Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases

Background

The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases.

Scope of review

In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof.

Major conclusions

Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity.

General significance

Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.     

In silico prediction of novel residues involved in amyloid primary nucleation of human I56T and D67H lysozyme

Background

Amyloidogenic proteins are most often associated with neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, but there are more than two dozen human proteins known to form amyloid fibrils associated with disease. Lysozyme is an antimicrobial protein that is used as a general model to study amyloid fibril formation. Studies aimed at elucidating the process of amyloid formation of lysozyme tend to focus on partial unfolding of the native state due to the relative instability of mutant amyloidogenic variants. While this is well supported, the data presented here suggest the native structure of the variants may also play a role in primary nucleation.

Results

Three-dimensional structural analysis identified lysozyme residues 21, 62, 104, and 122 as displaced in both amyloidogenic variants compared to wild type lysozyme. Residue interaction network (RIN) analysis found greater clustering of residues 112–117 in amyloidogenic variants of lysozyme compared to wild type. An analysis of the most energetically favored predicted dimers and trimers provided further evidence for a role for residues 21, 62, 104, 122, and 112–117 in amyloid formation.

Conclusions

This study used lysozyme as a model to demonstrate the utility of combining 3D structural analysis with RIN analysis for studying the general process of amyloidogenesis. Results indicated that binding of two or more amyloidogenic lysozyme mutants may be involved in amyloid nucleation by placing key residues (21, 62, 104, 122, and 112–117) in proximity before partial unfolding occurs. Identifying residues in the native state that may be involved in amyloid formation could provide novel drug targets to prevent a range of amyloidoses.

     

Is it possible to predict amyloidogenic regions from sequence alone?

Identification of potentially amyloidogenic regions in polypeptide chains is very important because the amyloid fibril formation can be induced in most normal proteins. In our work we suggest a new method to detect amyloidogenic regions in protein sequence. It is based on the assumption that packing is tight inside an amyloid and therefore regions which could potentially pack well would have a tendency to form amyloids. This means that the regions with strong expected packing of residues would be responsible for the amyloid formation. We use this property to identify potentially amyloidogenic regions in proteins basing on their amino acid sequences only. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Predictions of the regions which are responsible for the formation of amyloid fibrils in proteins unrelated to disease have been also done.     

Prediction of amyloidogenic and disordered regions in protein chains

The determination of factors that influence protein conformational changes is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for eight of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of amyloid fibril formation is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value with which one can predict both intrinsically disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.     

α-synuclein reactive antibodies as diagnostic biomarkers in blood sera of Parkinson's disease patients

Background

Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinson''s (PD) and Alzheimer''s diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimer''s disease and potentially other neurodegeneration ailments.

Methodology/Principal Findings

Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies – α-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance. We found significantly higher antibody levels towards monomeric α-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression (P<0.0001). This indicates potential protective role of autoimmunity in maintaining the body homeostasis and clearing protein species whose disbalance may lead to amyloid assembly. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards α-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression (P<0.0001). Pooled IgGs from PD patients and controls interacted also with the amyloid fibrils of Aβ (1–40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to the generic amyloid conformational epitope, displaying higher specificity towards human amyloid species associated with neurodegeneration.

Conclusions/Significance

Our findings may suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein – α-synuclein can be of value in the development of treatment and diagnostic strategies, especially during the early disease stages.     

A search for amyloidogenic regions in protein chain

We suggest a new method to detect amyloidogenic regions in a protein sequence. In the present work it is shown that regions enriched with amino acid residues which have a high expected packing density are responsible for the amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for 8 of 11 amyloid-forming proteins and peptides in which positions of amyloidogenic regions have been revealed experimentally.     

The contrasting effect of macromolecular crowding on amyloid fibril formation

Background

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.

Methodology/Principal Findings

As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.

Conclusions/Significance

We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.     

Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme

To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.     

Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

Background

Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated.

Methodology/Principal Findings

We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway.

Conclusions/Significance

Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.     

Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

Amyloidogenic sequences in native protein structures

Numerous short peptides have been shown to form β‐sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 proteins containing 45 sequences known to form amyloid, to see how the proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen‐bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form β structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a β structure. Secondly, amyloidogenic sequences that are found in β structure are usually buried within the protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to protein aggregation via assembly of the amyloidogenic regions. The use of α‐helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for β structure, is thus a widespread mechanism to avoid protein aggregation.     

Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain

Background

X11-family proteins, including X11, X11-like (X11L) and X11-like 2 (X11L2), bind to the cytoplasmic domain of amyloid β-protein precursor (APP) and regulate APP metabolism. Both X11 and X11L are expressed specifically in brain, while X11L2 is expressed ubiquitously. X11L is predominantly expressed in excitatory neurons, in contrast to X11, which is strongly expressed in inhibitory neurons. In vivo gene-knockout studies targeting X11, X11L, or both, and studies of X11 or X11L transgenic mice have reported that X11-family proteins suppress the amyloidogenic processing of endogenous mouse APP and ectopic human APP with one exception: knockout of X11, X11L or X11L2 has been found to suppress amyloidogenic metabolism in transgenic mice overexpressing the human Swedish mutant APP (APPswe) and the mutant human PS1, which lacks exon 9 (PS1dE9). Therefore, the data on X11-family protein function in transgenic human APP metabolism in vivo are inconsistent.

Results

To confirm the interaction of X11L with human APP ectopically expressed in mouse brain, we examined the amyloidogenic metabolism of human APP in two lines of human APP transgenic mice generated to also lack X11L. In agreement with previous reports from our lab and others, we found that the amyloidogenic metabolism of human APP increased in the absence of X11L.

Conclusion

X11L appears to aid in the suppression of amyloidogenic processing of human APP in brain in vivo, as has been demonstrated by previous studies using several human APP transgenic lines with various genetic backgrounds. X11L appears to regulate human APP in a manner similar to that seen in endogenous mouse APP metabolism.

     

Hacking the Code of Amyloid Formation

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by non-amyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our studies together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.     

Hacking the Code of Amyloid Formation: The Amyloid Stretch Hypothesis

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by nonamyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our conclusions together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.     

Conformational preferences of non-polar amino acid residues: an additional factor in amyloid formation

Amyloid consists of β-sheet polymers and is associated with disease and with functional assemblies. Amyloid-forming proteins differ widely in native structures and sequences. We describe here how conformational preferences of non-polar amino acid residues can affect amyloid formation. The most non-polar residues promote either β-strands (Val, Ile, Phe, and Cys, VIFC) or α-helices (Leu, Ala, and Met, LAM), while the most polar residues promote only α-helices. For 12 proteins associated with disease, the localizations of the amyloid core regions are known. Eleven of these contain segments that are biased for VIFC, but essentially lack segments that are biased for LAM. For the amyloid β-peptide associated with Alzheimer’s disease and an amyloidogenic fragment of the prion protein, observed effects of mutations support that VIFC bias favors formation of β-sheet aggregates and amyloid, while LAM bias prevents it. VIFC and LAM profiles combine information on secondary structure propensities and polarity, and add a simple criterion to the prediction of amyloidogenic regions.     

New strategy for the generation of specific D-peptide amyloid inhibitors

The conversion of a soluble protein into β-sheet-rich oligomeric structures and further fiber formation are critical steps in the pathogenesis of the group of human diseases known as amyloidoses. Drugs that interfere with this process may thus be able to prevent and/or cure these diseases. Recent results have shown that short amino acid stretches can provide most of the driving force needed to trigger amyloid formation of a protein. These evidence suggest that compounds that specifically bind to peptides synthesized upon the sequence of such amyloidogenic protein stretches might also be able to inhibit amyloid formation of the corresponding full-length protein and, likely, amyloid-induced cytotoxicity as well. Here we present a general strategy to obtain d-peptides that specifically interact with protein amyloid stretches. The screening of a d-peptide combinatorial library for inhibitors of an amyloidogenic peptide designed de novo has allowed us to extract a set of empirical rules for the design of d-peptide inhibitors of any six-residue amyloidogenic stretch. d-peptides generated on these bases prevent amyloid formation and disassemble preformed fibrils of different amyloid hexapeptides identified in human amyloid proteins. In addition, they are also specific for their target sequence. The d-peptide designed here for the Alzheimer's Aβ_1-42 peptide not only inhibits and disassembles amyloid material but also reduces Aβ_1-42 amyloid-induced cytotoxicity in cell culture.     

Increased expression and local accumulation of the Prion Protein,Alzheimer Aβ peptides,superoxide dismutase 1, and Nitric oxide synthases 1 &; 2 in muscle in a rabbit model of diabetes

Background  

Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP), and superoxide dismutase 1 (SOD1), as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies.     

A search for amyloidogenic regions in protein chains

A new method was developed for identifying amyloidogenic regions in protein chains. The formation of amyloid fibrils was attributed to protein regions enriched in residues with a high expected packing density. Predictions consistent with experimental findings were obtained for 8 out of 11 amyloid-forming proteins examined.     

Chain collapse of an amyloidogenic intrinsically disordered protein

Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.     

Fast dynamics perturbation analysis for prediction of protein functional sites

Background  

We present a fast version of the dynamics perturbation analysis (DPA) algorithm to predict functional sites in protein structures. The original DPA algorithm finds regions in proteins where interactions cause a large change in the protein conformational distribution, as measured using the relative entropy D _x . Such regions are associated with functional sites.