UNIVERSITA’ DEGLI STUDI DI TRIESTE
XXVI Ciclo del Dottorato di Ricerca in Scienze della Riproduzione e dello Sviluppo indirizzo Genetico Molecolare
Insights into the pathogenic mechanisms of Williams Beuren syndrome through the
functional analysis of TRIM50
MED/03 Genetica Medica
Ph.D. student Giuseppe Merla
Ph.D. program Director Prof. Giuliana Decorti Thesis Supervisor Prof. Paolo Gasparini Thesis Co-supervisor Dott. Leopoldo Zelante
ANNO ACCADEMICO 2012-2013
ABSTRACT ... 3
ABSTRACT (Italian) ... 4
Keywords ... 5
1. INTRODUCTION ... 5
1.1 Genomic Rearrangements and Mutational Mechanisms in Williams-Beuren syndrome . 5 1.2 Williams Beuren syndrome phenotype ... 7
1.3 Genotype-phenotype correlation studies: mouse models and atypical patients ... 10
1.4 TRIpartite Motif (TRIM) proteins and TRIM50 ... 11
1.5 Ubiquitination and related processes ... 12
2. MATERIALS AND METHODS ... 15
2.1 Plasmid generation ... 15
2.2 Cell culture and stable cell line production ... 15
2.3 Immunofluorescence Microscopy ... 16
2.4 Statistical analysis ... 17
2.5 Mass spectrometry analysis ... 17
2.6 Immunoprecipitation, GST pull-down and western blot ... 18
3. RESULTS ... 18
3.1 TRIM50 localizes into cytoplasmic bodies associated to aggresomes ... 18
3.2 TRIM50 interacts with and is acetylated by HDCA6 ... 22
3.3 HDAC6 deacetylates TRIM50 in a microtubule-dependent manner ... 26
3.4 p300 and PCAF are acetyltransferases of TRIM50 ... 28
3.5 TRIM50 acetylation antagonizes TRIM50 ubiquitination ... 28
3.6 RFP domain and acetylation are necessary for aggresome localization of TRIM50 ... 28
3.7 TRIM50 interacts with p62 ... 31
3.8 TRIM50 promotes the sequestration of ubiquitinated proteins into aggresome ... 31
4. DISCUSSION ... 35
5. CONCLUSION ... 38
6. REFERENCES ... 38
ABSTRACT
Interstitial deletion of 7q11.23 encompassing approximately 30 transcribed genes causes Williams-Beuren syndrome (WBS; OMIM 194050), a neurodevelopmental genomic disorder. Alongside cardiovascular symptoms and facial dysmorphism, the hallmark is a unique behavioural and cognitive profile that combines hypersociability with a form of mental retardation characterized by a severe impairment in visuospatial processing, counting and planning, pointing to this gene cluster as a central regulator of social behaviour and cognition.
The genetic dissection of the phenotypes associated with WBS syndrome have been inferred with complementary strategies including clinical, psychological and molecular analysis of affected individuals with atypical (shorter or larger) deletions, knockout mice model, and functional and biochemical studies on single gene(s).
Here we report insights on the function of TRIM50, one of the genes associated to WBS. By using a combination of biochemical and cellular approaches we show that TRIM50 forms cytoplasmic bodies that are aggresome precursors. Aggresome is an inclusion cytoplasmic body where damaged proteins are stored before being degraded through Proteasome or Autophagy-lysosome pathways. We demonstrate that the E3 ubiquitin ligase TRIM50 promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome and participates to their clearance.
In addition we identified and preliminarily characterize the first two interactors of TRIM50 protein, HDAC6 and p62, both involved in protein ubiquitination and degradation processes. Furthermore we report that TRIM50 is acetylated by PCAF and p300 and de- acetylated by HDAC6 in a microtubule-dependent manner and that acetylation and ubiquitination compete for the same lysine residue regulating the activity and stability of TRIM50.
Overall the data obtained during this PhD project suggest that TRIM50 is an emerging player of aggregation processes involving polyubiquitinated proteins and in the regulation of misfolded protein aggregates clearance. Interestingly enough, although extensive studies are required, this study opens the way to the interesting hypothesis that the proven haploinsufficiency of TRIM50 could account for certain WBS phenotypes through the accumulation of ubiquitinated proteins otherwise degraded.
ABSTRACT (Italian)
La microdelezione della regione cromosomica 7q11.23, che comprende circa 30 geni causa la sindrome di Williams-Beuren (WBS, OMIM 194050), una patologia genomica neurocomportamentale. Accanto ai sintomi cardiovascolari e ai dismorfismi facciali, il segno distintivo di questa sindrome è costituito dal profilo comportamentale e cognitivo unico che è caratterizzato da una forma moderata-grave di ritardo mentale associato a delle compromesse capacità visivo-spaziali.
La dissezione genetica dei fenotipi associati a questa sindrome è effettuata mediante diverse strategie tra cui l’analisi clinica, psicologica e molecolare di pazienti con delezioni definite “atipiche” (più corte o più lunghe), modelli animali (soprattutto murini) e studi funzionali e biochimici sui singoli geni associati alla sindrome.
In questo studio, utilizzando una combinazione di approcci biochimici e cellulari mostriamo che TRIM50 forma delle strutture citoplasmatiche (TRIM50 bodies) che rappresentano dei precursori degli Aggresomi. L’Aggresome è una struttura citoplasmatica che si forma a ridosso della membrana nucleare, in cui proteine danneggiate, misfolded e tossiche vengono conservate e modificate prima di essere degradate ed eliminate attraverso il Proteasoma o l’Autofagia.
Noi dimostriamo che la proteina TRIM50, mediante la sua attività di E3 ubiquitina
ligasi, promuove il reclutamento di proteine poliubiquitinate all’aggresoma e partecipa alla loro eliminazione.
Inoltre abbiamo identificato e preliminarmente caratterizzato due proteine che interagiscono con TRIM50, HDAC6 e p62, entrambe coinvolte nei processi di ubiquitinazione e degradazione proteica. Inoltre abbiamo dimostrato che TRIM50 è acetilata da PCAF e p300 e deacetilata da HDAC6 e che l'acetilazione e ubiquitinazione competono per lo stesso residuo di lisina regolando l'attività e la stabilità di TRIM50.
Nel complesso i dati ottenuti nel corso di questo progetto di dottorato suggeriscono che TRIM50 è una nuova proteina coinvolta nei processi cellulari di aggregazione ed eliminazione delle proteine danneggiate e non più utili alla cellula. Questo progetto di tesi, anche se studi più approfonditi sono necessari, apre la strada all’interessante ipotesi che la ridotta espressione di TRIM50, osservata nelle linee cellulari di pazienti con Sindrome di Williams, potrebbe causare alcuni fenotipi della Sindrome attraverso l’accumulo all’interno della cellula di proteine danneggiate e tossiche che non possono essere più degradate in modo corretto.
Keywords
TRIM50, Proteasome, Aggresome, Williams Beuren syndrome, 7q11.23
1. INTRODUCTION
1.1 Genomic Rearrangements and Mutational Mechanisms in Williams-Beuren syndrome
Copy number variants (CNVs, microduplications and microdeletions) are the most prevalent types of structural variations in the human genome (Iafrate et al., 2004; Sebat et al., 2004). Several studies found that CNVs make a substantial contribution to human diseases
and provide greater insight into the aetiology of phenotypes that result from complex genetic patterns of inheritance such as neurodevelopmental diseases, autism spectrum disorders, bipolar disorders, and schizophrenia (Beckmann et al., 2007). CNVs result in the alteration of a variable number of genes causing changes in gene dosages, which ultimately may lead to specific clinical phenotypes.
The disruption of regulatory regions within the CNVs can also result into altered dosage and function of genes outside the deleted or duplicated intervals (Merla et al., 2006;
Henrichsen et al., 2009).
Interstitial deletions of 7q11.23 encompassing approximately 30 transcribed genes cause Williams-Beuren syndrome (WBS; OMIM 194050), one of the best-characterized genomic disorder affecting 1/7,500 to 1/10,000 live births (Stromme et al., 2002). As with many other genomic disorders, the common recurrent 1.55 (95%) and 1.8 (2-3%) Mb microdeletions occurs by non-allelic homologous recombination (NAHR) (Stankiewicz and Lupski, 2002) between LCRs (Urban et al., 1996) flanking the deleted region (Bayes et al., 2003; Hillier et al., 2003) (FIGURE 1).
Figure 1. Schematic representation of 7q11.23 genomic rearrangements (not drawn to scale). The centromeric (c), middle (m), and telomeric (t) LCRs are shown as colored arrows with their relative orientation to each other. Please note that multicopy genes within the blocks are represented only once. In the middle, the common deletions of 1.5 and 1.8 Mb are depicted; breakpoints within the centromeric and the medial copy of LCR block B and within the centromeric and the medial copy of LCR block A are shown. Schematic representation of the 7q11.23 deletion and duplication are shown.
Because NAHR can generate both microdeletions and microduplications, it was suspected that reciprocal microduplication of microdeletion syndromes should also occur (Lupski, 1998). With the advent of diagnostic array comparative genomic hybridization (aCGH), this prediction was verified with the identification of patients with 7q11.23 duplications (OMIM 609757). The frequency of WBS microduplication resulted less documented for long time because the phenotypes seen in patients with 7q11.23 microduplications are quite unlike those seen with the common WBS microdeletion therefore it would have been difficult to predict the phenotype.
1.2 Williams Beuren syndrome phenotype
Since the first pictures of patients with WBS, reported in 1956 by Dr. Schlesinger, Dr.
Williams in 1961 and shortly thereafter by Dr. Beuren (Schlesinger et al., 1956; Williams et al., 1961; Beuren et al., 1962), the WBS phenotype has been extensively studied and the distinctive signs well characterized.
The typical facial dysmorphism includes a stellate or lacy pattern of the irides, broad forehead, periorbital fullness, epicanthal folds, flat nasal bridge, a short upturned nose, long philtrum and wide mouth with full lips, full cheeks, small jaw, malocclusion, a high prevalence of dental caries, oligodontia, microdontia, and aberrant tooth shape (FIGURE 2) (Axelsson et al., 2003; Pober, 2010).
Figure 2. Representative images of facial characteristics of WBS patients at different ages.
Other symptoms include weakness of connective tissue (hoarse/deep voice, hernias, bladder/bowel diverticulae, soft/lax skin, joint laxity or limitation) short stature, and mild to moderate intellectual disability (ID). Cardiovascular defects include mainly supravalvular aortic stenosis (SVAS), pulmonary artery stenosis, and coarctation or aortic arch hypoplasia (Merla et al., 2010). Additional clinical features are: growth retardation, hyperacusis, premature ageing, hypercalcemia, glucose intolerance and diabetes mellitus, renal anomalies, dental defects, gastrointestinal problems, urinary tract abnormalities, weakness in daily living skills, and motor abilities (Pober, 2010) (Table 1).
Table 1. List (with percentage) of the main phenotypes associated to Williams Beuren syndrome patient carrying classical (1.5 Mb) microdeletion.
The neurocognitive and behavioural profile is very characteristic as it includes relative strengths in verbal short-term memory and lexical comprehension, alongside severe weakness in visuospatial construction. WBS individuals have a unique cognitive and personality profile with areas of relative strength and weakness (Morris et al., 1988; Francke, 1999). The cognitive profile generally consists of mild to moderate mental retardation with Intelligence Quotients (IQ) in the high 50s to low 70s and it is characterized by a severe visuospatial construction deficit contrasting with a relative strength in verbal short-term memory and language.
Impairment in visuospatial construction is a hallmark of the WBS neurocognitive profile and is characterized by poor performance on tests of block design or pattern construction (Mervis and Klein-Tasman, 2000). WBS individuals may focus on the particular, while failing to appreciate the global aspects (Tassabehji, 2003). From a behavioural standpoint, striking features of individuals with WBS are their high sociability and empathy for others, leading them to engage in social interaction even with strangers (Klein-Tasman and Mervis, 2003).
They also appear to have impaired detection of social threat. Although the excessive drive to socialize is an hallmark of WBS, and individuals with WBS appear to be particularly drawn to faces (Wang et al., 1995; Gagliardi et al., 2003) their socialization is often “shallow”
with conversations focusing on their own interests rather than engaging in a more typical give-and-take (Einfeld et al., 1997). Intriguingly, their remarkable hypersociability is associated with general and anticipatory anxieties, phobia related to non-social objects, and depression (Stinton et al., 2010).
1.3 Genotype-phenotype correlation studies: mouse models and atypical patients The genetic dissection of the phenotypes associated with this syndrome have been inferred with complementary strategies including clinical, psychological and molecular
analysis of affected individuals with atypical deletions, knockout mice model, functional and biochemical studies on single genes and global gene expression studies. These studies suggest correlations between haploinsufficiency of some WBS genes and WBS phenotypic features.
Mouse models of LIMK1, CYLN2 and GTF2IRD1 suggest that hemizygosity of these genes might play a role in some aspects of this phenotype and/or in craniofacial anomalies (Hoogenraad et al., 2002; Morris et al., 2003; Gray et al., 2006) although no absolute association was established. On the contrary hemizygosity of the ELN gene was unequivocally linked to the SVAS phenotype (Curran et al., 1993). Reduced BAZ1B protein level has been linked to cardiac, craniofacial, and hypercalcemia defects.
Single gene mouse models provide valuable information about the function of an individual gene, but they are not true genetic models of WBS, which is by definition a contiguous gene deletion disorder. The high conservation of gene content and order across the WBS region with its syntenic region on mouse chromosome 5G, enabled the engineering of two half-deletions that together reproduced almost entirely the genetic configuration of WBS.
The proximal deletion (spanning Gtf2i through Limk1) resulted mainly in increased sociability and altered neuronal density in the somatosensory cortex, whereas the distal deletion (spanning Limk1 through Fkbp6) was associated with cognitive defects (Li et al., 2009).
Further genotype–phenotype correlations have been proposed following diagnosis of atypical patients presenting smaller or larger WBS microdeletions (Botta et al., 1999;
Tassabehji et al., 1999; Korenberg et al., 2000; Gagliardi et al., 2003; Heller et al., 2003;
Hirota et al., 2003; Karmiloff-Smith et al., 2003; Morris et al., 2003; Howald et al., 2006;
Schubert and Laccone, 2006; Edelmann et al., 2007; Ferrero et al., 2010; Fusco et al., 2013).
1.4 TRIpartite Motif (TRIM) proteins and TRIM50
TRIM50 is one of 30 hemizygous genes mapping to the WBS microdeletion. We
previously reported that TRIM50 encodes an E3-Ubiquitin ligase, like other TRIM proteins, that self-associates to form cytoplasmic bodies in the cell (Micale et al., 2008). TRIM50 is a stomach-specific member specifically expressed in gastric parietal cells and predominantly localized in the tubulovesicular and canalicular membranes. Trim50 knockout mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knockout parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells (Nishi et al., 2012).
The TRIpartite Motif (TRIM) proteins harbour from their amino- to their carboxy- terminal end, a RING (R), one or two B-boxes and a predicted coiled-coil (CC) (Reddy et al., 1992; Cao et al., 1997; Cao et al., 1998; Reymond et al., 2001). The TRIM motif is usually followed by either one or two C-terminal domains, which are specific for each member of the family. Alteration of these proteins is associated to pathological conditions that range from Mendelian diseases to cancer and viral infection. Moreover they are involved in a variety of cellular processes, including regulation of cell cycle progression, differentiation, development, oncogenesis, and apoptosis (Micale et al., 2012).
1.5 Ubiquitination and related processes
TRIM proteins act as E3 Ubiquitin ligases through the RING domain. Ubiquitination is a versatile post-translational modification process mediated by ubiquitin, a 76-residue polypeptide, used by eukaryotic cells to control protein level and remove non-functional, damaged, and/or misfolded proteins from the cell through the proteasome-mediated degradation. The covalent attachment of several ubiquitin molecules in the form of a multiubiquitin chain on lysine residues of target proteins proceeds via three sequential steps:
first the activation of the ubiquitin peptide by an activating E1 enzyme, second the transfer of the ubiquitin to an E2 conjugating enzyme, and third its transfer to the substrate facilitated by an E3 ubiquitin ligase (Glickman and Ciechanover, 2002) (FIGURE 3).
Figure 3. Schematic diagram of the ubiquitin-proteasome system (UPS). The UPS involves at least three enzymes (E1, E2, and E3) that catalyze the addition of ubiquitin to lysine residues on the substrate protein.
Polyubiquitinated substrates are then recognized by the proteasome or proteasome associating protein, and the ubiquitin removed by deubiquitinases and the substrate unfolded and translocated in the 20S core for proteolysis.
The ubiquitin proteasome system (UPS) is highly conserved. However, when the capacity of the proteasome is impaired, misfolded proteins cannot be properly cleared and they accumulate into the aggresome (Goldberg, 2003; Kawaguchi et al., 2003), an inclusion body localized in the proximity of the microtubule-organizing centre (MTOC) (Iwata et al., 2005; Pandey et al., 2007). Microtubule-associated histone deacetylase 6 (HDAC6) mediates this process (Matthias et al., 2008). Through its ubiquitin-binding BUZ finger domain,
HDAC6 binds to and facilitates the transport of polyubiquitinated misfolded proteins along microtubules to aggresome (Kawaguchi et al., 2003).
Protein lysine acetylation/deacetylation is as a key posttranslational modification (PTM) in gene expression regulation, protein stability, and signal transduction. Protein acetylation is a dynamic process controlled by the antagonistic actions of two large families of enzymes: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). The balance between the activities of these enzymes serves as a key regulatory mechanism for gene expression and governs numerous developmental processes and disease states. At least two independent HATs, namely p300 and PCAF, regulate the activity of non-histone proteins pointing out HATs as multifunctional factors (Ge et al., 2009).
Like HATs, histone deacetylases also possess substrate specificity and accumulating evidences suggest that many, if not all, HDACs can deacetylate also non-histone proteins (Peserico and Simone, 2011). Additionally, since lysines also provide specific sites for other PTMs, their acetylation would chemically lock the residue, conferring to this modification a regulatory potential with the ability to interfere with cellular functions relying on other lysine modifications, i.e., methylation, sumoylation, neddylation, biotinylation, and ubiquitination (Sadoul et al., 2008). Indeed cross-regulation between lysine acetylation and ubiquitination was found as a critical cellular regulatory mechanism.
Aggresome’s clearance is mediated by ubiquitin-binding proteins like p62/SQSTM1 (Gal et al., 2007; Kirkin et al., 2009). These adaptor proteins through their ubiquitin-binding domain (UBA) decide the fate of protein degradation either through UPS or autophagy- lysosome pathway (Komatsu et al., 2007; Komatsu et al., 2010).
Autophagy is another intracellular catabolic process, which plays an essential role in tissue homeostasis by removing damaged proteins and organelles. In this process, double-
membrane vesicles, termed autophagosomes, wrap around portions of cytosol and transport them to the lysosome for degradation. Selective forms of autophagy are responsible for the specific removal of aggregate-prone proteins, damaged or supernumerary organelles upon ubiquitination mediated by E3 ligase proteins (Mizushima et al., 2008).
2. MATERIALS AND METHODS 2.1 Plasmid generation
pcDNA3-EGFP wild-type and mutants TRIM50 were described in (Micale et al., 2008). Human β2-tubulin ORF was cloned into a pcDNA3 vector with FLAG as tag using a PCR based method with appropriate oligonucleotides followed by in-frame insertion into the vector. EGFP-TRIM50_K372R, EGFP-TRIM50_Del372-374 were generated by site-directed mutagenesis via QuickChange II kit (Stratagene) using EGFP-TRIM50. pENTR-EGFP, GST tagged p62 and GST-p62 mutants were a kind gift from Prof. T. Johansen (Institute of Medical Biology, University of Tromso, Norway), pcDNA3-Myc-p62 was a generous gift of Prof. Marie W. Wooten (Cellular and Molecular Biosciences Program, Auburn University, USA). pcDNA3-HA-HDAC6 mutants were a gift of Prof. Matthias (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland). FLAG-PCAF and HA-p300 were a gift of Prof. E. Seto (Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, USA).
2.2 Cell culture and stable cell line production
HEK293, HeLa cells, SH-SY5Y (all from ATCC, Manassas, USA), and MEF were maintained in DMEM with Glutamax medium supplemented with 10% fetal bovine serum and 1% antibiotics (Invitrogen, Carlsbad, CA). Hdac6 MEF (mouse embryo fibroblast) and Trim50 MEF cells were kindly provided by Prof. Joo-Yong Lee (Duke University, Durham, USA), and Prof. A. Reymond (University of Lausanne, Switzerland). Fugene 6 (Roche) was
used for transfection according to the manufacturers’ instructions. HEK293 and HeLa were transfected with pcDNA3-FLAG-TRIM50 or empty vector and selected for 2 weeks with 1 mg/ml G418 (Invitrogen, Carlsbad, CA) selective agent. The expressing colonies were expanded and then used for protein extract preparations following standard procedures.
HEK293 and HeLa cell lines were used since the low level expression of endogenous TRIM50 protein. Hereafter the stable cell lines will be referred to as FLAG-TRIM50#3 and FLAG#3 in HE293, and FLAG-TRIM50#12 and FLAG#4 in HE293 respectively.
2.3 Immunofluorescence Microscopy
For immunofluorescence analyses, the cells transfected with EGFP-TRIM50 and EGFP-TRIM50 mutants were fixed before their incubation with the primary and secondary antibodies of interest, mounted in mowiol and examined on a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). All confocal images were obtained using the necessary filter sets for GFP, Alexafluor 488 and 546, using a Zeiss Plan-Neofluor 63x oil immersion objective (NA 1.4), with the pinhole set to one Airey unit. HDAC6 antibody (Santa Cruz) was used at 1:200 dilution, where is indicated.
EGFP-TRIM50 transfected HeLa cells were grown on CELLocate coverslips with coordinated grid and then prepared for CLEM microscopy identification of TRIM50 structures according to (Polishchuk et al., 2000). Briefly, after visualization of EGFP- TRIM50 positive bodies by time-lapse confocal microscopy, the cells were fixed, labeled with an antibody against EGFP using the gold-enhance protocol, embedded in Epon-812, and cut in serial sections. Then region containing TRIM50-positive bodies were analyzed in serial thin sections under a Philips Tecnai-12 electron microscope (Philips, Einhoven, The Netherlands). EM images were acquired from the region of interest using an Ultra View CCD digital camera (Soft Imaging Systems, Munich, Germany).
2.4 Statistical analysis
All microscopy experiments were performed in triplicate. Approximately 40 cells were analyzed for each experimental condition using ImageJ program. All p-values were adjusted for multiple comparison following Tukey-Kramer’s method. A p-value <0.05 was considered for statistical significance. All statistical analyses and graphs were performed using SAS Release 9.1 (SAS Institute, Cary, NC, USA) and R (version 2.10.1) software, respectively.
2.5 Mass spectrometry analysis
TRIM50 complexes were isolated from HEK293 cells total extracts by immunoprecipitation. FLAG-TRIM50#3 and FLAG#3 cell lines were lysed in PBS, 0.5% NP- 40, 1 mM PMSF, and COMPLETE protease inhibitors (Roche) for 45 min under gently mixed. Total protein extracts were pre-cleared with unspecific Mouse IgG Agarose Beads (Sigma) overnight in lysis buffer. The protein extracts were recovered by centrifugation (3000 rpm for 5 min) and then incubated overnight, under gently agitation, onto M2 anti-FLAG agarose-conjugated antibody beads (Sigma) previously blocked with no fat milk treatment.
Unbound proteins were discarded and the beads were collected by centrifugation and extensively washed with lysis buffer supplemented with 150mM NaCl to eliminate non- specific bound proteins. Elution of the desired protein complexes was performed by competition with FLAG peptide in elution buffer. The eluted proteins were precipitated in methanol/chloroform and then loaded onto a 10% SDS-PAGE. The gel was stained with colloidal Coomassie blue (Pierce). Protein bands were excised from the gel, reduced, alkylated and digested with trypsin as described elsewhere (Zito et al., 2007). Peptide mixtures extracted from the gel were analyzed by nano-chromatography tandem mass spectrometry (nanoLC–MS/MS) on a CHIP MS Ion Trap XCT Ultra equipped with a capillary 1100 HPLC system and a chip cube (Agilent Technologies, Palo Alto, CA). Peptide
analysis was performed using data-dependent acquisition of one MS scan (mass range from 400 to 2000 m/z) followed by MS/MS scans of the three most abundant ions in each MS scan.
Raw data from nanoLC–MS/MS analyses were employed to query a non-redundant protein database using in house MASCOT software (Matrix Science, Boston, USA).
2.6 Immunoprecipitation, GST pull-down and western blot
Co-immunoprecipitation experiments were performed as previously described (Merla et al., 2004). Complexes were analyzed by western blotting using indicated antibodies.
Horseradish peroxidase conjugated anti-mouse and anti-rabbit antibodies (GE Healthcare) and the ECL chemiluminescence system (GE Healthcare) was used for detection. Where indicated the MG132 proteasome inhibitor (Calbiochem, USA) was added. GST-p62 (231-385) and GST-p62 (232-370) fusion proteins were purified using glutathione-Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. For the GST pulldown assay, 3µg of GST-recombinant proteins were mixed with 40 µg of total FLAG-TRIM50#3 cell lysate and incubated at 4°C for 2 h with rotation, and then incubated with FLAG antibody for 4 h. The binding fraction was washed four times and then loaded into a SDS 10% PAGE gel, and immunoblotted with anti-GST antibody (Santa Cruz). Soluble and insoluble fractions were obtained using RIPA buffer as described elsewhere (Muqit et al., 2006). Protein band densities were determined using densitometer (Kodak). The amount of the protein was calculated by the initial amount of FLAG-protein level and normalized with GAPDH. Cells treated with DMSO were used as control.
3. RESULTS
3.1 TRIM50 localizes into cytoplasmic bodies associated to aggresomes
Fluorescence microscopy showed that ectopically expressed and endogenous TRIM50 localizes mainly into cytoplasmic bodies heterogeneous in size and shape, with the intact
central region of the protein (B-Box and Coiled-Coil domains) indispensable for the proper localization (Micale et al., 2011; Fusco et al., 2012). Further characterization of TRIM50 bodies achieved by Correlative Light-Electron Microscopy (CLEM) revealed that the fluorescent bodies corresponded to heterogeneous in morphology TRIM50-containing protein aggregates, confirming the tendency of TRIM protein to self-associate into larger structures (FIGURE 4).
Figure 4. CLEM-ImmunoElectron microscopy of TRIM50 bodies. Representative immuno-EM images obtained from HeLa cells, trasfected with EGFP-TRIM50 and labelled with anti-GFP antibody. TRIM50-gold particles localizes to multivesicular structures like autophagosomes and into or surrounded membrane-free proteins aggregates.
Due to the intrinsic E3-Ubiquitin ligase activity of TRIM50 we treated neuroblastoma SH-SY5Y cells with the proteasome inhibitor MG132 and stained the cells with FK2, which recognizes polyubiquitinated proteins. Endogenous TRIM50 localized to perinuclear structures reminiscent the aggresome (FIGURE 5A). To explore the possibility of a link between TRIM50 and aggresome, we used a well-known established aggresome marker
HDAC6 (Kawaguchi et al., 2006) in co-localization experiments. We found that both endogenous and transfected TRIM50 partially located with HDAC6 under proteasome inhibition (FIGURE 5B). This cellular localization does not depend on the E3-ligase activity of the RING domain of TRIM50 as a mutant lacking the RING domain still retains the ability to localize to aggresome (FIGURE 5B).
Figure 5. Endogenous TRIM50 localizes in the aggresome. SH-SY5Y cells were stained with anti-TRIM50 antibody. Where indicated, the cells were treated with 25 mM MG132 for 6 h. (B) HeLa cells expressing EGFP- TRIM50 and EGFP-TRIM50ΔRING were stained with an anti-HDAC6 antibody. Where indicated, the cells were treated with 25 mM MG132 for 6 h.
In accordance with the central role of microtubule system in the formation of aggresome, nocodazole treatment of SH-SY5Y cells prevented the localization of TRIM50 to
A
B
aggresome (data not shown). Consistently we demonstrated that TRIM50 interacts with Tubulin beta 2B class IIb (Tubb2b), a microtubules component (FIGURE 6).
Figure 6. Fig.6 TRIM50 interacts with Tubulin beta 2b.The interaction between TRIM50 and beta tubulin was assayed in HEK293 cells transiently expressing FLAG-Tubb2b and EGFP-TRIM50. The cell lysates were immunoprecipitated with anti-FLAG and immunoblotted with anti-GFP antibody.
These findings suggest that TRIM50 bodies may be aggresome precursors that in response to proteasome inhibition move towards aggresome by a microtubule dependent movement.
Next we tested whether HDAC6 is required for the proper localization of TRIM50, by performing immunofluorescence microscopy assays in Hdac6 deficient mouse fibroblasts. We found that in Hdac6 wild-type cells, Trim50 bodies localize within FK2-ubiquitin-containing aggresomes upon MG132 treatment (FIGURE7, a-f). Conversely, when MG132 was added to Hdac6 knock out cells Trim50 bodies loose the ability to form whole aggresome, although they still continued to partially colocalize with ubiquitinated aggregates (FIGURE7, g-l).
Figure 7. TRIM50 bodies colocalize into aggresome by a Hdac6-dependent way. Hdac6 wild type and Hdac6 deficient mouse fibroblasts were transfected with EGFP-Trim50 followed by treatment with 25 mM MG132 for 6 h where indicated, and immunostained with FK2 antibody.
3.2 TRIM50 interacts with and is acetylated by HDCA6
We assessed whether the observed TRIM50-HDAC6 colocalization results also in their physical interaction. As reported in FIGURE 8, TRIM50 interacts with endogenous HDAC6; an interaction that strengthens in response to MG132 treatment (compare lane 1 to lane 2 in FIGURE 8A). To map the domain(s) of HDAC6 that binds TRIM50 we performed co-immunoprecipitation assays with different HDAC6 truncating mutants in presence of MG132. As reported in FIGURE 8 (B-D) only the two constructs containing the first deacetylase (CAT1) domain, HDAC6_DBUZ and HDAC6 (1-503), interact with TRIM50 indicating that CAT1 is necessary for TRIM50 interaction. Overall these results indicate that HDAC6, by interacting with TRIM50 is required for the proper localization of TRIM50 bodies and of ubiquitinated proteins within the aggresome.
Figure 8. TRIM50 interacts with HDAC6. (A) The interaction between TRIM50 and HDAC6 was assayed in HEK293 FLAG-TRIM50#3 cell line, treated with MG132. The cell lysates were immunoprecipitated (IP) with anti-FLAG and Immunoblot (IB) with HDAC6 antibody. Asterisk indicate IgG aspecific band. (B) Schematic representation of HDAC6 deletion mutants with the minimal region of interaction between TRIM50 and HDAC6 depicted in gray. (C–D) HEK293 transiently transfected with EGFP-TRIM50 and FLAG-HDAC6 deletion mutants were treated with 25µM ofMG132 for 6 h, immunoprecipitated with anti-FLAG and immunoblotted with anti-GFP. (E) FLAG-TRIM50#3 transiently transfected with HA-HDAC6 deletion mutants were treated with 25 µM of MG132 for 6 h, immunoprecipitated with anti-HA and immunoblottedwith anti-FLAG.
A
B C
D E
The association between TRIM50 and the catalytic HDAC6 domain suggests that TRIM50 may be a deacetylation target of HDAC6. To assess this, two TRIM50 constructs, tagged with FLAG and EGFP respectively, were immunoprecipitated and an anti-acetyl- lysine specific antibody was used for immunodetection of acetylated-TRIM50. Acetylated form(s) of TRIM50 were detected showing that TRIM50 is an acetylated protein (FIGURE 9A and 9C). Moreover a TRIM50 mutant construct lacking the C-Terminal region (EGFP-
TRIM50_Dom1) (schematized in FIGURE 9B) was unable of being acetylated, hinting that RFP domain is involved in acetylation (FIGURE 9C).
Next we searched for putative lysine residues of TRIM50 involved in acetylation.
Computational prediction revealed the presence of two evolutionarily conserved lysines, K372 and K374, as the more likely acetylation sites in the C-terminal TRIM50 region (FIGURE 9D). Therefore, to assess the acetylated residues in TRIM50 protein, by direct mutagenesis we generated the two EGFP-TRIM50_Del372-374 and EGFP-TRIM50_K372R constructs and performed immunoprecipitation assays using an EGFP antibody. These studies revealed that both TRIM50_Del372-374 and TRIM50_K372R mutant proteins did not undergo to acetylation modifications when compared to TRIM50 wild-type construct, hinting that K372 residue is a target of acetylation (FIGURE 9E).
Figure 9. TRIM50 is a deacetylation target of HDAC6. (A) FLAG-TRIM50#3 and FLAG-#4 empty vector, were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Ack. (B) Schematic representation of TRIM50 deletion mutants.(C)HEK293 transiently transfected with EGFP-TRIM50, EGFP- TRIM50_Dom1 and EGFP-empty vectorswere immunoprecipitated with anti-EGFP and anti-Ack and immunoblottedwith Ack and EGFP antibodies respectively. (D) PAIL bioinformatic analysis of TRIM50 sequence. (E) HEK293 transiently transfected with EGFP-TRIM50, EGFP-TRIM50 K372R and EGFP-TRIM50 Del372-374 vectors, were immunoprecipitated with anti-EGFP and immunoblotted with anti-Ack and anti-Ub antibodies.
A B
C D
3.3 HDAC6 deacetylates TRIM50 in a microtubule-dependent manner
Since the catalytic CAT1 domain of HDAC6 is involved in TRIM50 interaction and TRIM50 is acetylated, we asked whether HDAC6 deacetylases TRIM50. As shown in FIGURE 10A, the overexpression of HA-HDAC6 abrogates TRIM50 acetylation. To
confirm this result, we transfected Mouse Embryo Fibroblasts (MEFs) derived from Hdac6 +/+
and -/y mice with EGFP-TRIM50 and we found that, TRIM50 acetylation increased in Hdac6 -
/y cell lines, providing additional evidence for a role of HDAC6 in mediating TRIM50 deacetylation (FIGURE 10B). Finally, we used Trichostatin A (TSA), a deacetylase inhibitor of class I/II HDAC family enzymes (FIGURE 10 C-D). By immunoprecipitation assay we found that TSA increased TRIM50 acetylation level. Collectively, these data indicate that HDAC6, through its first catalytic domain, interacts with and regulates TRIM50 acetylation in K372 residue.Since HDAC6 and TRIM50 possess both a microtubule-binding capacity we assessed whether microtubules integrity is required for TRIM50 deacetylation. We treated the FLAG-TRIM50#3, a HEK293 cell line stably expressing a FLAG-TRIM50 construct, in ice for 1h, a treatment that leads to complete depolymerization of microtubule network (Guan et al., 2008). After microtubule depolymerization, by immunoprecipitating TRIM50 protein and blotting with the acetyl-lysine antibody we observed an increase of TRIM50 acetylation level.
This increase was persisting even in the presence of HDAC6 (FIGURE 10E). Overall, these results indicate that an intact microtubule network is required for HDAC6 to deacetylase TRIM50.
Figure 10. HDAC6 deacetylates TRIM50 in a microtubule-dependent manner. (A) FLAG-TRIM50#3 was transfected with HA-HDAC6, immunoprecipitated with anti-Ack antibody and immunoblotted with FLAG and anti-Ack antibodies. (B) HDAC6+/+ and HDAC6 −/Y MEFs were transfected with EGFP-TRIM50, immunoprecipatated with anti-EGFP and immunoblotted with Ack and EGFP antibodies, respectively. (C) HEK293 transiently transfected with EGFP-TRIM50 was treated with 5 µM of TSA for 1 h, immunoprecipitated with anti-EGFP antibody and immunoblotted with anti-Ack.(D) HEK293 transiently transfected with EGFP- TRIM50 was treated with TSA 5 µM for 1 h and/or cotransfected with FLAG-PCAF or HA-p300, immunoprecipitated with anti-Ack and immunoblotted with anti-Ack and anti-EGFP antibodies. (E) FLAG- TRIM50#3 treated or not 1 h in ice, staining with αTubulin, was immunoprecipitated and immunoblotted with anti-Ack, FLAG, HA and Ub antibodies.
A B
C D
E
3.4 p300 and PCAF are acetyltransferases of TRIM50
Acetylation Set Enrichment-Based software (ASEB;
http://202.38.126.151/hmdd/huac/) predicted CBP/p300 and PCAF as acetylase proteins associated to K372 residue of TRIM50. By immunoblotting assays we showed that ectopically expression of p300 and PCAF increased the level of acetylated TRIM50, although with different strength (FIGURE 10D).
3.5 TRIM50 acetylation antagonizes TRIM50 ubiquitination
Since acetylation can compete with the ubiquitination in targeting proteins we asked whether acetylation competes with and may inhibit TRIM50 ubiquitination. To assess this, we analysed the ubiquitination level of TRIM50 and of K372-TRIM50 mutant protein. A higher ubiquitination level of TRIM50 was observed in wild type construct when compared to K372R-TRIM50 proteins, suggesting that acetylation and ubiquitination compete for the same lysine residue (FIGURE 9E). Corroborating these data, we also observed that after microtubule depolymerization, which causes an increase of TRIM50 acetylation level, the ubiquitination of TRIM50 protein decreased (FIGURE 10E).
3.6 RFP domain and acetylation are necessary for aggresome localization of TRIM50
Since TRIM50 re-localizes into aggresome upon proteasome inhibition, we determined the contribution of TRIM50 domains to aggresome localization. As elsewhere described TRIM50 aggresome-localization does not depend on the E3 Ubiquitin ligase activity, as a mutant lacking the RING domain retains the ability to localize to aggresome (Fusco et al., 2013). Likewise, MG132 treatment was able to induce a main aggresome localization of the B-box or the C-terminal domains of TRIM50 (FIGURE 11). Conversely, deletion of RFP domain induced a significant localization of TRIM50 protein outside the
aggresome, in MG132 treated cells. Overall, these results substantiate the importance of RFP domain for TRIM50 aggresome localization. Next we asked whether the K372 residue has a role in TRIM50 aggresome localization. In untreated cells, ectopically expressed K372R- TRIM50 construct localized in cytoplasmic structures as the wild-type protein; after proteasome inhibition, approximately 40% of K372R-TRIM50 resulted in a complete aggresome localization, while around 50% of the protein localizes just partially in the aggresome with the remaining fraction retaining a diffuse localization (FIGURE 11C). Since elimination of K372 residue, involved in both ubiquitination and acetylation significantly affects the localization of TRIM50, we intimate that K372 contributes to drive TRIM50 aggresome localization.
Figure 11. Aggresome localization of TRIM50 domains. (A) HeLa cells transfected with the reported TRIM50 deletion mutants were stained using HDAC6 antibody and analysed at confocal microscopy.Where indicated the cells were treated with 25 µM MG132 for 6 h. (B) Schematic representation of TRIM50 deletion mutants used in immunofluorescence assays. (C) The diagram shows the percentage of TRIM50 localization of each mutant.
A
B C
3.7 TRIM50 interacts with p62
TRIM50-associated proteins were isolated by immunoprecipitation of total protein lysate from FLAG-TRIM50#3, a cell line that stably expresses TRIM50, followed by nano LC-MS/MS identification. Among the putative TRIM50 interactors, we focused on p62/Sequestosome1 because of its involvement in the formation of protein aggregates (Babu et al., 2005), its role as shuttling factor for the delivery of polyubiquitinated substrates to the proteasome (Gal et al., 2007), and for its importance at the intersection of proteasome and autophagy pathways (Pankiv et al., 2007). First we assessed whether TRIM50 and p62 interact. Total lysates from FLAG-TRIM50#3 cell line transfected with a plasmid that expresses Myc-tagged p62 were immunoprecipitated with an anti-FLAG and immunoblotted with an anti-Myc specific antibody. An anti-Myc reactive band was exclusively precipitated in the presence of FLAG-TRIM50 (FIGURE 12A). Consistently, we detected FLAG- TRIM50 in protein lysates immunoprecipitated with an anti-Myc and immunoblotted with an anti-FLAG antibody (FIGURE 12B), substantiating the interaction between TRIM50 and p62.
3.8 TRIM50 promotes the sequestration of ubiquitinated proteins into aggresome
We asked whether TRIM50 has any role in the recruitment and/or accumulation of polyubiquitinated proteins to the aggresome. Using mild detergent, we separated detergent soluble and insoluble protein fractions from FLAG-TRIM50#3 cell and analysed them by western blotting. MG132 treatment resulted in a prominent accumulation of higher- molecular-weight species constituted by polyubiquitinated proteins in the detergent insoluble fraction as showed by immunoblot with an anti-ubiquitin antibody (FIGURE 12C). These data were confirmed in Trim50 deficient mouse embryo fibroblasts where depletion of endogenous Trim50 resulted in a decrease of polyubiquitinated protein levels in MG132 treated cells (compare lane 4 to 8 in FIGURE 12D).
Figure 12. TRIM50 interacts with p62 (A-B) The interaction between TRIM50 and p62 was assayed in FLAG-TRIM50#3 transfected with Myc-p62. The cell lysates were immunoprecipitated with anti-FLAG (left) and with anti-Myc (right) antibodies, respectively. Immunoblot were done as indicated (asterisk indicates FLAG- TRIM50 coming from the first FLAG blotting.(C) Lysates from FLAG-TRIM50#3 and FLAG#3 cell lines, treated with vehicle (–) or MG132 (+) were separated in RIPA detergent-soluble (S) and detergent insoluble (I) fractions and immunoblotting with anti-FLAG and anti-ubiquitin antibodies. (D) Lysates from MEF Trim50 cell lines with different genotype (+/+, -/-), treated with vehicle (–) or MG132 (+) were separated in RIPA detergent- soluble (S) and detergent insoluble (I) fractions, analyzed by Western Blot and immunoblotting with anti- ubiquitin antibody.
A B
C D
Since the aggregates formation is a reversible process, to explore the effect of TRIM50 on the clearance of aggresome components, we analyzed the aggresome insolvency. MG132- pretreated MEF Trim50 cells (kindly provided by Dr. Reymond, University of Lausanne) were incubated in a free-drug media for 48h, and the FK2-aggresome positive cells were counted. Immediately after the removal of MG132, we found a significant decrease of the number of FK2-positive aggregates in Trim50 -/- cells compared to the Trim50 +/+
(FIGURE 13 A-B). More interestingly, 48 hours after the MG132 removal we observed a significantly higher number of FK2-positive aggregates in MEF Trim50 -/- compared to MEF Trim50 +/+ suggesting that Trim50 is required for the clearance of polyubiquitinated proteins included within aggresome (p=0.03, FIGURE 13C). Overall our analysis suggests that TRIM50 plays an active role in the sequestration of polyubiquitinated proteins in the aggresome.
Figure 13. TRIM50 promotes the accumulation of polyubiquitinated proteins into the aggresome. (A) MEF Trim50 cell lines were stained with anti-FK2 antibody. The area, perimeter and intensity of signal of three independent experiments of each genotype were estimated using imageJ program. (B) MEF Trim50 cell lines were treated with 10 mM MG132 over night and stained with anti-FK2 antibody. The area, perimeter and intensity of signal of three independent experiments of each genotype were estimated using imageJ program. (C) MEF Trim50 cell lines were treated with 10 mM MG132 over night, incubated o and 48 h with DMEM after MG132 wash out and stained with anti-FK2 antibody. The diagram shows the percentage of aggresome-positive
A
B
C
4. DISCUSSION
In this PhD program project we combined biochemical and cellular strategies to decipher the biological function of TRIM50 with the aim to investigate its possible role in determining some of the clinical phenotypes observed in Williams Beuren syndrome patients.
The experimental data achieved in this study provide evidence that TRIM50 inclusion bodies are aggresome precursors and that TRIM50 is a novel protein involved in the ubiquitination and aggregation process of misfolded and damaged proteins. These data come mainly from the following findings: i) the E3 ubiquitin ligase TRIM50 forms heterogeneous cytoplasmic bodies containing polyubiquitinated proteins that localizes into aggresome when the proteasome activity is inhibited, ii) localization of TRIM50 bodies does not depend on TRIM50 E3 ubiquitin ligase activity, iii) TRIM50 aggresome localization depend on HDAC6, vi) TRIM50 interacts with HDAC6 and p62, both involved in the clearance of polyubiquitinated and misfolded protein aggregates, vi) proteomic approaches identified many TRIM50 interactors that have been found associated with aggresome in previous studies (Song et al., 2008; Wilde et al., 2011).
In order to characterize the functional link between TRIM50 and HDAC6 we performed biochemical assays based on the known function of HDAC6 as deacetylase. We showed that i) TRIM50 is deacetylated by HDAC6 and that intact microtubules are necessary for TRIM50 deacetylation, ii) CBP/p300 and PCAF protein drive TRIM50 acetylation, and iii) Lysine 372 is a residue involved in both TRIM50 acetylation and auto-ubiquitination.
Overall these data allowed us to hypothesize that HDAC6 deacetylates TRIM50 at K372 favouring its ubiquitination and its subsequently transport into aggresome associated to polyubiquitinated proteins. Moreover by competing with the same lysine aminoacid residue, the acetylation interferes with TRIM50 ubiquitination probably enhancing its own stability.
Such acetylation and ubiquitination competition has been already reported for other
proteins. For instance Lysine acetylation controls SMAD7 stability and promotes SMAD7 ubiquitination and degradation (Kume et al., 2007). Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination and proteosomal degradation of Notch1 ICD (Popko-Scibor et al., 2011); finally p14ARF, increases the level of p53 acetylated at lysine 382 in a nuclear chromatin-rich fraction increasing its stability and its transcription factor activity and inhibiting its degradation (van Leeuwen et al., 2013).
In this study we identified and confirmed the interaction of TRIM50 with p62. p62 is a multifunctional adapter protein implicated in autophagy, cell signalling, receptor internalization, inflammation and protein turnover. p62 interacts with ubiquitinated proteins carrying them on the road to autophagy-mediated degradation. The TRIM50-p62 interacting region involves amino acids 302-370, a region that includes the LC3-Interacting Region (LIR) domain involved in the binding to LC3 (microtubule-associated protein 1A/1B light chain 3) a modifier protein that plays a pivotal role in autophagosome biogenesis (Kabeya et al., 2000).
In that way the observed interaction between TRIM50 and LC3 (data not shown) is intriguing and deserves more investigations.
Increasing evidences indicate that autophagy-related proteins are sequestrated into the aggresome as a selective mechanism to regulate their degradation (Lee et al., 2010). Our result showing that TRIM50 controls the recruitment and clearance of polyubiquitinated proteins to aggresome and that TRIM50 interacts with HDAC6 and p62 allow us to make some speculations about a possible model of TRIM50 action (FIGURE 14). During physiological cellular condition, TRIM50 ubiquitinates targeted proteins toward the Proteasome for their degradation. On the contrary, when the proteasome activity is impaired, TRIM50 ensures the sequestration of its targets to the aggresome via the association with HDAC6 and their subsequent likely removal by p62-mediated autophagy. However, further
studies, particularly the identification of TRIM50 specific substrates being followed, are needed to unequivocally assess the authenticity of this model.
Ubiqui&na&on*
E2-UBH7*
TRIM50*
Ub*Ub*
TRIM50* Ub*
P300/PCAF(
HDAC6( Ac*
Acetyla&on*
TRIM50*
Active
Proteasome Impaired
Proteasome
Misfolded and ubiquitinated
proteins
Proteasome Microtubule
Sequestration of proteins into AGGRESOMES Protein Degradation
Autophagy
E3 Ligase E3 Ligase
HDAC6
p62
*
TRIM50
TRIM50
TRIM50 TRIM50
p62*
Figure 14. TRIM50 model of action. See main text for details
5. CONCLUSION
Overall the data obtained during this PhD project suggest that TRIM50 is an emerging player of aggregation processes involved polyubiquitinated proteins and in the regulation of protein aggregates clearance. Our study highlights how TRIM50 acetylation is a fundamental posttranslational modification of this E3 Ubiquitin ligase protein. Our study points to the existence of a potential crosstalk between acetylation and ubiquitination in the regulation of TRIM50.
Interestingly, although extensive studies are required, this study opens the way to an interesting hypothesis that the proven haploinsufficiency of TRIM50 could account for some WBS phenotypes through the accumulation of ubiquitinated proteins otherwise degraded.
Accumulation of polyubiquitinated protein aggregates is a hallmark of several neurodegenerative disorders as well as of a number of other protein aggregation diseases affecting muscles, heart, liver and lung. Of note and in line with our hypothesis the deposition of ß/A4 amyloid in senile plaques accompanied by neocortical neurofibrillary tangles, two well-established changes in Alzheimer's disease, have been observed in WBS patients {Golden, 1995 #10333}. p62 has been identified as a component of inclusion bodies in several human diseases, such as neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis) and in liver diseases (e.g., alcoholic hepatitis, hepatic steatosis, and hepatocellular carcinoma). It will hence be interesting to investigate whether TRIM50 is also a component of such bodies and it could even be responsible for targeting p62 to these sites.
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