Page 1 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Mutations of profilin-1 associated with amyotrophic lateral sclerosis promote aggregation due to structural changes of the native states
Edoardo Del Poggetto1, Francesco Bemporad1, Francesca Tatini2, Fabrizio Chiti1*
1
Department of Biomedical Experimental and Clinical Sciences, Section of Biochemistry, University of Florence, Viale Morgagni 50, I-50134, Florence, Italy. 2
Institute of Applied Physics Nello Carrara, National Research Council, Via Madonna del Piano 10, 50019, Sesto Fiorentino (FI), Italy.
*
To whom correspondence should be addressed. e-mail:
[email protected]; tel.: 0039-055-
2751220
ACS Paragon Plus Environment
1
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 29
ABSTRACT
The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T and G118V, have been found in patients with familial forms of the disease and another, E117G, proposed to be a moderate risk factor for disease onset. In this work we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was not found to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism and intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behaviour is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.
ACS Paragon Plus Environment
2
Page 3 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Amyotrophic lateral sclerosis (ALS) is a neuropathological condition associated with the progressive degeneration of motoneurons in the brain, brainstem, and spinal cord1. It results in rapidly progressing muscle weakness, atrophy, fasciculation, spasticity, difficulty in moving, speaking, swallowing, breathing, with all these symptoms reflecting progressive degeneration of motoneurons of the primary motor cortex, corticospinal tracts, brainstem and spinal cord1, 2. At the histopathological level, ALS is generally characterised by the formation of intracellular cytoplasmatic inclusions of the TAR DNAbinding protein 43 (TDP-43) in the spinal cord, hippocampus and cortex3, 4, although a fraction of the genetic cases of ALS have inclusions of other proteins, such as superoxide dismutase 15, RNA binding protein FUS6 and C9orf727. Approximately 10% of ALS cases are familial (fALS), with autosomic dominant inheritance and involving mutations in one of ca. 20 genes so far identified8. PFN1 is one of such genes, coding for profilin-19. Using exome capture followed by sequencing of two large families, Wu and co-workers were able to identify two mutations of profilin-1 causing fALS, namely C71G and M114T9. In each of the two families the mutation co-segregated with the disease and the patients with fALS were heterozygous. By sequencing a database with 272 individuals suffering from fALS of unidentified genetic origin, five carriers of profilin-1 mutations were found. Two had the C71G substitution and for one of them the DNA was available for members of the same family, confirming that the C71G cosegregated with the disease and was indeed pathogenic. A third patient had the M114T mutation, which was also found in one relative with the disease (no other family members were investigated). The remaining two patients had the G118V and E117G mutations, respectively, but DNA was not available for other family members. The sequencing of the PFN1 gene within the NHLBI ESP Exome Variant Server, the 1000 Genome Project Database, a panel of 1089 healthy individuals, and a panel of 816 patients suffering with sporadic ALS, did not reveal the C71G, M114T and G118V mutations, lending support to the idea that they are all pathogenic. By contrast, the E117G mutation was found in three non-ALS cases, raising the hypothesis that this mutation is a benign polymorphism rather than a fALS causing mutation. By comparing the frequency of the mutation among the ALS and non-ALS cases, the authors concluded that the E117G is probably a moderate risk factor for developing fALS. Subsequent analyses suffragated this conclusion10-12. Other mutations were reported later, including T109M, A20T, Q139L and R136W10,
13, 14
. All of them are under investigation to assess whether they are pathogenic or
represent benign polymorphisms. Profilin-1 is a 139-residue, α+β protein belonging to the profilin-like structural family15. The NMR structure (PDB entry 1PFL) shows that it consists of an antiparallel, 7-stranded β-sheet packed ACS Paragon Plus Environment
3
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 29
between three amphipathic α-helices, two on one side of the sheet and one on the other side15. The Xray structure confirms this overall fold (DPB entry 1FIK) although a full scientific report has not yet appeared. Profilin-1 is known to bind to monomeric G-actin and act as a complex regulator of filamentous F-actin growth through its binding to monomeric G-actin and exchange of ADP with ATP on G-actin16, 17. Expression in neuroblastoma N2A cells and primary motoneurons of the four mutants of profilin-1 that were first identified resulted in the formation of profilin-1-positive, ubiquitin-positive cytoplasmic inclusions9. It was also shown that the mutations inhibit the binding of profilin-1 to Gactin in HEK293 cells and axon outgrowth in primary motoneurons, possibly resulting in the alteration of the cytoskeleton organisation9. Therefore, the aggregation of profilin-1 following mutation could be the indirect result of a decreased affinity for G-actin. Indeed, it has been shown for other systems, such as tau and apoA1, that the decrease of binding affinity for partner proteins or lipids following mutation is the cause of the aggregation of these systems18,
19
. It may well be that the decreased binding of
profilin-1 mutants to G-actin is a consequence of the aggregation of such mutants. Hence, the role of profilin-1 aggregation in this form of fALS, whether the mutations are able to induce aggregation of the protein intrinsically and independently of their effect on G-actin binding and the mechanism by which the mutations increase such an aggregation propensity, if any, are issues that remain to be addressed. In this work we have purified wild-type profilin-1 and the four mutants previously reported9. We have characterised all of them with an array of biophysical techniques to determine their structural features, conformational flexibility, overall conformational stability and propensity to aggregate in vitro. We will show that the four mutants have aggregation propensities that are higher, to various extents, than that of the wild-type protein and that such a propensity correlates with the degree of the changes of the overall folded structure and its conformational flexibility in this group of variants. The data have implications for elucidating why profilin-1 mutations induce PFN1-associated fALS and reveal the mechanisms through which the pathogenic effects of these mutations are mediated.
RESULTS AND DISCUSSION Wild-type profilin-1 forms fibrils We started our analysis by exploring suitable conditions for inducing aggregation of wild-type profilin1. The protein mother solution was filtered and centrifuged to eliminate any pre-existing aggregates and was then diluted to a concentration of 0.8mg/mL in 100mM Tris/HCl, 11mM NaCl, pH 7.4, 37°C. ACS Paragon Plus Environment
4
Page 5 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
At regular time points aliquots were withdrawn for the analysis with the thioflavin T (ThT) assay, the Congo red (CR) assay, transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD) and Fourier transform infrared spectroscopy (FTIR), in the absence of any further centrifugation and/or filtration. The protein sample was unable to increase significantly ThT fluorescence within the first 8-9 days (Figure 1a, inset). A rapid growth of ThT fluorescence was observed in the following days until a plateau was reached after 14-15 days (Figure 1a, inset). This kinetic profile is typical of a nucleated growth process where mature aggregates form rapidly after a few nuclei are formed.20 The TEM inspection showed the presence of oligomers and short curvilinear fibrils with a diameter of ~13nm after 4 days (Figure 1a). At the end of the lag phase the sample was still dominated by short curved fibrils, but these appear to have increased in length (Figure 1b). At the plateau the sample is dominated by straight fibrils having a diameter of ~13nm, often associating together to form bundles of fibrils with larger diameters (Figure 1c). A high magnification image of these fibrils shows that the 13nm fibrils were formed by a number of 3-4nm wide, yet discontinuous, protofilaments that associate laterally (Figure 1d). The ThT fluorescence spectra indicate that the native protein at day 0, as well as the first oligomers and short curvilinear fibrils present during the lag phase after 4 and 9 days did not bind ThT, whereas the straight fibrils present in the sample after 15 days did (Figure 1e). The absence of ThT binding during the lag phase, when aggregates are however present, indicates that profilin-1 aggregation occurs through a conformational nucleated conversion, in which the first aggregates lack a regular structure and need to reorganise to form structured nuclei competent for fast fibril growth20, 21. Albeit significant, the fluorescence increase occurring later, at the end of the growth phase, is small compared to well-structured fibrils studied for other protein systems. The CR assay shows no binding with CR at all time points, confirming the absence of a highly regular structure in the fibrils (Figure 1f). Far-UV CD spectra acquired after 4 and 9 days had small values of mean residue ellipticity relative to the spectrum at 0 days, indicating severe differential absorption flattening22, which limits our ability to interpret the spectra (Figure 1g). This phenomenon became even more severe after 15 days with a flat CD spectrum. Flat CD spectra of this type are normally found when large protein sediments are present23. Despite these limitations, the far-UV CD spectra appeared to be different before and after the mature fibrils are present, making far-UV CD a suitable probe to follow the formation of the mature, ThT-positive fibrils (see below). FTIR spectra of protein aggregates do not present problems of differential absorption flattening. The amide I region of the FTIR spectrum acquired after 15 days showed a composite band contour with many overlapping contributions. The second derivative of this spectrum showed three major bands at ACS Paragon Plus Environment
5
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 29
1626, 1650 and 1675cm-1, which can readily be assigned to intermolecular β-sheet structure, αhelical/unordered and β-turn structure, respectively (Figure 1h). The Fourier self-deconvolution of the original spectrum confirmed this result, with three major bands at 1627, 1650 and 1671cm-1 (data not shown). The second derivative FTIR spectrum of native profilin-1 (0 days) shows that the β-sheet component is at ca. 1638cm-1, representing intramolecular, as opposed to intermolecular24, β-sheet structure (Figure 1h). Hence, the FTIR analysis indicated the presence of intermolecular β-sheet structure for a limited portion of the polypeptide chain, with the rest of the sequence adopting β-turn and unordered structure. The weak increase of ThT fluorescence, the overall absence of CR binding and discontinuous morphology of the protofilaments visualised with TEM indicate the intermolecular β-sheet structure present in the fibrils lacks structural order.
Mutants of profilin-1 have very different aggregation propensities We then compared the aggregation rates of the mutants with that of the wild-type protein. In this set of experiments wild-type and mutant proteins were incubated as described above, but under constant agitation and in larger test-tubes to increase the water-air interface (see methods for details). Both conditions are known to accelerate aggregation25-28, making our tests more rapid, reproducible and repeatable. The ThT fluorescence time courses indicate remarkable differences in this group of mutants (Figure 2). The wild-type protein induces the maximum ThT fluorescence emission within 10 days and this value is 14.8±1.3a.u. above that of free ThT (blank). The E117G mutant reaches its maximum ThT fluorescence emission at day 3 with this value amounting to 18.1±1.6a.u. The M114T mutant has a higher aggregation propensity as it reaches the maximum ThT fluorescence at day 2 (30.5±2.5a.u.). In this rank the G118V follows, with a maximum ThT fluorescence of 53.7±3.5a.u. at day 2. The C71G is the most aggregating mutant with a maximum ThT fluorescence of 72.1±3.2a.u. reached after 1 day or less. The maximum ThT fluorescence measured in these experiments is proportional to the quantity of protein aggregates present in the sample, rather than the structural order present in the aggregates, as shown by quantification with the bicinchoninic acid (BCA) protein assay (Figure S1). The rank of aggregation propensity is therefore WT
