be also the cause of a dominant clinical course when the wild-type protein in complexes with the mutant protein is retained inside the cells. Examples for pathological gain-of-function pathogenesis-associated protein folding diseases are myocilin-caused open-angle glaucoma, familial hypophyseal diabetes insipidus, Parkinson’s disease and Huntington’s disease.
Many attempts have been made to at least partially correct the protein misfolding in order to overcome their trafficking defect and to alleviate ER stress. Among other approaches, small molecule synthetic chaperones have been used in order to shift the folding equilibrium of mutant proteins towards a more native state (Arakawa et al. 2006; Chaudhuri and Paul 2006; Cohen and Kelly 2003; Papp and Csermely 2006; Perlmutter 2002). Chemical chaperones include osmotically active substances such as DMSO, glycerol, polyols or deuterated water, and other compounds such as 4-phenylbutyric acid (Burrows et al. 2000; Lim et al. 2004; Liu et al. 2004; Pedemonte et al. 2005; Rubenstein and Zeitlin 2000; Tamarappoo and Verkman 1998; Tveten et al. 2007; Welch and Brown 1996). Other substances such as enzyme inhibitors (Fan et al. 1999; Matsuda et al. 2003; Sawkar et al. 2002) and receptor ligands or antagonists {Petäjä-Repo, 2002 #16211;Egan, 2002 #12283} have been shown to function as pharmacological chaperones.
Here, we have chosen two examples from our work to demonstrate how immunocytochemistry and microscopy in combination with biochemical analyses can be applied to demonstrate the functionality of a chemical and a pharmacological chaperone in rescuing the consequence of disease-causing protein misfolding. It has been mentioned above that open-angle glaucoma-causing mutant myocilin forms insoluble protein aggregates in the ER lumen (Russel bodies), which result in ER stress and apoptotic cell death (Yam et al. 2007b). Among the other tested chemical chaperones, treatment with sodium 4-phenylbutyrate significantly reduced the amount of intracellular detergent–insoluble myocilin aggregates and thereby the number of Russel bodies in the cells (Fig. 8a–c), diminished mutant myocilin interaction with calreticulin and restored the secretion of mutant myocilin. As a consequence, the ER stress was released and most interesting, the apoptosis rate was reduced close to levels observed in control cells expressing wild-type myocilin (Fig. 8d). Thus, sodium 4-phenylbutyrate exerts a beneficial effect by protecting the cells from the deleterious effects of mutant myocilin. Since sodium 4-phenylbutyrate is a tissue and cell-permeable molecule, it holds the potential for topical administration in the treatment of myocilin-caused primary open-angle glaucoma.
The second example concerns Fabry’s disease, a lysosomal storage disorder caused by a deficiency of alpha-Gal A activity in lysosomes that results in the accumulation of glycosphingolipid globotriosylceramide (Gb3). The lysosomal trafficking of mutant alpha Gal A is impaired because the enzyme is retained in the ER by the protein quality control (Yam et al. 2005). Others had demonstrated that the activity of mutant alpha-Gal A in vitro at neutral pH could be stabilized with the competitive enzyme inhibitor 1-deoxygalactonorijimycin (DGJ) (Fan et al. 1999). Treatment of cells expressing mutant alpha-Gal A with a non-inhibitory dose of DGJ enhanced the intracellular enzyme activity (Yam et al. 2005, 2006). In addition, we could demonstrate by immunofluorescence and quantitative immunogold labeling that the mutant enzyme was redistributed from the ER to lysosomes and that this trafficking was mannose 6-phosphate-dependent. The DGJ treatment resulted in release of mutant alpha-Gal A from the chaperone BiP and in its conversion in the mature lysosomal form. Double confocal immunofluorescence and immunogold labeling demonstrated that the lysosomal Gb3 storage was cleared and that the size of the lysosomes became normalized (Yam et al. 2005, 2006). Together, this demonstrated that DGJ exhibited a chaperone-like effect and induced the trafficking of ER-retained mutant alpha Gal A to lysosomes where the enzyme was catalytically active. Therefore, the pharmacological chaperone DGJ potentially offers a convenient and cost-efficient therapeutic alternative to enzyme replacement therapy.
References
Alanen A, Pira U, Lassila O, Roth J, Franklin R (1985) Mott cells are plasma cells defective in immunoglobulin secretion. Eur J Immunol 15:235–242
Anton L, Schubert U, Bacik I, Princiotta M, Wearsch P, Gibbs J, Day P, Realini C, Rechsteiner M, Bennink J, Yewdell J (1999) Intracellular location of proteasomal degradation of a viral antigen. J Cell Biol 146:113–124
Appenzeller-Herzog C, Hauri HP (2006) The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function. J Cell Sci 119:2173–2183
Arakawa T, Ejima D, Kita Y, Tsumoto K (2006) Small molecule pharmacological chaperones: from thermodynamic stabilization to pharmaceutical drugs. Biochimica Biophysic Acta Proteins Proteomics 1764:1677–1687
Aridor M, Hannan LA (2002) Traffic jams II: an update of diseases of intracellular transport. Traffic 3:781–790
Arvan P, Zhao X, RamosCastaneda J, Chang A (2002) Secretory pathway quality control operating in Golgi, plasmalemmal, and endosomal systems. Traffic 3:771–780
Bannykh SI, Balch WE (1997) Membrane dynamics at the endoplasmic reticulum–Golgi interface. J Cell Biol 138:1–4
Bannykh SI, Balch WE (1998) Selective transport of cargo between the endoplasmic reticulum and Golgi compartments. Histochem Cell Biol 109:463–475
Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin–proteasome system by protein aggregation. Science 292:1552–1555
Bennett EJ, Bence NF, Jayakumar R, Kopito RR (2005) Global impairment of the ubiquitin–proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell 17:351–365
Bhamidipati A, Denic V, Quan EM, Weissman JS (2005) Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol Cell 19:741–751
Bogaert R, Tiller GE, Weis MA, Gruber HE, Rimoin DL, Cohn DH, Eyre DR (1992) An amino acid substitution (Gly853→Glu) in the collagen alpha 1(II) chain produces hypochondrogenesis. J Biol Chem 267:22522–22526
Brada D, Dubach UC (1984) Isolation of a homogeneous glucosidase II from pig kidney microsomes. Eur J Biochem 141:149–156
Bukau B, Weissman J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125:443–451
Burns DM, Touster O (1982) Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis. J Biol Chem 257:9990–10000
Burrows JA, Willis LK, Perlmutter DH (2000) Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci USA 97:1796–1801
Buschhorn BA, Kostova Z, Medicherla B, Wolf DH (2004) A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins. FEBS Lett 577:422–426
Cabral CM, Liu Y, Moremen KW, Sifers RN (2002) Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs. Mol Biol Cell 13:2639–2650
Caldwell SR, Hill KJ, Cooper AA (2001) Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi. J Biol Chem 276:23296–23303
Canfield M, Tamarappoo B, Moses A, Verkman A, Holtzman E (1997) Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response. Hum Mol Genet 6:1865–1871
Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin–ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126:361–373
Chaudhuri TK, Paul S (2006) Protein-misfolding diseases and chaperone-based therapeutic approaches. FEBS J 273:1331–1349
Cohen FE, Kelly JW (2003) Therapeutic approaches to protein-misfolding diseases. Nature 426:905–909
Corboy MJ, Thomas PJ, Wigley WC (2005) Aggresome formation. Meth Mol Biol 301:305–327
Cox D (2001) α1-antitrypsin deficiency. In: Scriver C, Beaudet A, Sly W, Valle D (eds) The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp 5559–5578
Denic V, Quan EM, Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126:349–359
Desnick R, Ioannou Y, Eng C (2001) α-Galactosidase a deficiency: Fabry disease. In: Scriver C, Beaudet A, Sly W, Valle D (eds) The metabolic and molecular bases of inherited disease, vol III. McGraw-Hill, New York, pp 3733–3774
Dobson CM (2003) Protein folding and misfolding. Nature 426:884–890
Dong ZZ, Zuber C, Spiro MJ, Spiro RG, Roth J (2000) Immunohistochemical evaluation of endomannosidase distribution in rat tissues: evidence for cell type-specific expression. Histochem Cell Biol 114:461–467
Egan ME, GlocknerPagel J, Ambrose CA, Cahill PA, Pappoe L, Balamuth N, Cho E, Canny S, Wagner CA, Geibel J, Caplan MJ (2002) Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med 8:485–492
Eisele F, Braun B, Pfirrmann T, Wolf DH (2006) Mutants of the deubiquitinating enzyme Ubp14 decipher pathway diversity of ubiquitin–proteasome linked protein degradation. Biochem Biophys Res Commun 350:329–333
Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4:181–191
Fan JQ, Ishii S, Asano N, Suzuki Y (1999) Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat Med 5:112–115
Fan JY, Roth J, Zuber C (2007) Expression of mutant Ins2(C96Y) results in enhanced tubule formation causing enlargement of pre-Golgi intermediates of CHO cells. Histochem Cell Biol 128:161–173
Flura T, Brada D, Ziak M, Roth J (1997) Expression of a cDNA encoding the glucose trimming enzyme glucosidase II in CHO cells and molecular characterization of the enzyme deficiency in a mutant mouse lymphoma cell line. Glycobiology 7:617–624
Garcia-Mata R, Bebök Z, Sorscher EJ, Sztul ES (1999) Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. J Cell Biol 146:1239–1254
Gauss R, Jarosch E, Sommer T, Hirsch C (2006) A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery. Nat Cell Biol 8:849–854
Geuze HJ, Slot JW (1980) Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study. Eur J Cell Biol 21:93–100
Gilbert A, Jadot M, Leontieva E, Wattiaux D-C-S, Wattiaux R (1998) Delta F508 CFTR localizes in the endoplasmic reticulum–Golgi intermediate compartment in cystic fibrosis cells. Exp Cell Res 242:144–152
Gobeil S, Rodrigue MA, Moisan S, Nguyen TD, Polansky JR, Morissette J, Raymond V (2004) Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants. Invest Ophthalmol Vis Sci 45:3560–3567
Gregersen N, Bross P, Vang S, Christensen JH (2006) Protein misfolding and human disease. Annu Rev Gen Hum Genet 7:103–124
Hammond AT, Glick BS (2000) Dynamics of transitional endoplasmic reticulum sites in vertebrate cells. Mol Biol Cell 11:3013–3030
Hammond C, Braakman I, Helenius A (1994) Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci USA 91:913–917
Hebert DN, Foellmer B, Helenius A (1995) Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell 81:425–433
Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049
Hidvegi T, Schmidt BZ, Hale P, Perlmutter DH (2005) Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response. J Biol Chem 280:39002–39015
Hirano K, Zuber C, Roth J, Ziak M (2003) The proteasome is involved in the degradation of different aquaporin-2 mutants causing nephrogenic diabetes insipidus. Am J Pathol 163:111–120
Hirsch C, Blom D, Ploegh HL (2003) A role for N-glycanase in the cytosolic turnover of glycoproteins. EMBO J 22:1036–1046
Hirsch C, Jarosch E, Sommer T, Wolf DH (2004) Endoplasmic reticulum-associated protein degradation––one model fits all? Biochim Biophys Acta Mol Cell Res 1695:215–223
Hochstrasser M (1996) Ubiquitin-dependent protein degradation. Annu Rev Genet 30:405–439
Hosokawa N, Wada I, Hasegawa K, Yorihuzi T, Tremblay LO, Herscovics A, Nagata K (2001) A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation. EMBO Rep 2:415–422
Hosokawa N, Tremblay LO, You Z, Herscovics A, Wada I, Nagata K (2003) Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I. J Biol Chem 278:26287–26294
Hosokawa N, Wada I, Natsuka Y, Nagata K (2006) EDEM accelerates ERAD by preventing aberrant dimer formation of misfolded alpha 1-antitrypsin. Genes Cells 11:465–476
Hsu VW, Yuan LC, Nuchtern JG, Lippincott-Schwartz J, Hammerling GJ, Klausner RD (1991) A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules. Nature 352:441–444
Hughes H, Stephens D (2008) Assembly, organization, and function of the COPII coat. Histochem Cell Biol 127. doi:10.1007/s00418-007-0363-x
Ismail N, Ng DT (2006) Have you HRD? Understanding ERAD is DOAble! Cell 126:237–239
Jacobson N, Andrews M, Shepard A (2001) Nonsecretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshworl cells and in aqueous humor. Hum Mol Genet 10:117–125
Jakob CA, Burda P, Roth J, Aebi M (1998a) Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J Cell Biol 142:1223–1233
Jakob CA, Burda P, teHeesen S, Aebi M, Roth J (1998b) Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo. Glycobiology 8:155–164
Jakob CA, Bodmer D, Spirig U, Battig P, Marcil A, Dignard D, Bergeron JJ, Thomas DY, Aebi M (2001) Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast. EMBO Rep 2:423–430
Jarosch E, Taxis C, Volkwein C, Bordallo J, Finley D, Wolf DH, Sommer T (2002) Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat Cell Biol 4:134–139
Joe MK, Sohn S, Hur W, Moon Y, Choi YR, Kee C (2003) Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem Biophys Res Commun 312:592–600
Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883–1898
Johnston JA, Dalton MJ, Gurney ME, Kopito RR (2000) Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 97:12571–12576
Kabashi E, Durham HD (2006) Failure of protein quality control in amyotrophic lateral sclerosis. Biochim Biophys Acta 1762:1038–1050
Kanehara K, Kawaguchi S, Ng DT (2007) The EDEM and Yos9p families of lectin-like ERAD factors. Semin Cell Dev Biol. doi:10.1016/j.semcdb.2007.09.007
Katiyar S, Joshi S, Lennarz WJ (2005) The retrotranslocation protein derlin-1 binds Peptide: N-glycanase to the endoplasmic reticulum. Mol Biol Cell 16:4584–4594
Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A, Yao TP (2003) The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115:727–738
Kim PS, Arvan P (1998) Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones. Endocr Rev 19:173–202
Kim PS, Kwon OY, Arvan P (1996) An endoplasmic reticulum storage disease causing congenital goiter with hypothyroidism. J Cell Biol 133:517–527
Kim PS, Hossain SA, Park YN, Lee I, Yoo SE, Arvan P (1998) A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: a model of human endoplasmic reticulum storage diseases. Proc Natl Acad Sci USA 95:9909–9913
Kim PS, Ding M, Menon S, Jung CG, Cheng JM, Miyamoto T, Li BL, Agui T (2000) A missense mutation G2320R in the thyroglobulin gene causes nongoitrous congenital primary hypothyroidism in the WIC-rdw rat. Mol Endocrinol 14:1944–1953
Kopito RR (1999) Biosynthesis and degradation of CFTR. Physiol Rev 79:S167–173
Kopito R (2000) Conformation disease. Nat Cell Biol 2:E207–E209
Kopito RR, Sitia R (2000) Aggresomes and Russell bodies. Symptoms of cellular indigestion? EMBO Rep 1:225–231
Kostova Z, Wolf DH (2005) Importance of carbohydrate positioning in the recognition of mutated CPY for ER-associated degradation. J Cell Sci 118:1485–1492
Lehrman MA, Schneider WJ, Brown MS, Davis CG, Elhammer A, Russell DW, Goldstein JL (1987) The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum. J Biol Chem 262:401–410
Li GT, Zhao G, Zhou XK, Schindelin H, Lennarz WJ (2006) The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor. Proc Natl Acad Sci USA 103:8348–8353
Lilley BN, Ploegh HL (2004) A membrane protein required for dislocation of misfolded proteins from the ER. Nature 429:834–840
Lim M, McKenzie K, Floyd AD, Kwon E, Zeitlin PL (2004) Modulation of deltaF508 cystic fibrosis transmembrane regulator trafficking and function with 4-phenylbutyrate and flavonoids. Am J Respir Cell Mol Biol 31:351–357
Lindholm D, Wootz H, Korhonen L (2006) ER stress and neurodegenerative diseases. Cell Death Differ 13:385–392
Liu Y, Choudhury P, Cabral CM, Sifers RN (1999) Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome. J Biol Chem 274:5861–5867
Liu XL, Done SC, Yan K, Kilpelainen P, Pikkarainen T, Tryggvason K (2004) Defective trafficking of nephrin missense mutants rescued by a chemical chaperone. J Am Soc Nephrol 15:1731–1738
Lomas DA, Parfrey H (2004) Alpha1-antitrypsin deficiency. 4: molecular pathophysiology. Thorax 59:529–535
Lomas DA, Evans DL, Finch JT, Carrell RW (1992) The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 357:605–607
Lomas DA, Belorgey D, Mallya M, Onda M, Kinghorn KJ, Sharp LK, Phillips RL, Page R, Crowther DC, Miranda E (2004) Polymerisation underlies alpha1-antitrypsin deficiency, dementia and other serpinopathies. Front Biosci 9:2873–2891
Lubas WA, Spiro RG (1987) Golgi endo-alpha-d-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme. J Biol Chem 262:3775–3781
Lubas WA, Spiro RG (1988) Evaluation of the role of rat liver Golgi endo-alpha-d-mannosidase in processing N-linked oligosaccharides. J Biol Chem 263:3990–3998
Lucocq JM, Brada D, Roth J (1986) Immunolocalization of the oligosaccharide trimming enzyme glucosidase II. J Cell Biol 102:2137–2146
Lukacs GL, Mohamed A, Kartner N, Chang XB, Riordan JR, Grinstein S (1994) Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP. EMBO J 13:6076–6086
Matsuda J, Suzuki O, Oshima A, Yamamoto Y, Noguchi A, Takimoto K, Itoh M, Matsuzaki Y, Yasuda Y, Ogawa S et al (2003) Chemical chaperone therapy for brain pathology in GM1-gangliosidosis. Proc Natl Acad Sci USA 100:15912–15917
Mattioli L, Anelli T, Fagioli C, Tacchetti C, Sitia R, Valetti C (2006) ER storage diseases: a role for ERGIC-53 in controlling the formation and shape of Russell bodies. J Cell Sci 119:2532–2541
McCracken AA, Brodsky JL (2005) Recognition and delivery of ERAD substrates to the proteasome and alternative paths for cell survival. Curr Top Microbiol Immunol 300:17–40
McNaught KSP, Olanow CW, Halliwell B, Isacson O, Jenner P (2001) Failure of the ubiquitin–proteasome system in Parkinson’s disease. Nat Rev Neurosci 2:589–594
McNaught KS, Mytilineou C, JnoBaptiste R, Yabut J, Shashidharan P, Jenner P, Olanow CW (2002a) Impairment of the ubiquitin–proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures. J Neurochem 81:301–306
McNaught KS, Shashidharan P, Perl DP, Jenner P, Olanow CW (2002b) Aggresome-related biogenesis of Lewy bodies. Eur J Neurosci 16:2136–2148
McNaught KSP, Shashidharan P, Perl DP, Jenner P, Olanow CW (2002c) Aggresome-related biogenesis of Lewy bodies. Eur J Neurosci 16:2136–2148
McNaught KSP, Jackson T, JnoBaptiste R, Kapustin A, Olanow CW (2006) Proteasomal dysfunction in sporadic Parkinson’s disease. Neurology 66:S37–S49
Medeiros Neto G, Kim PS, Yoo SE, Vono J, Targovnik HM, Camargo R, Hossain SA, Arvan P (1996) Congenital hypothyroid goiter with deficient thyroglobulin––identification of an endoplasmic reticulum storage disease with induction of molecular chaperones. J Clin Invest 98:2838–2844
Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7:766–772
Molinari M, Calanca V, Galli C, Lucca P, Paganetti P (2003) Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science 299:1397–1400
Moore SE, Spiro RG (1990) Demonstration that Golgi endo-α-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins. J Biol Chem 265:13104–13112
Moore SE, Spiro RG (1992) Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells. J Biol Chem 267:8443–8451
Morello JP, Bichet DG (2001) Nephrogenic diabetes insipidus. Annu Rev Physiol 63:607–630
Nakatsukasa K, Nishikawa S, Hosokawa N, Nagata K, Endo T (2001) Mnl1p, an alpha-mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins. J Biol Chem 276:8635–8638
Neuber O, Jarosch E, Volkwein C, Walter J, Sommer T (2005) Ubx2 links the Cdc48 complex to ER-associated protein degradation. Nat Cell Biol 7:993–998
Oda Y, Hosokawa N, Wada I, Nagata K (2003) EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science 299:1394–1397
Oda Y, Okada T, Yoshida H, Kaufman RJ, Nagata K, Mori K (2006) Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol 172:383–393
Olanow CW, Perl DP, DeMartino GN, McNaught KS (2004) Lewy-body formation is an aggresome-related process: a hypothesis. Lancet Neurol 3:496–503
Olivari S, Galli C, Alanen H, Ruddock L, Molinari M (2005) A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation. J Biol Chem 280:2424–2428
Oyadomari S, Araki E, Mori M (2002a) Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis 7:335–345
Oyadomari S, Koizumi A, Takeda K, Gotoh T, Akira S, Araki E, Mori M (2002b) Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes. J Clin Invest 109:525–532
Palade G (1956) Intracisternal granules in the exocrine cells of the pancreas. J Biophys Biochem Cytol 2:417–422
Palade G (1975) Intracellular aspects of the process of protein biosynthesis. Science 189:347–358
Papp E, Csermely P (2006) Chemical chaperones: mechanisms of action and potential use. Handb Exp Pharmacol 172:405–416
Park SH, Bolender N, Eisele F, Kostova Z, Takeuchi J, Coffino P, Wolf DH (2007) The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin–proteasome system. Mol Biol Cell 18:153–165
Parodi AJ (2000) Protein glucosylation and its role in protein folding. Annu Rev Biochem 69:69–93
Parodi AJ, Mendelzon DH, Lederkremer GZ (1983) Transient glucosylation of protein-bound Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 in calf thyroid cells. A possible recognition signal in the processing of glycoproteins. J Biol Chem 258:8260–8265
Pathak RK, Merkle RK, Cummings RD, Goldstein JL, Brown MS, Anderson RGW (1988) Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex. J Cell Biol 106:1831–1841
Pavelka M, Roth J (2005) Functional ultrastructure. An Atlas of tissue biology and pathology. Springer, Vienna
Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS (2005) Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest 115:2564–2571
Pelletier MF, Marcil A, Sevigny G, Jakob CA, Tessier DC, Chevet E, Menard R, Bergeron JJ, Thomas DY (2000) The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo. Glycobiology 10:815–827
Perlmutter DH (2002) Chemical chaperones: a pharmacological strategy for disorders of protein folding and trafficking. Pediat Res 52:832–836
Petäjä-Repo U, Hogue M, Laperrière A, Walkers P, Bouvier M (2000) Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human δ opiod receptor. J Biol Chem 275:13727–13736
Petäjä-Repo UE, Hogue M, Bhalla S, Laperriere A, Morello JP, Bouvier M (2002) Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor maturation. EMBO J 21:1628–1637
Rabouille C, Spiro RG (1992) Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells. J Biol Chem 267:11573–11578
Raposo G, van S-HM, Leijendekker R, Geuze HJ, Ploegh HL (1995) Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER–Golgi intermediate compartment. J Cell Biol 131:1403–1419
Ravid T, Kreft SG, Hochstrasser M (2006) Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. Embo J 25:533–543
Riordan JR (1999) Cystic fibrosis as a disease of misprocessing of the cystic fibrosis transmembrane conductance regulator glycoprotein. Am J Hum Genet 64:1499–1504
Roth J (2002) Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem Rev 102:285–303
Roth J, Zuber C, Guhl B, Fan JY, Ziak M (2002) The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control. Histochem Cell Biol 117:159–169
Roth J, Ziak M, Zuber C (2003) The role of glucosidase II and endomannosidase in glucose trimming of asparagine-linked oligosaccharides. Biochimie 85:287–294
Rubinsztein DC (2006) The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443:780–786
Rubenstein RC, Zeitlin PL (2000) Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. Am J Physiol Cell Physiol 278:C259–267
Saraste J, Palade G, Farquhar M (1987) Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein. J Cell Biol 105:2021–2029
Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, Kelly JW (2002) Chemical chaperones increase the cellular activity of N370S beta-glucosidase: a therapeutic strategy for Gaucher disease. Proc Natl Acad Sci USA 99:15428–15433
Sayeed A, Ng DT (2005) Search and destroy: ER quality control and ER-associated protein degradation. Crit Rev Biochem Mol Biol 40:75–91
Schuberth C, Buchberger A (2005) Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation. Nat Cell Biol 7:999–1006
Schubert U, Anton LC, Gibbs J, Norbury CC, Yewdell JW, Bennink JR (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404:770–774
Schweizer A, Fransen JA, Bachi T, Ginsel L, Hauri HP (1988) Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J Cell Biol 107:1643–1653
Selkoe DJ (2003) Folding proteins in fatal ways. Nature 426:900–904
Shults CW (2006) Lewy bodies. Proc Natl Acad Sci USA 103:1661–1668
Sifers RN, Brashears-Macatee S, Kidd VJ, Muensch H, Woo SL (1988) A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum. J Biol Chem 263:7330–7335
Sitia R, Braakman I (2003) Quality control in the endoplasmic reticulum protein factory. Nature 426:891–894
Sohn S, Hur W, Joe MK, Kim JH, Lee ZW, Ha KS, Kee C (2002) Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities. Invest Ophthalmol Vis Sci 43:3680–3685
Soto C (2003) Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci 4:49–60
Sousa M, Parodi AJ (1995) The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. EMBO J 14:4196–4203
Spiro RG (2000) Glucose residues as key determinants in the biosynthesis and quality control of glycoproteins with N-linked oligosaccharides. J Biol Chem 275:35657–35660
Swanson R, Locher M, Hochstrasser M (2001) A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. Gen Dev 15:2660–2674
Szathmary R, Bielmann R, Nita-Lazar M, Burda P, Jakob CA (2005) Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19:765–775
Taatjes DJ,Sobel BE, Budd RC (2008) Morphological and cytochemical determination of cell death by apoptosis. Histochem Cell Biol. doi:10.1007/s00418-007-0356-9
Tamarappoo BK, Verkman AS (1998) Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones. J Clin Invest 101:2257–2267
Tamm E (2002) Myocilin and glaucoma: facts and ideas. Prog Retin Eye Res 21:395–428
Taxis C, Vogel F, Wolf DH (2002) ER–Golgi traffic is a prerequisite for efficient ER degradation. Mol Biol Cell 13:1806–1818
Teckman JH, Perlmutter DH (1996) The endoplasmic reticulum degradation pathway for mutant secretory proteins alpha 1-antitrypsin Z and S is distinct from that for an unassembled membrane protein. J Biol Chem 271:13215–13220
Torossi T, Fan JY, Sauter-Etter K, Roth J, Ziak M (2006) Endomannosidase processes oligosaccharides of alpha1-antitrypsin and its naturally occurring genetic variants in the Golgi apparatus. Cell Mol Life Sci 63:1923–1932
Trombetta ES, Parodi AJ (2003) Quality control and protein folding in the secretory pathway. Annu Rev Cell Dev Biol 19:649–676
Trombetta ES, Simons JF, Helenius A (1996) Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit. J Biol Chem 271:27509–27516
Tsai B, Ye YH, Rapoport TA (2002) Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3:246–255
Turner GC, Varshavsky A (2000) Detecting and measuring cotranslational protein degradation in vivo. Science 289:2117–2120
Tveten K, Holla OL, Ranheim T, Berge KE, Leren TP, Kulseth MA (2007) 4-Phenylbutyrate restores the functionality of a misfolded mutant low-density lipoprotein receptor. FEBS J 274:1881–1893
Valetti C, Grossi CE, Milstein C, Sitia R (1991) Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum. J Cell Biol 115:983–994
Vashist S, Ng DT (2004) Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J Cell Biol 165:41–52
Vashist S, Kim W, Belden WJ, Spear ED, Barlowe C, Ng DT (2001) Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J Cell Biol 155:355–368
Vidair C, Huang R, Doxsey S (1966) Heat shock causes protein aggregation and reduced protein solubility at the centrosome and other cytosolic locations. Int J Hyperther 12:681–695
Wang J, Takeuchi T, Tanaka S, Kubo SK, Kayo T, Lu D, Takata K, Koizumi A, Izumi T (1999) A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. J Clin Invest 103:27–37
Ward CL, Kopito RR (1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J Biol Chem 269:25710–25718
Welch WJ, Brown CR (1996) Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1:109–115
Wigley WC, Fabunmi RP, Lee MG, Marino CR, Muallem S, DeMartino GN, Thomas PJ (1999) Dynamic association of proteasomal machinery with the centrosome. J Cell Biol 145:481–490
Wojcik C, Schroeter D, Wilk S, Lamprecht J, Paweletz N (1996) Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome. Eur J Cell Biol 71:311–318
Wolf DH, Hilt W (2004) The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta Mol Cell Res 1695:19–31
Yam GH, Zuber C, Roth J (2005) A synthetic chaperone corrects the trafficking defect and disease phenotype in a protein misfolding disorder. FASEB J 19:12–18
Yam GH, Bosshard N, Zuber C, Steinmann B, Roth J (2006) Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants. Am J Physiol Cell Physiol 290:C1076–1082
Yam GH, Gaplovska-Kysela K, Zuber C, Roth J (2007a) Sodium 4-phenylbutyrate acts as a chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 48:1683–1690
Yam GH, Gaplovska-Kysela K, Zuber C, Roth J (2007b) Aggregated myocilin induces Russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma. Am J Pathol 170:100–109
Yam GHF, GaplovskaKysela K, Zuber C, Roth J (2007c) Aggregated myocilin induces Russell bodies and causes apoptosis––implications for the pathogenesis of myocilin-caused primary open-angle glaucoma. Am J Pathol 170:100–109
Ye Y, Meyer HH, Rapoport TA (2003) Function of the p97–Ufd1–Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J Cell Biol 162:71–84
Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429:841–847
Younger JM, Chen L (2006) Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 126(3):571–582
Zhang K, Kaufman RJ (2006) Protein folding in the endoplasmic reticulum and the unfolded protein response. Handb Exp Pharmacol :69–91
Ziak M, Meier M, Etter KS, Roth J (2001) Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells. Biochem Biophys Res Commun 280:363–367
Zuber C, Spiro MJ, Guhl B, Spiro RG, Roth J (2000) Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control. Mol Biol Cell 11:4227–4240
Zuber C, Fan JY, Guhl B, Parodi A, Fessler JH, Parker C, Roth J (2001) Immunolocalization of UDP-glucose: glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control. Proc Natl Acad Sci USA 98:10710–10715
Zuber C, Fan JY, Guhl B, Roth J (2004) Misfolded proinsulin accumulates in expanded pre-Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice. FASEB J 18:917–919
Zuber C, Cormier JH, Guhl B, Santimaria R, Hebert DN, Roth J (2007) EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites. Proc Natl Acad Sci USA 104:4407–4412
Zwickl P, Seemuller E, Kapelari B, Baumeister W (2002) The proteasome: a supramolecular assembly designed for controlled proteolysis. In: Richards FM, Eisenberg DS, Kuriyan J (eds) Protein folding in the cell, vol 59. Academic Press Inc., San Diego, pp 187–222
Acknowledgments
The work of the authors has received financial support by the Swiss National Science Foundation, the Wolfermann-Nägeli Foundation, Zurich, the Velux Foundation, Zurich, the Kamillo Eisner Foundation, Hergiswil, the Bonizzi-Theler Foundation, Zurich and the Canton of Zurich.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License ( https://creativecommons.org/licenses/by-nc/2.0 ), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
About this article
Cite this article
Roth, J., Yam, G.HF., Fan, J. et al. Protein quality control: the who’s who, the where’s and therapeutic escapes. Histochem Cell Biol 129, 163–177 (2008). https://doi.org/10.1007/s00418-007-0366-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-007-0366-7