Polyalanine Stretch Research Articles

Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination

Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

P.355 - Mitochondrial dysfunction in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by ptosis, dysphagia and weakness of proximal limbs. OPMD is caused by the expansion of polyalanine in poly(A)-binding protein, nuclear 1 (PABPN1). The molecular pathogenesis is still poorly understood. As for today, mitochondrial abnormality has been proposed as the possible etiology. The aim of our study is to evaluate mitochondrial function in animal and cell models of OPMD, and to realize the involvement of mitochondrial dysfunction in the pathogenesis of OPMD. First, we employed transgenic mice expressing high level of expanded human PABPN1 with 13-alanine stretch driven by cytomegalovirus early enhancer element/ chicken beta actin (CAG) promoter. Immunofluorescence staining of skeletal muscles showed that PABPN1 co-localized with mitochondria. PABPN1 was detected in mitochondria fraction from the skeletal muscle, and PABPN1 located at the cytosolic face of outer membrane of mitochondria. Expression levels and enzymatic activities of respiratory chain complex proteins were reduced in the skeletal muscle of transgenic mice, compared with non-transgenic littermates. Next, we used cell model expressing PABPN1 with 10-alanine and 18-alanine stretch. We also observed mitochondrial dysfunction in cells with expanded polyalanine stretch. We proposed that expansion of polyalanine in PABPN1 may cause mitochondrial dysfunction, and it may be a possible mechanism of myodegeneration in OPMD.

MeCP2_E1 N-terminal modifications affect its degradation rate and are disrupted by the Ala2Val Rett mutation

Methyl CpG-binding protein 2 (MeCP2), the mutated protein in Rett syndrome (RTT), is a crucial chromatin-modifying and gene-regulatory protein that has two main isoforms (MeCP2_E1 and MeCP2_ E2) due to the alternative splicing and switching between translation start codons in exons one and two. Functionally, these two isoforms appear to be virtually identical; however, evidence suggests that only MeCP2_E1 is relevant to RTT, including a single RTT missense mutation in exon 1, Ala2Val. Here, we show that N-terminal co- and post-translational modifications differ for MeCP2_E1 and MeCP2_E1-Ala2Val, which result in different protein degradation rates in vitro. We report complete N-methionine excision (NME) for MeCP2_E1 and evidence of excision of multiple alanine residues from the N-terminal polyalanine stretch. For MeCP2_E1-Ala2Val, we observed only partial NME and N-acetylation (NA) of either methionine or valine. The localization of MeCP2_E1 and co-localization with chromatin appear to be unaffected by the Ala2Val mutation. However, a higher proteasomal degradation rate was observed for MeCP2_E1-Ala2Val compared with that for wild type MeCP2_E1. Thus, the etiopathology of Ala2Val is likely due to a reduced bio-availability of MeCP2 because of the faster degradation rate of the unmodified defective protein. Our data on the effects of the Ala2Val mutation on N-terminal modifications of MeCP2 may be applicable to Ala2Val mutations in other disease genes for which no etiopathological mechanism has been established.

Common PHOX2B poly-alanine contractions impair RET gene transcription, predisposing to Hirschsprung disease

HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.

Association of FOXE1 gene polymorphism with idiopathic premature ovarian failure

To assess the influence of length of the alanine tract of forkhead box E1 (FOXE1) gene on genetic susceptibility to idiopathic premature ovarian failure (POF). Totally 110 patients with idiopathic POF were recruited between February 2009 and December 2012 at the Affiliated Shenzhen City Maternity and Child Healthcare Hospital of Southern Medical University. Controls (n = 110) were individuals with normal menstrual cycles, normal FSH concentrations. The polyalanine tract and flanking sequence of FOXE1 were screened using the multiplex ligation-dependent probe amplification (MLPA) technique and direct sequence technique. The most frequent of FOXE1 polyalanine stretch length was 14 residues in both groups. The length of FOXE1 polyalanine reported in this study varied from 12 to 16 alanines, and three variants of FOXE1-polyalanine length, containing 12, 14, or 16 alanine residues, and 5 different genotypes were identified. The most common genotypes were 14/14 homozygote, occurring with the frequency of 81.8% (90/110) in the POF group, while 96.4% (106/110) in control subjects, respectively. The incidence of 14/14 genotypes of FOXE1-polyalanine was significantly lower in patients with POF (χ² = 119.730, P = 0.001) in comparison to the controls. There were significantly higher frequencies of the 16/16 genotypes in cases with POF [10.0% (11/110) versus 0; χ² = 3.403, P = 0.001], as compared with the controls. The FOXE1 14 alanine allele was significantly less common in the POF patient group than the controls [84.5% (186/220) versus 98.2% (216/220); χ² = 25.923, P = 0.001]. The FOXE1 16 alanine allele was significantly more common in the POF patient group than the controls [12.7% (28/220) versus 1.8% (4/220); χ² = 19.412, P = 0.001]. The polymorphism of the polyalanine tract of FOXE1 gene have a certain relevance for the genetic aetiology of idiopathic POF.

Functional classification and mutation analysis of a synpolydactyly kindred.

The aim of the present study was to analyze a congenital syndactyly/polydactyly kindred and propose a new functional classification method of clinical significance. The modes of inheritance and mutational mechanisms were also determined using genetic analyses. Hand and foot anatomy and functions were measured using photographic images, X-ray imaging and grip ability tests. Genetic analysis comprised the genotyping of polymorphic microsatellite markers at known polydactyly-associated loci and the sequencing of the candidate gene. A functional classification system was devised to divide the clinical features into three types, which included mild, moderate or severe deformity. The family was concluded to have syndactyly type II with autosomal dominant inheritance. The microsatellites, D2S2310 and D2S2314, at the 2q31–32 chromosome, which have previously been associated with synpolydactyly type I, were found to be associated with the disorder in the current family. A 27-bp insertion mutation was identified in the affected individuals in the HOXD13 gene at this locus. The insertion added a further nine alanine residues to the polyalanine stretch within the encoded protein. In conclusion, the functional classification method described in the present study may be used to guide surgical approaches to treatment. A family was identified in whom expansion of the polyalanine tract in the HOXD13 gene causes autosomal dominant hereditary synpolydactyly.

Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins

Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n⩾2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-β structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-β-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into β-sheet structures, which is necessary for nucleation and beneficial for fibril growth.

The E3 ubiquitin ligase TRIM11 mediates the degradation of congenital central hypoventilation syndrome-associated polyalanine-expanded PHOX2B

Expansions of a polyalanine (polyA) stretch in the coding region of the PHOX2B gene cause congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by the absence of adequate control of autonomic breathing. Expansion of polyA in PHOX2B leads to protein misfolding and accumulation into inclusions. The mechanisms that regulate mutant protein degradation and turnover have been poorly elucidated. Here, we investigate the regulation of degradation of wild-type and polyA-expanded PHOX2B. We show that expanded PHOX2B is targeted for degradation through the ubiquitin-proteasome system, resulting in lowered levels of the mutant protein relative to its wild-type counterpart. Moreover, we show that mutant PHOX2B forms ubiquitin-positive inclusions, which sequester wild-type PHOX2B. This sequestration correlates with reduced transcriptional activity of endogenous wild-type protein in neuroblastoma cells. Finally, we show that the E3 ubiquitin ligase TRIM11 plays a critical role in the clearance of mutant PHOX2B through the proteasome. Importantly, clearance of mutant PHOX2B by TRIM11 correlates with a rescue of PHOX2B transcriptional activity. We propose that CCHS is partially caused by a dominant-negative effect of expanded PHOX2B due to the retention of the wild-type protein in pathogenic aggregates. Our results demonstrate that TRIM11 is a novel modifier of mutant PHOX2B toxicity and represents a potential therapeutic target for CCHS.

Comprehensive genotype–phenotype analysis in 230 patients with tetralogy of Fallot

BackgroundTetralogy of Fallot (ToF), the most frequent cyanotic congenital heart disease, is associated with a wide range of intra- and extracardiac phenotypes. In order to get further insight into genotype–phenotype...

The first missense mutation causing Rett syndrome specifically affecting the MeCP2_e1 isoform

We report the identification of the first de novo mutation at a highly conserved residue within the polyalanine stretch in the N-terminal region of the brain-dominant protein isoform MeCP2_e1 in a girl with classical Rett syndrome. The missense mutation, p.Ala2Val, leads to severe developmental delay, microcephaly, no language, severe epilepsy, and cognitive impairment. To evaluate the pathogenic potentials of the MECP2 mutation specific to the MeCP2_e1 isoform detected in this patient, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to generate a fusion protein with c-myc, and constructs were transfected in COS7 cells. In vitro studies demonstrated that, like wild-type MeCP2e_1, the N-terminal mutant is localized in the nucleus. Neither transcriptional nor translational effect on the MeCP2_e2 isoform was observed in fibroblasts from the p.Ala2Val patient, suggesting that MeCP2_e1 is involved in other functional process. These data suggest the important involvement of the N-terminus in the function of MeCP2 protein, and provide further evidence for the major impact of a specific MeCP2e_1 deficiency in the development of intellectual processing.

P2X5 Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.

Prevention of oculopharyngeal muscular dystrophy-associated aggregation of nuclear poly(A)-binding protein with a single-domain intracellular antibody

Oculopharyngeal muscular dystrophy (OPMD) belongs to the group of protein aggregation disorders and is caused by extensions of the N-terminal polyalanine stretch of the nuclear polyA-binding protein 1 (PABPN1). The presence of PABPN1-containing intranuclear aggregates in skeletal muscle is unique for OPMD and is also observed in transgenic mouse and cell models for OPMD. These models consistently support a direct role for the protein aggregation in OPMD pathogenesis. We have isolated and characterized a diverse panel of single-domain antibody reagents (VHH), recognizing different epitopes in PABPN1. The antibody reagents specifically detect endogenous PABPN1 in cell lysates on western blot and label PABPN1 in cultured cells and muscle sections. When expressed intracellularly as intrabodies in a cellular model for OPMD, aggregation of PABPN1 was prevented in a dose-dependent manner. More importantly yet, these intrabodies could also reduce the presence of already existing aggregates. Given the domain specificity of VHH-mediated aggregation interference, this approach at least allows the definition of the nucleation kernel in aggregation-prone proteins, thus facilitating etiological insight into this and other protein aggregation disorders, and ultimately, it may well provide useful therapeutic agents.

Molecular consequences of PHOX2B missense, frameshift and alanine expansion mutations leading to autonomic dysfunction

Heterozygous mutations of the PHOX2B gene account for a broad variety of disorders of the autonomic nervous system, either isolated or combined, including congenital central hypoventilation syndrome (CCHS), tumours of the sympathetic nervous system and Hirschsprung disease. In CCHS, the prevalent mutation is an expansion of a 20-alanine stretch ranging from +5 to +13 alanines, whereas frameshift and missense mutations are found occasionally. To determine the molecular basis of impaired PHOX2B function, we assayed the transactivation and DNA binding properties of wild-type and mutant PHOX2B proteins. Furthermore, we investigated aggregate formation by proteins with polyalanine tract expansions ranging from +5 to +13 alanines using immunofluorescence of transfected cells and gel filtration of in vitro translated proteins. We found that transactivation of the dopamine beta-hydroxylase promoter by PHOX2B proteins with frameshift and missense mutations was abolished or severely curtailed, as was in vitro DNA binding although the proteins localized to the nucleus. The transactivation potential of proteins with polyalanine tract expansions declined with increasing length of the polyalanine stretch, and DNA binding was affected for an expansion of +9 alanines and above. Cytoplasmic aggregation in transfected cells was only observed for the longest expansions, whereas even the short expansion mutants were prone to form multimers in vitro. Such a tendency to protein misfolding could explain loss of transactivation for alanine expansion mutations. However, additional mechanisms such as toxic gain-of-function may play a role in the pathogenic process.

Poly‐(L‐alanine) expansions form core β‐sheets that nucleate amyloid assembly

Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.

In vivo aggregation properties of the nuclear poly(A)-binding protein PABPN1

A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a dominantly inherited disease caused by (GCG) repeat expansions in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions. Here we show that normal PABPN1 is inherently aggregation-prone when exogenously expressed in either HeLa or myogenic C2 cells. Similar deposits of insoluble PABPN1 are formed by variant forms of the protein containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the mutation responsible for OPMD is not essential for formation of PABPN1 inclusions. In contrast, interfering with any of the protein domains required for stimulation of poly(A) polymerase prevents the formation of inclusions. Most surprisingly, photobleaching experiments reveal that both normal and expanded PABPN1 molecules are not irreversibly sequestered into aggregates, but rather move rapidly in and out of the inclusions. These findings have important implications for the interpretation of OPMD model systems based on exogenous expression of PABPN1.

Oculopharyngeal muscular dystrophy-like nuclear inclusions are present in normal magnocellular neurosecretory neurons of the hypothalamus.

Intranuclear inclusions composed of tubular filaments constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD). Autosomal dominant OPMD is caused by (GCG) repeat expansions in the gene that encodes for poly(A) binding protein nuclear 1 (PABPN1). The mutation results in the expansion of a polyalanine stretch in the N-terminus of the protein. It has been proposed that mutated PABPN1 induces protein aggregation, which in turn causes the formation of the filamentous nuclear inclusions. Here we report the presence of intranuclear inclusions composed of tubular filaments in oxytocin-producing neurons from normal rat hypothalamus. Like OPMD inclusions, the filamentous structures in neurosecretory neurons accumulate PABPN1, poly(A) RNA, ubiquitin and proteasomes. These inclusions do not contain members of Hsp40 and HDJ-2/DNAJ families of chaperones. The proportion of oxytocin-producing neurons that contain inclusions decreases during parturition and lactation (when synthesis and release of oxytocin is maximal) and increases at 1 day post-weaning (when occurs a drastic reduction in the production of the hormone). Thus, PABPN1 filaments in normal neurons are dynamic structures, the appearance of which correlate with changes in cellular activity. These data provide the first physiological evidence that polyalanine expansions are not essential to induce polymerization of PABPN1 into filamentous nuclear inclusions.

Differential role of the loop region between helices H6 and H7 within the orphan nuclear receptors small heterodimer partner and DAX-1.

The orphan nuclear receptors small heterodimer partner (SHP) and dosage-sensitive sex-reversal adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1 (DAX-1) contain extra amino acids between helices H6 and H7 of LBD, and here we investigated a possible role of these additional amino acids. Transient transfection assay demonstrated that, in contrast to wild type, in mutant SHP Delta128-139 deletion of 12 extra amino acids in H6-H7 failed to repress the transactivity of orphan nuclear receptors such as estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Interestingly, yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that wild-type and SHP Delta128-139 have similar abilities to interact with estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Unexpectedly, in wild-type DAX-1 and mutant DAX-1 Delta338-362, deletion of 25 extra amino acids in H6-H7 had no significant difference in the interaction and repression of steroidogenic factor 1 transactivation. Mutant SHP that contains DAX-1 extra amino acids or polyalanine stretch in H6-H7 showed indistinguishable pattern of repression from wild-type SHP. Interestingly, the swapped SHP mutant with DAX-1 extra amino acids interacted with EID-1 (E1A-like inhibitor of differentiation 1), which is characterized as an SHP-interacting corepressor. However, interaction between SHP Delta128-139 and EID-1 was significantly diminished. Moreover, SHP-mediated repression of constitutive androstane receptor transactivation was significantly released by down-regulation of EID-1 expression with EID-1 small interfering RNA. The present study suggests that H6-H7 loop regions of SHP and DAX-1 play a different role in the repression of nuclear receptor transactivation.

Caspase 8 mediated apoptotic cell death induced by β-sheet forming polyalanine peptides

Expansion of a polyalanine stretch from 10 to 12–17 residues in the N-terminus of the protein PABP2 has been implicated in the genetically acquired disease oculopharyngeal muscular dystrophy, characterized by nuclear protein deposits. Here we report a correlation between the structural properties and cell toxicity of two peptides mimicking the N-terminal domain of PABP2: one containing seven and the other 11 uninterrupted alanine residues. Consistent with earlier observations, the longer peptide (11-ala) was found to adopt β-sheet structure while the shorter one (7-ala) formed α-helix over a wide range of concentrations (∼20–500 μM). We observed that treatment with 11-ala resulted in significantly enhanced death of Chinese hamster V79 cells, compared to the effect of treatment with 7-ala, via the cytochrome c mediated apoptotic pathway. Increases in caspase 8 and caspase 3 activity were also observed in human cells (K562) treated with 11-ala. These results indicate that the toxicity of pathogenic peptides is directly linked to their β-sheet structure and also support recent observations that small oligomeric species of peptides and proteins are the key toxic elements in causing protein aggregation diseases.

HnRNP A1 and A/B interaction with PABPN1 in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)6 repeat to (GCG) in the PABPN1 gene. The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1. The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death. Human fetal brain cDNA library was used to look for PABPNI binding proteins using yeast two-hybrid screen. The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays. Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions. We identify two PABPNI interacting proteins, hnRNP A1 and hnRNP A/B. When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B co-localize with mPABPN1 in the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death.

Progress in understanding the pathogenesis of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping (ptosis), swallowing difficulties (dysphagia), and proximal limb weakness. The autosomal dominant form of this disease is caused by expansions of a (GCG)6 repeat to (GCG)8-13 in the PABPN1 gene. These mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminal domain of PABPN1. Mutated PABPN1 (mPABPN1) induces the formation of muscle intranuclear inclusions that are thought to be the hallmark of this disease. In this review, we discuss: 1) OPMD genetics and PABPN I function studies; 2) diseases caused by polyalanine expansions and cellular polyalanine toxicity; 3) mPABPN1-induced intranuclear inclusion toxicity; 4) role of oligomerization of mPABPNI in the formation and toxicity of OPMD intranuclear inclusions and; 5) recruitment of subcellular components to the OPMD inclusions. We present a potential molecular mechanism for OPMD pathogenesis that accounts for these observations.