ITCH regulates degradation of mutant glucocerebrosidase: implications to Gaucher disease

Abstract

Inability to properly degrade unfolded or misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response. This is particularly important in cases of diseases in which the mutant proteins undergo ER-associated degradation (ERAD), as in Gaucher disease (GD). GD is a genetic, autosomal recessive disease that results from mutations in the GBA1 gene, encoding the lysosomal enzyme acid β-glucocerebrosidase (GCase). We have shown that mutant GCase variants undergo ERAD, the degree of which is a major determinant of disease severity. Most ERAD substrates undergo polyubiquitination and proteasomal degradation. Therefore, one expects that mutant GCase variants are substrates for several E3 ubiquitin ligases in different cells. We tested the possibility that ITCH, a known E3 ubiquitin ligase, with a pivotal role in proliferation and differentiation of the skin, recognizes mutant GCase variants and mediates their polyubiquitination and degradation. Our results strongly suggest that ITCH interacts with mutant GCase variants and mediates their lysine 48 polyubiquitination and degradation.

INTRODUCTION

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a multi-step process (1,2) that includes recognition of misfolded proteins, attempts to refold them, their retrotranslocation through the ER membrane in a sec61- or derlin-dependent manner, their polyubiquitination and proteasomal degradation (3). This process ensures maturation of folded proteins and elimination of unfolded molecules in the secretory pathway (4). However, it has an immense effect on the processing of mutant molecules which are misfolded (5). There is a growing number of diseases in which the causative mutant proteins undergo ERAD (6,7), one of which is Gaucher disease (GD).

GD is characterized by accumulation of glucosylceramides mainly in monocyte-derived cells (8,9). This results from decreased catabolism of the substrate, performed by the lysosomal acid β-glucocerebrosidase (GCase), in the presence of the activator, saposin C (10,11), which is encoded by the prosaposin gene.

Following their synthesis on ER-bound polyribosomes, GCase molecules enter the ER, where they undergo N-linked glycosylation on four asparagines (12,13). If correctly folded, they are transported through the ER to the Golgi. Endocytic transport of GCase from the Golgi to the lysosomes, at least in fibroblasts, depends on the membrane-associated lysosomal protein LIMP-2 (14,15).

Several laboratories documented ERAD of mutant GCase variants (6,16–18). We have shown that the level of ERAD of mutant GCase is one of the factors that determine the severity of GD. High levels of ERAD of a given mutant GCase variant lead to lower amount of folded GCase molecules that reach the lysosomes and are capable of some catabolic activity. This, in turn, leads to higher accumulation of the substrate, which seems to affect the entire cell and its normal metabolism (19).

An association has been documented between GD and Parkinson disease (PD). Namely, there is a significantly higher propensity to develop PD among GD patients and carriers of GD mutations (20–25). Since carriers of GD mutations do not accumulate glucosylceramide, we tested the possibility that ERAD of mutant GCase accounts for this association. We could show that parkin, an E3 ubiquitin ligase in dopaminergic cells, mutations in which cause PD, interacts with mutant GCase variants but not with the normal counterpart. Parkin, but not its kinase-dead mutants (T240R, P347L), mediates their lysine 48 polyubiquitination and degradation (26). Other groups documented that α-synuclein accumulates in brains of PD patients who were carriers of GD mutations as well as in several animal models for GD (27,28). Cullen et al. (29) showed that overexpression of mutant GCase variants in neuroblastoma M17 cells led to α-synuclein accumulation.

GD is a heterogeneous disease and therefore has been subdivided into type 1, with no primary neurological signs, and types 2 and 3, which involve a neurological disease, with an early onset in type 2 and a later manifestation in type 3 GD (30,31).

Type 2 GD, the most severe form of the disease, has an early onset and death at early childhood. At a neonate stage, it is also associated with a skin abnormality, namely ichthyosis (32). The ichthyotic skin derived from type 2 patients demonstrated an increased epidermal glucosylceramide-to-ceramide ratio (33), abnormal stratum corneum ultrastructure and an increase in transepidermal water loss (33). It has been speculated that this phenotype results from abnormal intracellular cohesion or induction of abnormal skin permeability barrier leading to hyperproliferation (34–36). Some of the skin pathology observed in type 2 GD patients was recapitulated in some knockout mouse models, and in a mouse model homozygous for the N370S mutation (37,38).

One of the key regulators in skin differentiation is the E3 ubiquitin ligase ITCH. ITCH, known also as atrophin-1-interacting protein 4, is a member of the homologous to E6-AP C terminus (HECT)-type family of E3 ubiquitin ligases (39). It consists of an N-terminal calcium-dependent phospholipid-binding C2-domain, a module containing four conserved double tryptophans (WW) and a C-terminal HECT domain (40). The HECT domain interacts with an E2 ubiquitin-conjugating enzyme, after which it is ubiquitinated in a conserved cysteine residue and transfers the ubiquitin molecule to the substrate (40). ITCH plays a key role as a modulator of skin differentiation by ubiquitinating and downregulating ΔNp63 and notch-encoding genes (41). ITCH controls the capacity of ΔNp63, a transcription factor, to direct expansion of the basal epidermal layer of the skin (42). The TP63 gene, encoding the ΔNp63 transcription factor, encodes several RNA species, which result from the use of two promoters and alternative splicing. ΔNp63 (N-terminal-deleted isoform) RNA is transcribed from a promoter that resides within intron 3. The ΔNp63 isoform accounts for most TP63-encoded proteins in epidermal tissue (41).

The ΔNp63 isoform is responsible for maintaining the proliferative potential of the basal layer, whereas TAp63, another TP63 isoform, is responsible for the stratification and maturation of the spinous layer (43). Reduction in ΔNp63 level during keratinocyte differentiation parallels accumulation of ITCH. Thus, by mediating ΔNp63 (and Notch) degradation, ITCH regulates specification of the spinous layer and its differentiation (44).

In a transcriptome analysis of samples from brain tissue, isolated from mouse homozygotes for the GD-associated V394L point mutation, combined with hypomorphic expression of the prosaposin transgene (4L/PS-NA) (45), several E3 ubiquitin ligases were significantly elevated, suggesting their participation in physiological processes that occur in the mutant animals. One of them was ITCH.

In the present study, we examined the interaction between ITCH and mutant GCase variants as well as ITCH-mediated polyubiquitination and proteasomal degradation of mutant GCase. Our results show that ITCH interacts with mutant GCase variants and mediates their lysine 48 polyubiquitination and proteasomal degradation.

RESULTS

ITCH interacts with mutant GCase

Ichthyotic skin represents disruption of the homeostasis between proliferation and differentiation in keratinocytes (46). One of the key modulators of this homeostasis is the E3 ubiquitin ligase ITCH. The ichthyotic skin in type 2 GD patients may reflect the occupation of ITCH in ubiquitination and degradation of mutant GCase, leading to accumulation of ΔNp63, and a breach of the homeostasis between proliferation and differentiation in the skin. Should this theory be correct, ITCH and mutant GCase interact with each other.

We first tested whether ITCH is expressed in skin fibroblasts, which are the skin cells available from GD patients. As evident from the results, presented in Figure 1A, ITCH is expressed in comparable levels in skin fibroblasts and HeLa cells, as well as in a keratinocyte-derived cell line (HaCat). Based on these results, coimmunoprecipitation analysis was performed on type 2 GD skin fibroblasts to test for possible interaction between ITCH and mutant GCase. ITCH-containing complexes were immunoprecipitated using anti-ITCH antibody, and the corresponding blot was reacted with anti-GCase antibodies. The results (Fig. 1B) showed that mutant GCase, but not normal GCase, interacts with ITCH, though mutant and normal fibroblasts contained comparable amount of ITCH (Fig. 1B).

Figure 1.

ITCH associates with mutant GCase. (A) Cells were lysed and subjected to western blot analysis and interaction with anti-ITCH antibody. (B) Cell lysates were prepared from human skin fibroblasts that derived from normal and GD patients. ITCH-containing complexes were subjected to western blot analysis and interaction with anti-GCase antibody. (C) HEK293 cells were transfected with myc-tagged ITCH and myc-his-GCase-expressing plasmids. Forty-eight hours later, GCase-containing complexes were isolated on nickel beads (Ni-NTA). Complexes were subjected to western blot analysis and interaction with anti-myc-antibody.

To further extend the experiment, we examined whether this interaction could be recapitulated between transfected ITCH and mutant GCase. To do that, myc-His-GCase-containing complexes were isolated from HEK293T cells, cotransfected with plasmids expressing mutant GCase and ITCH, on Ni-NTA beads. The corresponding blot was reacted with anti-myc antibody. As evident from the results (Fig. 1C), the transfected myc-His-mutant GCase interacted with myc-ITCH.

ITCH mediates degradation of mutant GCase

To determine whether the interaction between mutant GCase and ITCH leads to ITCH-mediated degradation of mutant GCase, we tested the effect of ITCH expression on GCase levels in transfected cells. HEK293T cells were transiently transfected with plasmids expressing myc-tagged WT or mutant ITCH (the C830A, inactive ITCH) (46) and myc-His-tagged WT, D409H or V394L mutant GCase. The V394L (c.1297G<A from the first ATG of the cDNA) mutation leads to severe type 1 GD with a pronounced visceral disease but no neurological signs (47–49). It has been listed as the fifth most common mutation among Ashkenazi Jewish patients, and it is rare among non-Jewish patients (50). The D409H (c.1343G<C) mutation was first described in Arab patients who suffered from oculomotor apraxia and a valvular heart disease (51,52), and is associated with early-onset variable diseases of the viscera and the CNS. The mutation is found mostly in patients with types 2 and 3 GD (48,53).

The results, presented in Figure 2, showed that increasing amounts of WT ITCH reduced the amount of mutant GCase in a dose-dependent manner, whereas the C830A ITCH mutant failed to do so and even stabilized mutant GCase variants (Fig. 2C–F). This stabilization reflects, most probably, abortive interaction between mutant ITCH and mutant GCase. However, this interaction did not lead to ubiquitination and degradation of the latter, but rather to its protection from the activity of other cellular E3 ubiquitin ligases. Nevertheless, WT ITCH did not affect the amount of WT GCase (Fig. 2A and B). These results indicate that WT ITCH is involved in the recognition of mutant, but not normal, GCase variants.

Figure 2.

ITCH mediates degradation of mutant GCase. HEK293T cells were cotransfected with WT (A), V394L (C) or D409H (E) myc-His-tagged Gcase, and the indicated amounts of WT or mutant (C830A) myc-tagged ITCH. Forty-eight hours later, cell lysates, containing the same amount of protein, were subjected to western blot analysis and the blot was reacted with anti-myc antibody and anti-actin antibody, as a protein-loading marker. (B, D and F). To quantify the results, the blots were scanned and GCase intensity at each lane was divided by that of actin. The value obtained for any variant GCase in the absence of ITCH was considered 1. The results represent the mean ± SEM (error bars) of three independent experiments.

To confirm the results, showing an effect of ITCH on the stability of mutant GCase variants, cycloheximide (CHX) chase was performed. CHX blocks de novo synthesis of proteins and, thus, allows uncovering their stability (54). HEK293T cells were transfected with plasmids expressing myc-His-tagged WT or mutant GCase and myc-tagged ITCH. We tested the effect of ITCH on the stability of several mutations: N370S, D409H and the V394L. The N370S (c.1226A<G) mutation is a mild mutation, prevalent among Ashkenazi Jews, leading to type 1 GD (55). Cell lysates were prepared after different times of incubation with CHX and analyzed using western blotting. The results (Fig. 3) showed that, whereas the stability of WT GCase was very slightly affected by the presence of ITCH (Fig. 3A and B), mutant GCase forms were less stable in the presence of ITCH than in its absence (Fig. 3C–H), indicating that ITCH mediates degradation of mutant GCase.

Figure 3.

ITCH affects steady-state amounts of mutant GCase. HEK293 cells were cotransfected with myc-tagged WT ITCH and myc-his-tagged WT GCase (A), V394L GCase (C), D409H GCase (E) and N370S GCase (G)-expressing vectors. Twenty-four hours later, cells were incubated with CHX (100 μg/ml), and at the indicated time points, cells were lysed and subjected to western blot analysis. The blots were reacted with anti-myc antibody and either anti-ERK or anti-actin antibody, as a loading control. To quantify the results, the blots were scanned, and GCase intensity at each lane was divided by that of actin. The value obtained for any GCase variant in the absence of chase (time 0) was considered 1. The results [(B) WT GCase, (D) V394L GCase, (F) D409H GCase, (H) N370S GCase] represent the mean ± SEM (error bars) of three independent experiments, as percentage of the protein level at time 0, which was considered 1.

ITCH mediates lysine 48 polyubiquitination of mutant GCase variants

Ubiquitin undergoes polyubiquitination. Since ubiquitin contains seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) (56), they can all undergo polyubiquitination (57). Lysine 48 and lysine 63 are the most characterized polyubiquitination forms (58). Lysine 48-linked polyubiquitin chains represent a signal for proteasomal degradation (59). Lysine 63-linked polyubiquitin chains are known to signal in four pathways: DNA damage, the inflammatory response, protein trafficking and ribosomal synthesis (57). Lysine 29 polyubiquitination has been linked to lysosomal targeting (60). The fact that ITCH interacted with, and reduced the stability of, mutant GCase variants, led us to test whether ITCH mediates their polyubiquitination, thus leading to their proteasomal degradation. ITCH has been shown to mediate different forms of ubiquitination. Lysine 29 polyubiquitination of Deltex, the mammalian homolog of the Drosophila Notch ligand, delta, was reported (60), as well as lysine 48 polyubiquitinations of Notch and ΔNp63 (61). ITCH undergoes self-polyubiquitination, mediated by lysine 63 (62).

To study whether ITCH mediates polyubiquitination of mutant GCase variants, HEK293T cells were transiently transfected, in the presence of MG132, with myc-His tagged WT or mutant GCase variants, HA-tagged WT or K48R/K63R mutant ubiquitin, and with or without myc-tagged WT or C830A mutant ITCH. GCase-containing complexes were isolated from cell lysates with nickel beads and subjected to western blot analysis. The blots were reacted with anti-myc antibody to detect GCase and ITCH and with anti-HA antibody to follow polyubiquitination of GCase. As shown in Figure 4, in the absence of MG132, the steady-state levels of mutant GCase were reduced, indicating that it underwent proteasomal degradation. The results also showed that the D409H and the N370S mutant GCase variants underwent significant polyubiquitination in the presence of MG132, WT ITCH and WT-ubiquitin (Fig. 4A and C). In the absence of ITCH or the presence of its mutant form, this polyubiquitination was significantly reduced, indicating the importance of functional ITCH for polyubiquitination of GCase.

Figure 4.

ITCH mediates lysine 48-linked polyubiquitination of mutant GCase. HEK293 cells were cotransfected with plasmids expressing WT, D409H (A) or N370S (C) mutant myc-his-tagged GCase variants, WT or mutant (C830A) myc-tagged ITCH and HA-tagged WT/K48R/K63R ubiquitin. Thirty-six hours later, after 18 h of treatment with 25 μm MG132, cells were lysed under denaturing conditions and incubated with nickel beads. Complexes were subjected to western blot analysis, and the corresponding blots were reacted with either anti-HA or anti-myc antibodies. (B and D) An amount of 2.5% of total cell lysates were subjected to western blot analysis and reacted with anti-HA and anti-myc antibodies.

Ubiquitin, mutated at its lysine 48 (K48R ubiquitin), failed to mediate polyubiquitination of mutant GCase variants, whereas lysine 63 mutant ubiquitin (K63R ubiquitin) did not affect the polyubiquitination pattern of mutant GCase, implying that mutant GCase undergoes lysine 48-mediated polyubiquitination in the presence of WT ITCH. WT GCase showed lower level of interaction with ITCH and it was hardly ubiquitinated, which support the assumption that ITCH interacts with and mediates lysine 48 polyubiquitination of mutant GCase variants.

Competition between mutant GCase and a known ITCH substrate

We assume that ITCH-mediated polyubiquitination and degradation of mutant GCase lead to ‘negligence’ of its natural substrates. If this is so, natural substrates of ITCH, like ΔNp63 (44), should be elevated in the presence of mutant GCase. ΔNp63 controls the proliferative potential of skin keratinocytes, and its absence leads to accelerated aging (43). ITCH plays a major role in controlling ΔNp63 levels and, therefore, has a significant impact on the development of the skin (63).

HEK293T cells were cotransfected with increasing amounts of myc-His-tagged WT or mutant GCase, and equal amounts of GFP-ΔNp63. Cell lysates were prepared and analyzed by western blotting. The results, presented in Figure 5, show that with elevated amounts of mutant GCase in the cells, the level of ΔNp63 increased by 150%, compared with elevated amounts of WT GCase, which caused only 50% increase on ΔNp63 level. These results strongly indicate that mutant GCase and ΔNp63 compete for ITCH as their E3 ubiquitin ligase and imply that the resulting accumulation of ΔNp63 in the cells contributes to the skin abnormalities seen in type 2 GD patients.

Figure 5.

Increasing amounts of mutant GCase elevate ΔNp63 levels in the cells. HEK293 cells were cotransfected with increasing amounts of either WT or D409H mutant myc-his-tagged GCase, and equal amounts of GFP-tagged ΔNp63. Forty-eight hours later, cell lysates containing the same amount of protein were subjected to western blot analysis and the blot was reacted with anti-myc, anti-GFP antibodies and anti-ERK antibodies as a protein-loading marker. To quantify the results, the blots were scanned and ΔNp63 intensity was divided by that of ERK. The value obtained for ΔNp63 in the absence of a GCase variant (WT GCase or D409H GCase) was considered 1. The results represent the mean ± SEM (error bars) of three independent experiments.

Elevated levels of ITCH in type-2-derived fibroblasts

The fact that ITCH mediates polyubiquitination and proteasomal degradation of mutant GCase variants, along with the fact that ITCH was elevated in a transcriptome analysis of samples from mouse homozygotes for the V394L point mutation combined with hypomorphic expression of the prosaposin transgene (4L/PS-NA) (45), led us to test whether there was an elevation in Itch mRNA levels in GD-derived skin fibroblasts. To this end, mRNA was extracted from GD-derived or normal human fibroblasts, and quantitative real-time PCR was performed, using Itch-specific primers. The results, presented in Figure 6A, indicated that while in fibroblasts that derived from type 1 or type 3 GD patients there was a small increase in Itch mRNA level (<2-fold), in type-2-derived GD fibroblasts there was at least 3-fold increase in Itch mRNA. A parallel increase in ITCH protein level was observed (Fig. 6B and C), with the highest rise in cells that derived from type 2 GD patients.

Figure 6.

Elevation of ITCH expression in GD-derived fibroblasts. (A) mRNA was isolated from normal or GD-derived skin fibroblasts, and subjected to quantitative real-time PCR analysis, using specific primers for Itch transcript. The amount of mRNA was evaluated by the C_t value and is presented as fold of increase over that detected in normal fibroblasts. (B) Protein extracts were prepared and subjected to western blot analysis and interaction with anti-ITCH antibody. (C) To quantify the results, the blots were scanned and ITCH intensity was divided by that of actin values. Values represent the mean ± SEM (error bars) of three independent experiments (**P < 0.01, *P < 0.05). The double dagger indicates that the patients are brothers carrying the same mutations but presenting different clinical manifestations described in (81). The hash indicates severely affected brother.

DISCUSSION

The results presented in this work strongly suggest that the E3 ubiquitin ligase ITCH interacts with and mediates polyubiquitination and degradation of mutant GCase. More specifically, we show that there is an interaction between endogenous ITCH and mutant GCase. More so, ITCH, but not its C830A mutant, mediates lysine 48 polyubiquitination and proteasomal degradation of mutant GCase variants. To show the generality of the interaction between ITCH and mutant GCase, we chose three mutant GCase variants: (i) N370S, associated with type 1 GD (55); (ii) V394L (64), associated in compound heterozygosity with type 2 and 3 GD (65,66) and (iii) D409H, associated in homozygosity with neuronopathic, type 3 GD (52), and in heterozygosity with type 2 GD (67). The D409H mutant variant undergoes extensive ERAD, whereas N370S presents variable levels of low ERAD (19). These differences are not very obvious in transfection experiments (68,69), due, most probably, to overexpression and overwhelming of the ER quality control machinery.

We assume that ITCH interacts with all mutant GCase variants; however, only the interaction between ITCH and severe mutant GCase variants contributes to the development of ichthyosis in neonate type 2 GD patients by violating the delicate balance between skin differentiation and proliferation in the patients' cells. Ichthyosis is a heterogeneous group of genetic disorders of cornification, whose common characteristics are variable and include generalized scaling of the skin (70). The term ‘ichthyosis’ was first used by Robert Willan in 1808 to denote generalized disorders of cornification and derives from ‘ichthys’, the Greek definition for ‘fish’ (71).

The skin comprises three major elements: epidermis, dermis and hypodermis, from surface to the deepest layer. The epidermis has evolved to provide a physical and permeability barrier to the body (72). This barrier continuously regenerates by terminally differentiating keratinocytes. The differentiation process, termed ‘cornification’ (keratinization), involves movement of keratinocytes from the proliferative basal cell layer, through the spinous and granular layers, to the cornified layer that harbors flattened, dead cell remnants. Maintaining the integrity of the skin depends on cell proliferation and differentiation (73). These two processes are controlled by a large number of regulators, one of which is the E3 ubiquitin ligase ITCH (44).

ITCH modulates epidermal keratinocyte differentiation by controlling the capacity of ΔNp63 and notch (39,41,74). ΔNp63 and ITCH are expressed in opposing gradients in the human epidermis. Thus, in the basal layer, where ITCH levels are the lowest, the level of ΔNp63 is high, and it directs proliferation of cells. In the granular layer, where ITCH levels are highest, there is ITCH-mediated degradation of ΔNp63 and, therefore, low ΔNp63 levels. This leads to arrest in cell proliferation and to differentiation (44,75) (Fig. 7). In the absence of ITCH (itchy mutant), the expression of ΔNp63 changes such that there is hyperproliferation with less differentiation (76). This resembles the phenotype observed in type 2 GD.

Figure 7.

A model describing the involvement of mutant GCase in abnormal skin differentiation. (A) Under normal conditions, correctly folded GCase is transported from the ER to the lysosomes, where it catalyzes the hydrolysis of its substrate GlcCer. ITCH mediates polyubiquitination and proteasomal degradation of its natural substrate ITCH. (B) Mutant GCase is recognized as misfolded in the ER and is retrotranslocated to the cytoplasm, where it undergoes ITCH-mediated polyubiquitination and proteasomal degradation. ITCH, occupied by GCase, ‘abandons’ its natural substrate ΔNp63, leading to its accumulation in the skin. This causes abnormal proliferation of the spinous layer and aberrant skin development. Part of the illustration was modified from Melino et al. (44).

A very recent publication (77) described a reconstructed human epidermal keratinization model, which was employed as a tool to characterize ceramide metabolism in the stratum corneum. The authors found that addition of 3 mm conduritol-β-epoxide (a non-competitive inhibitor of GCase), during 7 days, reduced ceramide levels in this model in parallel to a remarkable elevation of glucosylceramide. However, this substrate accumulation was not accompanied by any structural changes in the epidermis (77). These results imply that accumulation of glucosylceramide is not the sole cause of structural changes in the epidermis. Based on our results, showing occupation of ITCH in ubiquitination and degradation of mutant GCase, we suggest that this occupation leads to less ITCH-mediated degradation of ΔNp63 and thus, to hyperproliferation with less differentiation, manifesting itself as ichthyotic skin.

Considering our findings, we propose a mechanism (Fig. 7) in which the interaction between ITCH and mutant GCase contributes to the development of skin abnormalities in type 2 GD neonates. Mutant GCase variants, carrying severe mutations, are recognized as misfolded in the ER and undergo retrotranslocation to the cytoplasm. In the cytoplasm, they are recognized by ITCH, which mediates their lysine 48 polyubiquitination and proteasomal degradation. This occupation of ITCH molecules decreases its pool, available for ΔNp63, thus leading to elevation of the latter, which causes hyperproliferation and contributes to the abnormalities in the skin of type 2 GD neonates.

Interaction between mutant GCase and other E3 ubiquitin ligases has already been documented. Thus, Ron et al. (26) showed that mutant GCase interacts with parkin, which mediates its lysine 48 polyubiquitination and proteasomal degradation. We hypothesize that this interaction leads to accumulation of other parkin substrates such as PARIS (78) and ARTS (79), which are deleterious to cells. This accumulation accounts, at least in part, for the development of PD among GD patients and carriers of GD mutations. Lu et al. (80) showed that c-Cbl is an E3 ubiquitin ligase that interacts with mutant GCase.

In conclusion, we suggest that extreme ERAD of mutant GCase variants contributes to the abrogation of the homeostasis in the skin, leading to more proliferation and less differentiation, manifesting itself as ichthyosis in neonates who suffer from type 2 GD.

MATERIALS AND METHODS

Materials

The following primary antibodies were used in this study: mouse monoclonal anti-myc (9B11, Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-GFP, rabbit polyclonal anti-HA (Santa Cruz Biotechnology, CA, USA), rabbit polyclonal anti-ERK (Santa Cruz Biotechnology), mouse monoclonal anti-actin (MP Biomedicals, OH, USA), rabbit polyclonal anti-GCase, (Sigma-Aldrich, Rehovot, Israel), mouse monoclonal anti-ITCH (BD Bioscience, Israel).

Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit (Jackson ImmunoResearch Laboratories, PA, USA).

Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) was purchased from Calbiochem (CA, USA). Restriction enzymes were purchased from several companies and employed according to the manufacturers' recommendations. CHX, leupeptin, phenylmethylsulfonyl fluoride (PMSF) and aprotinin were from Sigma-Aldrich. Absolute Blue qPCR SYBR Green ROX Mix was from TAMAR Laboratory Supplies (Mevaseret Zion, Israel).

Cell lines

HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Beit Haemek, Israel). Human primary skin fibroblasts were grown in DMEM supplemented with 20% FBS. All cells were grown at 37°C in the presence of 5% CO₂.

Construction of plasmids

Construction of all myc-His-GCase plasmids was described elsewhere (26). Prk5 myc-Itch and its mutant prk5 myc-C830A Itch were a gift of Prof. Y. Shaul (The Weizmann Institute of Science, Rehovot, Israel). HA-ubiquitin and its K48R and K63R mutants were kindly provided by Prof. Y. Yarden (The Weizmann Institute of Science, Rehovot, Israel). PcDNA3-ΔNp63 was a gift of Prof. Moshe Oren (The Weizmann Institute of Science, Rehovot, Israel). A HindIII–BamHI fragment isolated from the pcDNA3-ΔNp63 vector was cloned between the HindIII–BamHI sites of pEGFPC3 plasmid (Clontech Laboratories, Inc., CA, USA) to produce GFP-ΔNp63.

SDS–PAGE and western blotting

Cell monolayers were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed at 4°C in lysis buffer (10 mm HEPES, pH, 8.0, 100 mm NaCl, 1 mm MgCl₂ and 1% Triton X-100) containing 10 µg/ml aprotinin, 0.1 mm PMSF and 10 µg/ml leupeptin. Lysates were incubated on ice for 30 min and centrifuged at 10 000g for 15 min at 4°C. Samples containing the same amount of protein were electrophoresed through 10% SDS–PAGE and electroblotted onto a nitrocellulose membrane (Schleicher and Schuell BioScience, NH, USA). Membranes were blocked with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS) for 1 h at room temperature (RT) and incubated with the primary antibody for 1–2 h at RT or overnight at 4°C. The membranes were then washed three times in 0.1% Tween-20 in TBS and incubated with the appropriate secondary antibody for 1 h at RT. After washing, membranes were reacted with ECL detection reagents (Santa Cruz Biotechnology) and analyzed by luminescent image analyzer (Kodak X-OMAT 2000 Processor Kodak, NY, USA).

Transfections

HEK293T cells were transfected using calcium phosphate solutions. A mixture of DNA in 250 μl of 250 mm CaCl₂ was dropped into a tube containing HBS*2 solution (50 mm Hepes, 280 mm NaCl, 1.5 mm Na₂HPO₄, pH 7.09) and incubated for 20 min at RT. The mixture was then added dropwise to subconfluent cells for 36–48 h.

Immunoprecipitation

Cells were washed three times with ice-cold PBS and lysed at 4°C in 1 ml of lysis buffer (10 mm HEPES, pH 8, 100 mm NaCl, 1 mm MgCl₂ and 0.5% NP-40) containing 10 µg/ml aprotinin, 0.1 mm PMSF and 10 µg/ml leupeptin. Following incubation on ice for 30 min and centrifugation at 10 000g for 15 min at 4°C, the supernatants were pre-cleared for 1–2 h at 4°C with protein A-agarose (Roche Diagnostic, Germany). Samples were centrifuged at 15 000g for 1 min at 4°C, and the supernatants were incubated overnight at 4°C with antibodies, immobilized on protein A-Sepharose (Sigma-Aldrich). Following four washes with 1 ml of lysis buffer containing protease inhibitors, proteins were eluted for 5 min at 100°C in 5× Laemmli loading buffer, electrophoresed through 10% SDS–PAGE and blotted. The corresponding blot was interacted with the appropriate antibodies.

Nickel beads (Ni-NTA) precipitation

Forty-eight hours after transfection, cells were lysed with lysis buffer or as specified under ‘in tissue culture’ ubiquitination conditions (see what follows), containing protease inhibitors as mentioned earlier. Following incubation on ice for 30 min and centrifugation at 10 000g for 15 min at 4°C, the supernatants were pre-cleared for 1 h at 4°C with 20 μl of protein A-agarose. Samples were centrifuged at 15 000g for 1 min at 4°C, and the supernatants were incubated with 30 μl of Ni-NTA agarose (Qiagen, GmbH, Hilden, Germany) for 3 h at 4°C. Following four washes with wash buffer (5% sucrose, Tris, pH 7.5, 50 mm NaCl, 0.5% NP40, 20 mm imidizole), the Ni-NTA precipitates were eluted for 5 min at 100°C with 5× Laemmli loading buffer and subjected to SDS–PAGE and western blot analysis.

CHX chase experiments

Twenty-four hours after transfection, cells were treated with CHX (100 mg/ml) to inhibit de novo protein synthesis. At different times, cell lysates were prepared, and the same amount of lysates were subjected to western blot analysis using anti-myc (to follow myc-GCase variants) and anti-actin antibodies, to follow the amount of actin as a loading control.

Quantitation

The blots were scanned using the Image Scan scanner (Amersham Pharmacia Biotech, Buckinghamshire, UK) and the intensity of each band was measured by the Image Master 1D Prime densitometer (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Ubiquitination in tissue culture

Twenty-four hours after transfection, MG132 (25 μm) in 0.05% DMSO or 0.05% DMSO (vehicle only) was added to the cells. Following an overnight incubation, they were harvested and lysed in 200 μl of denaturing buffer (1% SDS, 50 mm Tris, pH7.4, 140 mm NaCl) by boiling for 10 min after vigorous vortexing. Eight hundred microliters of renaturation buffer (2% Triton X-100, 50 mm Tris, pH 7.4, 140 mm NaCl) was added to the lysate. After centrifugation for 15 min at 10 000g at 4°C, the supernatant was collected for Ni-NTA precipitation, as mentioned earlier, and subjected to western blot analysis using anti-myc and anti-HA antibodies.

Proteasome inhibition

Cells were treated for 24 h with 25 μm MG132, after which they were processed according to the experiment performed (western blotting with or without immunoprecipitation).

RNA preparation

Total RNA was isolated using the EZ-RNA kit (Biological Industries, Beit Haemek, Israel), according to the manufacturer's instructions.

RT–PCR analysis

Two micrograms of RNA were reverse-transcribed with M-MLV reverse transcriptase (Promega Corporation, CA, USA), in the presence of 1 μg of oligo dT primer in a total volume of 20 µl, at 42°C for 60 min. Reactions were stopped by incubation at 70°C for 15 min. One to 2 µl of the resulting cDNA was amplified by quantitative real-time PCR as described.

Quantitative real-time PCR

One microliter of cDNA was used for real-time PCR. PCR was performed using the Power SYBR Green QPCR Mix reagent kit (Applied Biosystems, CA, USA) in a Rotor-Gene 6000 (Corbett Life Sciences). The reaction mixture contained 50% QPCR mix, 300 nm of forward primer (5′-ATCTGAAGGAGCAACATCTGG-3′) and 300 nm of reverse primer (5′-CACGGGCGAGTTTACTATGTAG-3′) in a final volume of 10 µl. Thermal cycling conditions were 95°C (10 min), and 40 cycles of 95°C (10 s), 60°C (20 s) and 72°C (20 s). Relative gene expression was determined by the C_t value.

FUNDING

This work was partially supported by grants from the Israel Ministry of Health and the Israel Science Foundation (to M.H.) and by grants from the Italian Health Department ‘Finanziamento Ricerca Corrente (contributo per la ricerca intramurale)’ (to M.F.).

ACKNOWLEDGEMENTS

We would like to thank Prof. Y. Shaul, Prof. Y. Yarden and Prof. M. Oren (The Weizmann Institute of Science, Rehovot, Israel) for their kind gifts of plasmids and the ‘Cell Line and DNA Biobank from Patients Affected by Genetic Diseases’ (G. Gaslini Institute)—Telethon Genetic Biobank Network (Project No. GTB07001A) for cells.

Conflict of Interest statement. None declared.