Monitoring fibril formation of the N-terminal domain of PABPN1 carrying an alanine repeat by tryptophan fluorescence and real-time NMR

Intranuclear fibrils due to poly-alanine expansions in the N-terminal domain of the poly(A) binding protein PABPN1 correlate with the disease oculopharyngeal muscular dystrophy (OPMD). For monitoring fibril formation by fluorescence and real-time NMR spectroscopy, tryptophans were introduced either into the middle or C-terminal of the poly-alanine segment. The kinetics of fibril formation which were monitored by fluorescence spectroscopy were matched by real-time NMR kinetics. Our results show that fibril formation is concomitant with the burial of the tryptophans in the fibrillar core. Since no soluble pre-fibrillar intermediate(s) was detected, fibril formation of this domain may be regarded as a two state conversion from an unfolded soluble into folded insoluble species.     

Effect of oculopharyngeal muscular dystrophy-associated extension of seven alanines on the fibrillation properties of the N-terminal domain of PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that usually manifests itself within the fifth decade. The most prominent symptoms are progressive ptosis, dysphagia, and proximal limb muscle weakness. The disorder is caused by trinucleotide (GCG) expansions in the N-terminal part of the poly(A)-binding protein 1 (PABPN1) that result in the extension of a 10-alanine segment by up to seven more alanines. In patients, biopsy material displays intranuclear inclusions consisting primarily of PABPN1. Poly l-alanine-dependent fibril formation was studied using the recombinant N-terminal domain of PABPN1. In the case of the protein fragment with the expanded poly l-alanine sequence [N-(+7)Ala], fibril formation could be induced by low amounts of fragmented fibrils serving as seeds. Besides homologous seeds, seeds derived from fibrils of the wild-type fragment (N-WT) also accelerated fibril formation of N-(+7)Ala in a concentration-dependent manner. Seed-induced fibrillation of N-WT was considerably slower than that of N-(+7)Ala. Using atomic force microscopy, differences in fibril morphologies between N-WT and N-(+7)Ala were detected. Furthermore, fibrils of N-WT showed a lower resistance against solubilization with the chaotropic agent guanidinium thiocyanate than those from N-(+7)Ala. Our data clearly reveal biophysical differences between fibrils of the two variants that are likely caused by divergent fibril structures.     

A folded and functional protein domain in an amyloid-like fibril

The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role.     

The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

Background

Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results

In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions

Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.     

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.     

Trinucleotide expansions leading to an extended poly-L-alanine segment in the poly (A) binding protein PABPN1 cause fibril formation

The nuclear poly(A) binding protein (PABPN1) stimulates poly(A) polymerase and controls the lengths of poly(A) tails during pre-mRNA processing. The wild-type protein possesses 10 consecutive Ala residues immediately after the start methionine. Trinucleotide expansions in the coding sequence result in an extension of the Ala stretch to maximal 17 Ala residues in total. Individuals carrying the trinucleotide expansions suffer from oculopharyngeal muscular dystrophy (OPMD). Intranuclear inclusions consisting predominantly of PABPN1 have been recognized as a pathological hallmark of the genetic disorder. To elucidate the molecular events that lead to disease, recombinant PABPN1, and N-terminal fragments of the protein with varying poly-L-alanine stretches were analyzed. As the full-length protein displayed a strong tendency to aggregate into amorphous deposits, soluble N-terminal fragments were also studied. Expansion of the poly-L-alanine sequence to the maximal length observed in OPMD patients led to an increase of alpha-helical structure. Upon prolonged incubation the protein was found in fibrils that showed all characteristics of amyloid-like fibers. The lag-phase of fibril formation could be reduced by seeding. Structural analysis of the fibrils indicated antiparallel beta-sheets.     

A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic toxicity of PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Polyalanine-independent conformational conversion of nuclear poly(A)-binding protein 1 (PABPN1)

Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.     

Conformational stability of the RNP domain controls fibril formation of PABPN1

The disease oculopharyngeal muscular dystrophy is caused by alanine codon trinucleotide expansions in the N‐terminal segment of the nuclear poly(A) binding protein PABPN1. As histochemical features of the disease, intranuclear inclusions of PABPN1 have been reported. Whereas the purified N‐terminal domain of PABPN1 forms fibrils in an alanine‐dependent way, fibril formation of the full‐length protein occurs also in the absence of alanines. Here, we addressed the question whether the stability of the RNP domain or domain swapping within the RNP domain may add to fibril formation. A variant of full‐length PABPN1 with a stabilizing disulfide bond at position 185/201 in the RNP domain fibrillized in a redox‐sensitive manner suggesting that the integrity of the RNP domain may contribute to fibril formation. Thermodynamic analysis of the isolated wild‐type and the disulfide‐linked RNP domain showed two state unfolding/refolding characteristics without detectable intermediates. Quantification of the thermodynamic stability of the mutant RNP domain pointed to an inverse correlation between fibril formation of full‐length PABPN1 and the stability of the RNP domain.     

Oculopharyngeal muscular dystrophy: potential therapies for an aggregate-associated disorder

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disease caused by the abnormal expansion of a polyalanine tract within the coding region of poly(A) binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. The mechanism by which the polyalanine expansion mutation in PABN1 causes disease is unclear. However, the mutation is thought to confer a toxic gain-of-function on the protein. Despite controversy over the role of aggregates, it has been consistently shown that agents that reduce aggregate load in cell models of OPMD also reduce levels of cell death. Recently generated animal models of OPMD will help elucidate the mechanism of disease and allow the trial of potential therapeutics. Indeed, administration of known anti-aggregation drugs attenuated muscle weakness in an OPMD mouse model. This suggests that anti-aggregation therapies may be beneficial in OPMD.     

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis

Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.     

Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG _D = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG _D = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.     

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.     

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8-13, leading to a stretch of 12-17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.     

Oculopharyngeal muscular dystrophy (OPMD): analysis of the PABPN1 gene expansion sequence in 86 patients reveals 13 different expansion types and further evidence for unequal recombination as the mutational mechanism

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset neuromuscular degenerative disease characterised by proximal muscle weakness, ptosis and swallowing difficulty. The causative genetic abnormality is an expansion consisting of 2–7 additional base triplets in a repeat sequence in exon 1 of the PABPN1 (PABP2) gene and results in an increase in length of the polyalanine tract in the PABPN1 protein from 10 to 12–17 residues. The expansions are stable through meiosis and mitosis suggesting a different mechanism of mutation from that of most other triplet repeat mutations. Most reports describe OPMD expansions as consisting of multiples of a GCG sequence. However, some studies have detected GCA interspersions. We have analysed 86 OPMD patients with a PABPN1 gene expansion, including three compound heterozygotes, and have identified 13 different types of expansion mutation, six of which contain GCA and GCG and almost all of which are consistent with a mutational mechanism of unequal recombination.     

Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1

Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity.     

Sequence determinants regulating fibrillation of human alpha-synuclein

α-Synuclein is a neural protein that comprises the fibrillar core of Lewy bodies, a histologically defining lesion of Parkinson’s disease. To investigate the role of each specific residue of the α-synuclein molecule in fibril formation, amino acid substitutions were introduced throughout the molecule. Incorporation of proline, especially in the region spanning residues 37-89, drastically retarded fibril formation. Substitutions with polar residues showed that the hydrophobicity of the central hydrophobic region is also important in fibrillation regulation. In the N-terminal repeated region, increasing the number of negative charges interfered with fibrillation. In contrast, single amino acid substitutions in the C-terminal acidic region of α-synuclein had only minimal effects on fibrillation. More than 20 different single amino acid substitutions that were sufficient to prevent fibrillation of α-synuclein were obtained, and most of them were impaired in both nucleation and fibril elongation. Identification of sequence determinants regulating fibrillation of amyloidogenic proteins may provide valuable information for designing peptide analog drugs to prevent protein amyloidosis.