Patent Application
20150307573
Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. Studies from many laboratories have suggested that the molecular agents (toxic entities) in amyloid-related conditions are not the associated protein fibrils that have long been taken as the defining feature of these disorders, but instead are lower molecular weight entities, often termed “small amyloid oligomers” (1-7). These oligomers are not generally stable aggregates; they appear as transient species during the conversion of their monomeric precursors to more massive, stable fibrils, and sometimes they appear as an ensemble of sizes and shapes. This polymorphic and time-dependent nature of small amyloid oligomers has made it difficult to pin down their assembly pathways, their stoichiometries, their atomic-level structures, their relationship to fibrils, and their pathological actions (1, 8-10). What has emerged is a consensus, minimal definition of small amyloid oligomers: they are non-covalent assemblies of several identical chains of proteins known also to form amyloid fibrils; the oligomers exhibit greater cytotoxicity than either the monomer or fibrils formed from the same protein; in many cases the oligomer is recognized by a “conformational” antibody (A11) that binds oligomers but not fibrils, regardless of the sequence of the constituent protein (5). This suggests that oligomers display common conformation features that differ from those of fibrils (11).
There is a need to better define small amyloid oligomers which are important etiologic agents for amyloid diseases or conditions and/or to isolate artificial versions of them which mimic the properties of the small amyloid oligomers, in order to devise reagents and assays for identifying putative agents which reduce toxicity of the small amyloid oligomers. Such agents would be expected to be useful for treating diseases or conditions which are mediated by the small amyloid oligomers.
The patent or application file contains at least one drawing executed in color. It is noted that many of these color drawings are present in the publication, Laganowsky et al. (2012), Atomic view of a toxic small oligomer, Science 335, 1228-31, doi: 10.1126.
This application relates, e.g., to the design, isolation and characterization of stable, artificially generated small amyloid oligomers named “cylindrins,” to methods of designing and making them, and to methods of using them to isolate putative agents which inhibit the cell toxicity (cytotoxicity) of the cylindrins.
A “cylindrin” of the invention, as used herein, is a non-covalent assembly of substantially identical chains of an amyloid or amyloid-related protein,
wherein each chain has a length of about 10-100 amino acid residues and comprises
wherein the cylindrin is a curved beta sheet formed from anti-parallel out-of-register extended protein strands, which is substantially filled with packed side chains.
The cylindrins of the present invention, which are artificially derived, differ from naturally occurring cylindrins, at least because the cylindrins of the present invention are produced synthetically (e.g. by chemical synthesis or by expression from a synthetic or recombinant gene) rather than being naturally occurring; are less complex and more homogenous (at least ⅔ of the sequences are cylindrin-forming sequences, in repeated copies, whereas naturally occurring cylindrins contain many additional regions, which are not involved in the aggregation required for the formation of cylindrins); generally are considerably smaller (the chains having a total length of only about 10-100 amino acids, compared to the lengths of the chains of naturally occurring cylindrins, which are often significantly larger); and often are considerably more stable.
Cylindrins of the present invention also differ from the “steric zippers” which have previously been described for amyloid or amyloid-related proteins, at least because the two types of molecular assemblies have completely different structures. For example, cylindrins are cylindrical whereas steric zippers are nearly flat. Furthermore, cylindrins are not adhesive, whereas steric zippers are adhesive and form fibrils.
One aspect of the invention is a cylindrin (an artificially derived cylindrin), as defined above. In one embodiment of the invention (e.g. the ABC cylindrin structure shown herein), the curved beta sheet is a cylindrical barrel formed from antiparallel protein strands. In another embodiment of the invention (e.g., the SOD1 cylindrin structure shown herein), the curved beta sheet is an antiparallel beta-sheet corkscrew.
Another aspect of the invention is a method for making a cylindrin, comprising
identifying a cylindrin-forming segment from a amyloid or an amyloid-like protein of interest, by using the cylindrin structure of a known cylindrin as a profiled structure in a method of 3D profiling,
synthesizing copies of the cylindrin-forming segment (e.g. as individual or as tandem copies), and
allowing the copies to form oligomers (e.g., in solution),
thereby forming a cylindrin.
Not all of the preceding steps need be carried out in order to make a cylindrin. For example, in some embodiments of the invention, the sequence of the cylindrin-forming segment has already been determined, and/or a cylindrin has already been synthesized, before the copies of the cylindrin-forming segments are allowed to form oligomers in solution.
The preceding methods of making a cylindrin can further comprise (a) testing the cylindrin for properties of a cylindrin, e.g. for its ability to inhibit cylindrin-mediated cell toxicity; and/or (b) crystallizing the cylindrin and/or characterizing (determining the 3D structure of) the cylindrin by X-ray crystallography.
Other aspects of the invention include: a polynucleotide encoding a cylindrin-forming segment or tandem copies thereof; an expression vector, comprising the polynucleotide, operably linked to a regulatory control sequence (e.g., a promoter or an enhancer); a cell comprising the expression vector; and a method of making a cylindrin or segment of cylindrin, comprising cultivating the cell and harvesting the polypeptide thus generated.
Another aspect of the invention is a method for identifying (designing, selecting, screening for) a putative agent that inhibits or reduces cylindrin-mediated cell toxicity, comprising
contacting cells with a cytotoxic cylindrin and with a putative inhibitory agent, and
measuring (determining) viability of the cells which were contacted with the putative agent compared to the viability of control cells which were not contacted with the putative inhibitory agent,
wherein a putative agent that results in a statistically significantly greater viability of the cells that were contacted with the putative agent than the control cells is a candidate for an agent that inhibits cylindrin-mediated toxicity.
In some embodiments of the invention, a 3D structure of a cylindrin determined by a method of the invention (e.g., the SOD1 structure described herein) can be used as a profiled structure for identifying cylindrin-forming sequences of amyloid or amyloid-related proteins.
Another aspect of the invention is a computer-readable medium, providing the structural representation of a cylindrin of the invention, as described herein.
Another aspect of the invention is a kit for making and/or characterizing a cylindrin, or for carrying out a method of the invention, such as method for making a cylindrin or a method screening for cylindrin inhibitors.
In initial studies, the present inventors chose to work with alphaB crystallin (ABC), a protein that is a chaperone (12-14) which forms amyloid fibrils (15). This protein was selected because the fibrils form more slowly than those of, e.g., the Amyloid beta peptide (Abeta) of Alzheimer's disease or Islet Amyloid polypeptide (IAPP), so that the oligomeric state may be trapped prior to the onset of fibrillization. The inventors first identified a segment of this amyloid-forming protein which forms an oligomeric complex exhibiting properties of other amyloid oligomers: beta-sheet-rich structure, cytotoxicity, and recognition by an anti-oligomer antibody. That is, the structures satisfy the definition of a small amyloid oligomer set forth in the Background section above. The ABC cylindrin binds to a conformational antibody which also binds to Abeta oligomers, indicating that the two have similar conformations.
The X-ray-derived atomic structure of this artificially derived ABC amyloid oligomer reveals a cylindrical barrel, formed from six anti-parallel, protein strands. This ABC structure is representative of the generic class of structures which the inventors have named cylindrins. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers. These cylindrins, which are small toxic protein oligomers (toxic agents), are believed to be the etiologic agents of several amyloid diseases, including Alzheimer's, Parkinson's, diabetes type 2, and the prion conditions. The peptide elements which form the chains of an oligomeric cylindrin complex are sometimes referred to herein as “cylindrin-forming segments” or “cylindrin-forming sequences” or “cylindrin-forming peptides.” Example I discusses the design, isolation and characterization of the ABC cylindrin.
The inventors subsequently identified cylindrin-forming segments (sequences) of Abeta, using the 3D structure (e.g. the molecular coordinates of the 3D structure) of the ABC cylindrin as a profiled structure in the 3D profiling method described in Bowie et al. (1991) Science 253, 164-170, and showed that the cylindrin structure for ABC is compatible with the sequence segment from the Abeta protein. These studies are presented in Example II.
In a further expansion of the method, the inventors, again using the ABC cylindrin structure as a profiled structure, identified cylindrin-forming segments from superoxide dismutase I (SODI), a protein which has been implicated in Amyotrophic lateral sclerosis. The cylindrins formed from these segments exhibit a structure similar to, but somewhat different from, the ABC cylindrin 3D structure. The SOD1 cylindrin structure is an antiparallel beta-sheet corkscrew. These studies are presented in Example III.
Using comparable techniques, based, e.g., on the 3D structure described herein of the ABC cylindrin, one of skill in the art can readily identify cylindrin-forming sequences for a variety of other amyloid or amyloid-related proteins. For example, Example IV shows cylindrin-forming sequences determined for the additional, representative, amyloid proteins IAPP, prion protein (PrP), α-synuclein, Tau, and TDP43. Using conventional techniques, including some described herein, a skilled worker can readily synthesize (or express) such peptides, generate cylindrins from them; confirm that they exhibit toxic properties; and use them to identify putative agents which inhibit cylindrin-mediated functions, such as cell toxicity. Furthermore, 3D structures determined as described herein can be used as profiled structures for identifying cylindrin-forming sequences from additional amyloid or amyloid-like proteins.
A cylindrin comprises substantially identical chains of an amyloid or an amyloid-related protein. A cylindrin, as used herein, comprises a cylindrin-forming segment of an amyloid or of an amyloid-related protein. In some places herein, the terms “amyloid” and “amyloid-related” are used interchangeably. It is to be understood that a cylindrin from an amyloid-related protein has similar properties to a cylindrin from an amyloid protein.
An “amyloid-related protein,” as used herein, refers a polypeptide having the common properties of an amyloid protein, but not yet officially recognized by the Nomenclature Committee of the International Society of Amyloidosis, who define amyloid diseases and proteins. An “amyloid protein,” as used herein, is one of a class of proteins having common properties, including, e.g., the ability to polymerize to form a cross-beta structure, in vivo, or in vitro. Many of these amyloid and amyloid-related proteins exhibit classic histopathological characteristics such as Congo red birefringence. Inappropriately folded (misfolded) versions of the proteins interact with one another or other cell components to form insoluble fibrils (e.g. plaques or tangles). A skilled worker will recognize a wide variety of amyloid or amyloid-related proteins that can be used to derive cylindrins by a method as described herein and/or to carry out a method of the invention, such as a method to select inhibitors of cylindrins. These proteins have been implicated in the etiology of a variety of diseases or conditions, including neurodegenerative ones, and include, e.g., beta amyloid (Alzheimer's disease, cerebral amyloid angiopathy), tau (Alzheimer's disease and a large number of tauopathies, including frontotemporal dementia and progressive supranuclear palsy), amylin (diabetes type 2), Prion protein (PrP-Creutzfeldt-Jacob Disease, fatal familial insomnia, other prior-based conditions), Superoxide dismutase1 (SOD1-ALS), TAR DNA-binding protein-43 (TDP-43-ALS), RNA-binding protein FUS (Fused in Sarcoma (FUS-ALS), alpha-synuclein (Parkinson's disease), p53 (many cancers), transthyretin (several different amyloidosis conditions), beta 2 microglobulin (dialysis related amyloidosis), insulin (injection amyloidosis) and lysozyme (lysozyme amyloidosis). For additional amyloid and amyloid-related proteins from which a skilled worker can derive cylindrins according to a method of the invention, and diseases or conditions mediated by them, see Sipe et al. (2012) Amyloid 19(4), 167-170, which is incorporated by reference herein. It is to be understood that the discussion herein with regard to amyloid-related proteins, e.g., in the context of cylindrins derived from them, is also applicable to amyloid proteins, and vice versa. Such conditions are sometimes referred to herein as “amyloid-mediated” or “cylindrin-mediated” conditions or diseases. A disease or condition that is “mediated” by, or “associated with” an amyloid or cylindrin is one in which the amyloid or cylindrin plays a biological role. The role may be direct or indirect, and may be necessary and/or sufficient for the manifestation of the symptoms of the disease or condition. It need not necessarily be the proximal cause of the disease or condition.
“Fibrillation” or “fibrillization” refers to the aggregation of amyloid molecules to form fibers.
A “cylindrin-forming segment” (sometimes referred to herein as a cylindrin-forming peptide or cylindrin-forming sequence) of an amyloid or amyloid-related protein is a segment of about 7-15 amino acids of the amyloid or amyloid-related protein which self-aggregates or aggregates with a complementary sequence to form a cylindrin structure, in vitro or in vivo.
“Complementary,” as used herein, is defined as follows: A cylindrin formed from two distinct segments, a first segment and a second segment, which are complementary to each other, will have an alternating pattern of these segments, and the side chains of these segments will pack to mostly fill the internal space of the cylindrin. Thus, the second, complementary sequence is one of similar length as the first segment, whose side chains can pack with those of the first sequence to substantially fill the internal space of the cylindrin.
Each “chain” of an amyloid or amyloid-related protein in a cylindrin has a length of about 10-100 amino acid residues. In embodiments of the invention, these peptide chains contain, for example, a single copy of a cylindrin-forming segment; or tandem adjacent copies of a cylindrin-forming sequence; or adjacent copies of a first cylindrin-forming segment and a second complementary segment of the first cylindrin-forming segment. In embodiments of the invention, tandem sequences are separated by suitable spacer sequences.
In embodiments of the invention, the chain comprises one copy, or 2, 3, 4, 5, 6 or more tandem copies, of a cylindrin-forming segment of the invention. For example, in the case of an 11 amino acid cylindrin-forming segment in a cylindrin having the shape of a cylindrical barrel, the cylindrin can contain 6 chains of the 11 amino acid cylindrin-forming segment; 3 chains of an about 25 amino acid peptide comprising two adjacent tandem copies of the 11 amino acid cylindrin-forming segment, optionally separated by suitable spacers, or one copy of the segment adjacent to a complementary copy of the segment, optionally separated by suitable spacers; 2 chains of an about 45 amino acid peptide consisting of a three adjacent tandem copies of the 11 amino acid cylindrin-forming segment, optionally separated by spacers, or alternating complementary copies, optionally separated by suitable spacers; or 1 chain containing six adjacent tandem copies of the 11 amino acid cylindrin-forming segment, or alternating complementary copies, optionally separated by suitable spacers. Some such structures are described herein. The chains are arranged in an anti-parallel fashion, so the chains containing two copies of a segment, for example, contain one segment in one orientation and a second segment in the opposite (complementary) orientation; when the two segments fold to form a hairpin-like structure, the two segments line up in an antiparallel fashion.
In some embodiments of the invention, in which more than one (e.g., two) different cylindrin-forming segments are identified in an amyloid or amyloid-related protein, a chain can comprise both of these segments, for example arranged in tandem. See, e.g., some of the SOD1 peptides shown in Table 7. In artificial cylindrins comprising, e.g., two different cylindrin-forming segments, the same sorts of arrangements of segments can be formed as described above. In addition, a chain can have mixtures of the two types of segments, e.g., two copies of one segment and four copies of the other.
Suitable spacers will be evident to a skilled worker who is familiar with structural biology. One function of the spacers is to allow portions of the chains (e.g., a segment and a complementary segment) to fold back upon one another to allow the formation of antiparallel strands in a cylindrin. When a tight turn is desired, such as for the formation of a cylindrical barrel, amino acids which allow for great flexibility can be used. These include, e.g., various combinations of at least two amino acids selected from, e.g., glycine, asparagine and proline. Typical spacers include Gly-Gly, Gly-Pro and Asn-Gly. When looser turns are possible, such as in the corkscrew structures formed by SOD1 cylindrins, larger “loops” of as many as about 20 amino acids intervening between cylindrin-forming segments can be tolerated. See, e.g., some of the SOD1 peptides in Table 7. The upper case letters represent the cylindrin-forming segments which are involved in the formation of the cylindrin, whereas the lower case letters represent spacers, including such loops.
The chains in a cylindrin are “substantially” identical. By “substantially identical” is meant that the chains contain identical cylindrin-forming segments, but may differ in other respects. For example, when tandem copies or complementary segments of cylindrin-forming segments are present, amino acid spacers between them may differ in the chains forming the cylindrin. In general, such variation will not significantly affect the structure of the cylindrin. Spacers, including variant spacers, are selected so that properties of the cylindrins, such as their cytotoxicity, are not negatively impacted. The chains of a cylindrin “consist essentially of” the cylindrin-forming segments. The additional sequences, such as intervening sequences and/or spacers, do not materially affect the basic and novel characteristic(s) of the cylindrin, such as its cytotoxicity.
In a cylindrin of the invention, at least about ⅔ of the amino acid residues in each chain are cylindrin-forming segments, which can be repeated in a regular, symmetric, fashion. This is one of the features which distinguishes the artificially generated cylindrins of the invention from naturally occurring cylindrins. In naturally occurring cylindrins, the cylindrin-forming segments are buried within a longer protein sequence, including many regions which are not related to the formation of a cylindrin; so the naturally occurring cylindrins are far more complex than the artificially generated cylindrins of the invention.
Some of the cylindrins of the invention, such as the ABC cylindrin described herein, contain six chains of a substantially identical cylindrin-forming sequence. In other embodiments of the invention, such as the beta sheet corkscrew of the SOD1 cylindrin, the number of chains is potentially infinite. Other cylindrins of the invention can have intermediate numbers of chains.
A cylindrin is formed from anti-parallel out-of-register extended protein strands. By “extended” is meant that the peptide backbone is nearly flat, e.g. that the cylindrin-forming segments adopt a beta-strand structure in which the backbone torsion angles approach ±180°. (In an “ideal” antiparallel beta-strand, the phi angle is roughly −140°, and the psi angle is roughly +140°, but in real beta-strands, such as those in a cylindrin, these values can vary by)±40°.
A cylindrin is a “curved” beta sheet. By “curved” is meant that the sheet is closed or partially closed on itself, such that amino acid side chains from one face of the sheet are at least partially buried and shielded from the solvent.
A cylindrin of the invention is “substantially” filled with packed side chains. As used herein, this term means that water molecules do not occupy more than about 15% of the volume of the interior of the cylindrin.
In many cylindrins of the invention, there is an important glycine (Gly) residue which occupies a central location in the cylindrin-forming segment and points toward the interior of the cylindrin (the interior of the curvature). The importance of the Gly residue being in this position is that glycine's lack of a side chain provides space for other side chains to pack without overlapping. A residue other than glycine would take up too much space, forcing other side chains apart, and reducing or eliminating the curvature of the beta-sheet (disrupting the cylindrin) in the center of the barrel or corkscrew, which is important for the toxicity of the cylindrins. Support for the importance of the glycine residue is provided by the mutagenesis studies of ABC discussed herein, and by the studies of SOD1 in patients with ALS, which show that although a wide variety of mutations are observed among the patient population, the glycine residue at this position is invariantly present.
In one embodiment of the invention, a cylindrin-forming segment, chain or cylindrin is isolated or purified, using conventional techniques such as the methods described herein. By “isolated” is meant separated from components with which it is normally associated, e.g., components present after the cylindrin is synthesized. A “purified” cylindrin can be, e.g., greater than 90%, 95%, 98% or 99% pure.
In embodiments of the invention, the cylindrin is detectably labeled. Labeled cylindrins can be used, e.g., to better understand the mechanism of action and/or the cellular location of cylindrins. Suitable labels which enable detection (e.g., provide a detectable signal, or can be detected) are conventional and well-known to those of skill in the art. Suitable detectable labels include, e.g., radioactive active agents, fluorescent labels, and the like. Methods for attaching such labels to a protein, or assays for detecting their presence and/or amount, are conventional and well-known.
A method of the invention can comprise using the molecular structure of a known cylindrin (e.g. the ABC cylindrin or the SOD1 cylindrin described herein) as a profiled structure in a method of 3D profiling, in order to identify a cylindrin-forming segment from another amyloid or amyloid-related protein of interest.
A skilled worker, after having become aware of the cylindrin structures and methods described herein, can identify cylindrin-forming segments of any amyloid or amyloid-related protein of interest, using, e.g., the 3D profiling method described in the paper of Bowie et al (1991) “A method to Identify Protein Sequences That Fold into a Known Three-Dimensional Structure” Science 253, 164-170. A computer program to carry out this procedure is available from the inventors' laboratory. Other methods for identifying cylindrin-forming segments from scratch might be molecular modeling and calculating energies, or running molecular dynamics simulations, but the method of Bowie et al is preferred.
Briefly: If a given 3D structure (e.g. the atomic coordinates) is known, one can use a 3D profile method to find amino acid sequences which are compatible with that 3D structure. The 3D structure is referred to herein as a “profiled structure.” That is, these sequences, which may be segments of full proteins, can fold into the given profiled structure. In the profiling method of Bowie et al., amino acid sequences are identified which are most compatible with the environments of the residues in the 3D structure. These environments can be described by: (i) the area of the residue buried in the protein and inaccessible to solvent; (ii) the fraction of side-chain area that is covered by polar atoms (0 and N); and (iii) the local secondary structure.
For steric zippers, the procedure of Thompson et al. (2006) Proc Natl Acad Sci USA 103, 4074-4078 was one application of 3D profiling. In that case, the profiled structure was the first steric zipper structure described, which consists of two sheets, each a stack of 6-residue segments which are self-complementary. Using this structure as the profile, other six residue segments were found that were predicted to form the same sort of steric-zipper structure, but that have different amino acid sequence.
For cylindrins, the same procedure is used, but now the profiled structure is a cylindrin structure described herein (such as a cylindrin from alpha B crystalline). This profiling procedure can be used to identify cylindrin-forming sequences from amyloid proteins, such as, e.g., Abeta, tau, SOD1, alpha-synuclein, and IAPP. Example II shows the application of this method for AP; Example III shows its application to SOD1; and Example IV shows its application to a variety of other amyloid proteins. Many other cylindrin-forming segments can be identified from other amyloid proteins by one of skill in the art, using comparable procedures.
In the representative method described in Example III, the profiled structure is the ABC cylindrin 3D structure. This can be found, for example, at the world wide web site rcsb.org/pdb/files/3SGO.pdb.; the atomic coordinates are provided in Table 5. In another embodiment of the invention, the profiled structure is the SOD1 cylindrin structure that is determined herein. This can be found, for example, at the world wide web site kv11_corkscrew_new_asu.pdb; the atomic coordinates are provided in Table 6.
Once the sequence of a cylindrin-forming segment has been determined, a peptide comprising that sequence, or multiple copies of the peptide as described elsewhere herein, can be synthesized (e.g., chemically or by recombinant expression in a suitable host cell) by any of a variety of art-recognized methods. In order to generate sufficient quantities of cylindrins for use in a method of the invention, such as for use in a cell toxicity assay, a practitioner can, for example, using conventional techniques, generate nucleic acid (e.g., DNA) encoding the peptide and insert it into an expression vector, in which the sequence is under the control of an expression control sequence such as a promoter or an enhancer, which can then direct the synthesis of the peptide. For example, one can (a) synthesize the DNA de novo, with suitable linkers at the ends to clone it into the vector; (b) clone the entire DNA sequence into the vector; or (c) starting with overlapping oligonucleotides, join them by conventional PCR-based gene synthesis methods and insert the resulting DNA into the vector. Suitable expression vectors (e.g., plasmid vectors, viral, including phage, vectors, artificial vectors, yeast vectors, eukaryiotic vectors, etc.) will be evident to skilled workers, as will methods for making the vectors, inserting sequences of interest, expressing the proteins encoded by the nucleic acid, and isolating or purifying the expressed proteins.
Peptides synthesized as above (e.g. individual, single copy cylindrin-forming segments, or chains comprising one or more cylindrin-forming segments) can be purified by conventional techniques such as the exemplary ones described herein. Generally, the peptides are lyophilized before storage.
In order to form cylindrins from the peptides, in some embodiments of the invention the peptides are reconstituted by dissolving the lyophilized peptide in water or buffer and are allowed to form oligomers in solution, under conditions in which the aggregates spontaneously form. The conditions for forming particular cylindrins can vary, depending on the cylindrin. Suitable conditions can be determined readily by a skilled worker, using empirical procedures. In some embodiments, the peptides are incubated under close to physiological conditions (e.g., a temperature between about 20-37° C., about neutral pH, mM or μM monovalent and/or divalent salts). In other embodiments, more “extreme” conditions are required (e.g., a temperature as low as 4° C. or as high as 65° C. or more; pH as low as about 2 or as high as about 11). Depending on the cylindrin and the conditions employed, a cylindrin may form within a matter of minutes, or it may take a considerably longer period of time, e.g., days, weeks or months. Some typical conditions for forming cylindrins are shown in the Examples.
Cylindrins of the invention can be tested for cell toxicity, using conventional methods that are well-known to those of skill in the art. Assays include those for measuring a variety of different markers that indicate the number of dead cells (cyototoxicity assay), the number of live cells (viability assay), the total number of cells, or the mechanism of cell death (e.g. apoptosis, necrosis, membrane leakage, etc.) In one embodiment of the invention, cylindrin-mediated cell toxicity is monitored by assaying for cell viability. For example, protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity (Niles et al. (2007) Anal. Biochem. 366, 197-206). Cytotoxicity can also be monitored using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. This assay is described in the Examples herein. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATP content as a marker of viability (Riss et al. (2004) Assay Drug Dev Technol 2, 51-62). Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction (Fan et al. (2007) Assay Drug Dev Technol 5, 127-36). Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay. A label-free approach to follow the cytotoxic response of adherent animal cells in real-time is based on electric impedance measurements when the cells are grown on gold-film electrodes. This technology is referred to as electric cell-substrate impedance sensing (ECIS). Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays. Other suitable assays will be evident to those of skill in the art.
The cells used in assays for cylindrin toxicity can be any of a variety of cell types which will be evident to a skilled worker. For example the cells can be eukaryotic, vertebrate, mammalian, such as the four mammalian cell lines discussed in the Examples (HeLa, HEK293, PC-12, and SH SY5Y), or other types of cells that will be evident to a skilled worker. In one embodiment, the cell type which is used is appropriate for the particular cylindrin being tested. For example, pancreatic cells or cell lines can be used to test for toxicity of the IAPP cylindrin. In some embodiments of the invention, neuronal cell lines are used. In other embodiments, motor neurons are generated by differentiating iPS cells (or other stem cells) with suitable agents.
In one embodiment of the invention, expression vectors in which cylindrins are expressed are introduced (e.g., with a phage or other viral vector) into cells, such as motor neurons; cylindrins are allowed to form in vivo, and toxicity of the cylindrins is assessed. These cells can also be used to assay for putative agents that inhibit cytotoxicity of cylindrins.
In some embodiments, cells are proliferating when they are contacted with the cylindrin.
In some embodiments, cells are cultured in a suitable culture medium, e.g. as is described in Example I, and after a suitable period of time, a cylindrin which has been allowed to form in solution is added to the culture medium. In other embodiments, a peptide or chain comprising one or more copies of a cylindrin-forming segment is added directly to the culture medium and is allowed to form a cylindrin in the medium or after it has entered a cell.
Cylindrins which have been shown to be cytotoxic can be used in screening assays to design and/or select (screen for) putative agents (drugs) which inhibit or reduce their toxic effects. These agents are sometimes referred to herein as “cylindrin-inhibitors” or “cylindrin-inhibitory agents.”
One aspect of the invention is a method for identifying (designing, selecting, and/or screening for) a putative agent that inhibits or reduces cylindrin-mediated cell toxicity, comprising contacting a cell with both a cylindrin of interest and a putative inhibitory agent, and determining if the agent inhibits or reduces the cytotoxicity brought about by the cylindrin to a statistically significant degree compared to the cytotoxicity when the putative agent is not contacted with the cell.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, “a” cell, as used above, can be two or more cells.
The cell can be contacted with the putative inhibitory agent concomitantly, after, or before it is contacted with the cylindrin. The timing and relative order of the addition of the cylindrin and the putative agent, and of the measurement of cytotoxicity, can be optimized empirically, following conventional procedures.
Suitable controls will be evident to a skilled worker. For example, cells can be cultivated in parallel with the treated cells, but not contacted with a cylindrin, to determine a base line for cell viability in the absence of a toxic cylindrin. Furthermore, instead of being contacted with a putative cylindrin inhibitor (a test substance), cells which are contacted with a cylindrin can also be contacted with a control substance, such as water, buffer, or cell culture medium which is known not to inhibit cylindrin-mediated toxicity, or they cannot be contacted (treated) at all.
Cytotoxicity can be measured by any of a variety of conventional assays, including those discussed above. In embodiments of the invention, the method comprises measuring (determining) the viability of cells which were contacted with the putative agent compared to the viability of control cells which were not contacted with the putative inhibitory agent.
Assays other than toxicity assays can also be used to determine if a putative agent inhibits a function of a cylindrin. These assays can measure, e.g., the ability of a putative agent to bind specifically (preferentially compared to a control) to a cylindrin; or the ability of a putative agent to alter the distribution of oligomer sizes in a cell. In other embodiments, the assay can measure the ability of a putative agent to bind specifically to a nucleic acid encoding a cylindrin segment or chain; to inhibit the synthesis of a cylindrin peptide or chain, or a nucleic acid encoding it; to inhibit or enhance aggregation of cylindrin-forming segments into a cylindrin, directly or indirectly; to cleave or otherwise inactivate the protein or nucleic acid; or to otherwise interfere with (inhibit) an activity that is responsible for, or contributes to, symptoms or other manifestations of the amyloid disease or condition.
A putative agent which results in a statistically significant amount of inhibition of one or more functions of a cylindrin (e.g. the inhibition or reduction cylindrin-mediated or induced cellular toxicity, leading to greater viability; the specific binding to a cylindrin molecule, etc.) compared to a suitable control which lacks such inhibitory activity, is a candidate for an agent that inhibits cylindrin-mediated toxicity. Conventional methods for statistical analysis can be used.
Any of a variety of types of putative agents can be tested in a method of the invention. Because many amyloid or amyloid-related conditions are neurodegenerative conditions or diseases, it is desirable that the agents can cross the blood-brain barrier. Small molecules are particularly suitable in this respect. The term “small molecule” refers to a low molecular weight organic compound, e.g. having a molecular weight of less than about 800 Daltons (e.g. <700, 600, 500, 400, 300 Daltons). As used throughout this application, “about” means plus or minus 5% of a value.
The test compounds may be known compounds or based on known compounds. Suitable libraries of small molecule compounds will be evident to a skilled worker. These include, for example, the ZINCPharmer (world wide web site zincpharmer.csb.pitt.edu) library, which is an online interface for searching for purchasable compounds; or the following libraries: BioMol (world wide web site mssr.ucla.edu/biomol.html), Chem Div (chemdiv.com), SPECS (specs.net), Chembridge (chembridge.com) or combinatorial libraries from ChemRx (combi.chemlab.com).
Other agents that can be tested include peptides, such as circular peptides, conformational antibodies, etc.
The invention also includes computer-related embodiments, such as a computer-readable medium, providing the structural representation of a cylindrin of the invention, or for storing and/or evaluating the assay results s described herein.
Another aspect of the invention is a kit for carrying out any of the methods described herein (e.g., methods for identifying cylindrin-forming peptides, for screening for compounds which inhibit cylindrin-mediated cellular toxicity, etc).
The storage medium (computer readable medium) in which the cylindrin structural representation is provided may be, e.g., random-access memory (RAM), read-only memory (ROM e.g. CDROM), a diskette, magnetic storage media, hybrids of these categories, etc. The storage medium may be local to the computer, or may be remote (e.g. a networked storage medium, including the internet). The present invention also provides methods of producing computer readable databases containing coordinates of 3-D cylindrin structures of the invention; computer readable media embedded with or containing information regarding the 3-D structure of a cylindrin of the invention; a computer programmed to carry out a method of the invention (e.g. for characterizing the structure of a cylindrin, or for designing and/or selecting small molecule cylindrin binders or inhibitors), and data carriers having a program saved thereon for carrying out a method as described herein.
Any suitable computer can be used in the present invention.
Another aspect of the invention is a kit for carrying out any one of the methods described herein (e.g., methods for identifying cylindrin-forming segments, for screening for compounds that inhibit cylindrin-mediated cellular toxicity, etc.)
The kit may comprise a suitable amount of a cylindrin of the invention; reagents for generating a cylindrin (e.g. oligonucleotides, primers, vectors, cells etc.); reagents for assays to measure cylindrin-mediated functions or activities, such as cylindrin cytotoxicity, or to screen for agents that inhibit or reduce such activities; or the like. Kits of the invention may comprise instructions for performing a method, such as a method for screening for inhibitors. Other optional elements of a kit of the invention include suitable buffers, media components, or the like; a computer or computer-readable medium for providing profiled structures for identifying cylindrn-forming segments, or for storing and/or evaluating the assay results; containers; or packaging materials. Reagents for performing suitable controls may also be included. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in single reaction form for screening assays.
In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
Synthetic peptides were purchased from CS BIO (Menlo Park, Calif.). All peptides were filtered through a 0.22 μm Ultrafree-MC centrifugal filter device (AMICON, Bedford, Mass., USA) prior to crystallization in hanging drop plates. All crystallization was performed at room temperature. KVKVLGDVIEV (SEQ ID NO:3) (K11V, see Table 1) was dissolved in water to a final concentration of 10 mM and mixed with 5 mM OrangeG (Product No. 861286, Sigma-Aldrich, St. Louis, Mo.), for a final concentration of 4 mM K11V and 3 mM OrangeG. This peptide mixture was crystallized in 0.1 M BIS-TRIS (pH 6.5), 45% 2-methyl-2,4-pentanediol (MPD), 0.2 M ammonium acetate (Index #51, Hampton Research, Aliso Viejo, Calif.). K11V-Br2, (2-Bromoallyl)-glycine substitution at position 2, was dissolved in water at 15 mg/mL, crystallized in 0.1 M TRIS (pH 7.0), 35% MPD, 0.2 M sodium chloride (Wizard #24, Emerald BioSystems, Bainbridge Island, Wash.) and crystals appeared in 1-3 days. K11V-Br8, (2-bromoallyl)-glycine substitution at position 8, was dissolved in water at 15 mg/mL and crystallized in 0.1 M HEPES (pH 7.5), 30% MPD, 0.2 M sodium citratre tribasic dihydrate (Crystal Screen #5, Hampton Research, Aliso Viejo, Calif.). KLKVLGDVIEV (SEQ ID NO:3) (K11VV2L) was dissolved in water at 10-15 mg/mL and crystallized in 0.1M TRIS (pH 7.0), 35% MPD, 0.2M sodium chloride (Wizard #24, Emerald BioSystems, Bainbridge Island, Wash.). GDVIEV (G6V) was dissolved in water at 6 mg/mL and crystallized in 2.1 M DL-Malic acid pH 7.0 (JCSG+#68, Qiagen, Valencia, Calif.).
A tandem repeat beta cylindrin peptide, K11V-TR, synthetic gene, codon optimized for E. coli, was designed using DNAWorks (1) and constructed using PCR-based gene synthesis as described (1). The synthetic gene was PCR amplified with Platinum Pfx polymerase (Invitrogen, Carlsbad, Calif.) with the N-terminal primer containing a Sad restriction and TEV protease site, and a C-terminal primer containing a stop codon and XhoI restriction site. Agarose gel purified PCR product, K11V-TR, was extracted using the QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.). Gel purified PCR product and custom vector, p15-MBP (described below), were digested with Sad and XhoI according to manufacturer's protocol (New England Biolabs, Ipswich, Mass.). The p15-MBP custom vector is a chimera constructed from the NdeI and XhoI digestion products pET15b (Novagen, Gibbstown, N.J.), and the maltose binding protein (MBP) gene from pMAL-C2X (New England Biolabs, Ipswich, Mass.), resulting in an N-terminal His-tag MBP fusion vector. Digested vector products were gel purified and extracted (as described above). DNA concentrations were determined using BioPhotometer UV/VIS Photometer (Eppendorf, Westbury, N.Y.). A ligation mixture was performed using a Quick Ligation kit (New England Biolabs, Ipswich, Mass.) according to manufacturer protocol and transformed into E. coli cell line TOP10 (Invitrogen, Carlsbad, Calif.). Several colonies were grown overnight, and plasmid containing the synthetic K11V-TR gene were purified using QIAprep Spin Miniprep Kit (Qiagen, Valencia, Calif.). The final construct p15-MBP-K11V-TR was sequenced prior to transformation into E. coli expression cell line BL21 (DE3) gold cells (Agilent Technologies, Santa Clara, Calif.).
All mutations in the DNA sequence were performed on the p15-MBP-K11V-TR plasmid using a Site-Directed Mutagenesis kit (QuickChange XL, Stratagene, La Jolla, Calif.) with site-directed primers designed using manufacturers QuickChange Primer Design Program available on-line (Stratagene, La Jolla, Calif.) according to manufacturer's protocol. The K11VV2L construct was achieved by mutation of the first glycine residue coding sequence in the linker region to a stop codon. The K11VV4W-TR was achieved by two-rounds of site-directed mutagenesis. The final constructs were sequenced prior to transformation into E. coli expression cell line BL21 (DE3) gold cells (Agilent Technologies, Santa Clara, Calif.).
A single colony was inoculated into 50 mL LB Miller broth (Fisher Scientific, Pittsburgh, Pa.) supplemented with 100 μg/mL ampicillin (Fisher Scientific, Pittsburgh, Pa.) and grown overnight at 37° C. One liter of LB Miller supplemented with 100 μg/mL ampicillin in 2 L shaker flasks was inoculated with 7 mL of overnight culture and grown at 37° C. until the culture reached an OD600˜0.6-0.8 using a BioPhotometer UV/VIS Photometer (Eppendorf, Westbury, N.Y.). IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and grown for 3-4 hours at 34° C. Cells were harvested by centrifugation at 5,000×g for 10 minutes at 4° C. The cell pellet was frozen and stored at −80° C.
The cell pellet was thawed on ice and re-suspended in buffer A (50 mM sodium phosphate, 0.3 M sodium chloride, 20 mM imidazole, pH 8.0) supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, Ill.) at 50 mL per 2 L of culture volume. The re-suspended culture was incubated on ice for 15 minutes prior to sonication. Crude cell lysate was clarified by centrifugation at 14,000×g for 25 minutes at 4° C. The clarified cell lysate was filtered through a 0.45 μm syringe filtration device (HPF Millex-HV, Millipore, Billerica, Mass.) before loading onto a 5 mL HisTrap-HP column (GE Healthcare, Piscataway, N.J.). The HisTrap-HP column was washed with five column volumes of buffer A and protein eluted with linear gradient to 100% in four column volumes of buffer B (50 mM sodium phosphate, 0.3 M sodium chloride, 500 mM imidazole, pH 8.0). Protein eluted around 50-70% buffer B and peak fractions pooled. A final concentration of 5 mM beta-mercaptoethanol (BME) and 1 mM ethylenediaminetetraacetic acid (EDTA) was added to the pooled sample prior to transferring to a Slide-A-Lyzer 10,000 MWCO dialysis cassette (Pierce, Thermo Fisher Scientific, Rockford, Ill.), and dialyzed against buffer C (25 mM sodium phosphate pH 8.0, 20 mM imidazole, 200 mM sodium chloride) at room temperature overnight. The dialyzed sample was pooled and 1/500 volume of TEV protease stock (2) was added. The TEV protease reaction was incubated overnight at room temperature before loading over a 5 mL HisTrap-HP column equilibrated in buffer A. The flow through was collected, containing the recombinant beta cylindrin peptide with an additional N-terminal glycine residue resulting from TEV protease cleavage. Pooled recombinant beta cylindrin peptide was 0.22 μm filtered (Steriflip, Millipore, Billerica, Mass.) and further purified by reverse phase high performance liquid chromatography (RP-HPLC) on a 2.2×25 cm Vydac 214TP101522 column equilibrated in buffer RA (0.1% trifluroacetic acid (TFA)/water) and eluted over a linear gradient from 0% to 100% buffer RB (Acetonitrile/0.1% TFA) in 40 minutes at a flow rate of 9 mL/min. Absorbance at 220 nm and 280 nm were recorded using a Waters 2487 dual A absorbance detector (Waters, Milford, Mass.). Peak fractions containing peptide were assessed for purity by either a MALDI-TOF mass spectrometry (Voyager-DE-STR, Applied Biosystems, Carlsbad, Calif.) or direct infusion nanoelectrospray mass spectrometry using a hybrid linear ion-trap/FT-ICR mass spectrometer (7T, LTQ FT Ultra, Thermo Scientific, Bremen, Germany). Pooled fractions were frozen in liquid nitrogen and lyophilized. Dried peptide powders were stored in desiccant jars at −20° C.
One to five milligrams of lyophilized peptide was dissolved in 1 mL of water and filtered through a 0.22 or 0.45 μm Centrex MF filter (Whatman, Florham Park, N.J.). Filtered samples were injected on a 21.5 mm×60 cm Tosohaas G3000SW column (Tosoh Bioscience, King of Prussia, Pa.) equilibrated in SEC buffer (25 mM sodium phosphate, 100 mM sodium sulfate pH 6.5) at a flow rate of 3 mL/min. Absorbance at 220 nm and 280 nm were recorded using a Waters 2487 dual λ absorbance detector (Waters, Milford, Mass.). Protein standards were monitored by absorbance at 280 nm, and cylindrin peptides monitored by absorbance at 220 nm. For native nanoelectrospray mass spectrometry experiments the SEC buffer was changed to 200 mM ammonium acetate, pH adjusted to 6.5 with acetic acid.
Crystals of K11V-TR were grown in hanging drop VDX plates (Hampton Research, Aliso, Viejo, Calif.) from either (i) purified oligomeric complexes or (ii) freshly dissolved peptide preparations. i) Peak fractions from SEC-HPLC in SEC buffer containing the oligomeric K11V-TR complex was concentrated using a 3,500 MWCO concentrator (Millipore, Billerica, Mass.) at 4° C. The concentrated K11V-TR buffer was exchanged by several washes in buffer (100 mM sodium chloride, 20 mM HEPES pH 7.5) followed by concentration. The buffer exchanged K11V-TR complex was concentrated to a concentration of ˜2.5 mg/mL, as judged by the Bradford assay (Bio-Rad, Hercules, Calif.) using known solutions of K11V-TR for a standard curve. ii) A microfuge tube containing a pre-weighed quantity of K11V-TR, usually a few milligrams, was chilled on ice. A given volume of ice cold water was gently added to yield a final peptide concentration of 2.5 mg/mL, and stored on ice until dissolution of peptide was complete without disturbance. Both preparations of the K11V-TR complex were either used immediately or stored at 4° C. prior to use. Crystals of K11V-TR were grown using ice cold components of a K11V-TR preparation with crystallization solution 30% MPD, 0.2M magnesium acetate, 0.1M sodium cacodylate pH 6.5 (Crystal Screen #21, Hampton Research, Aliso Viejo, Calif.). Crystallization was carried out at 10° C. Crystals from either starting preparations displayed similar X-ray diffraction quality.
All data were collected at 100K at Advanced Light Source (Berkeley, Calif.) beam line 8.2.1, Advanced Photon Source (Chicago, Ill.) beam lines 24-ID-C and 24-ID-E, and in-house on a Rigaku Raxis-IV++ imaging plate detector using Cu K(alpha) radiation from a Rigaku FRE+ rotating anode generator with confocal optics (Table 2). Single crystals were mounted with CrystalCap HT Cryoloops (Hampton Research, Aliso Viejo, Calif.). K11V, K11VV2L, K11V-Br2, K11V-Br8, and K11V-TR crystals were flash frozen in liquid nitrogen prior to data collection. For experimental phases, K11V-TR crystals were soaked briefly in a mother liquor solution containing potassium iodide and flash frozen in liquid nitrogen. G6V crystals were cryoprotected in mother liquor solution containing 20% glycerol and flash frozen in liquid nitrogen.
All data were processed using DENZO (3) and SCALEPACK (3) or XDS (4). G6V initial phases were found by molecular replacement of a poly-alanine beta sheet template peptide. K11V-Br2 and K11V-Br8 were phased using HKL2MAP (5), and models built using COOT (6). K11V and K11VV2L were phased by molecular replacement using PHASER (7) with the K11V-Br2 structure. Low resolution (˜2.9 Å) experimental phases for K11V-TR was obtained from iodo soaked crystal diffraction data collected in-house using HKL2MAP (5), and followed by model building using COOT (6). All model refinement was done using REFMAC (8) and PHENIX (9).
Surface area (10) and shape complementarity (11) calculations were performed with AREAIMOL and SC programs distributed by CCP4 (12).
Peak fractions containing the K11V-TR complex from SEC-HPLC in buffer (0.2M ammonium acetate, pH 6.5) were analyzed by direct nanospray injection (for review see (13)). Fractions were individually loaded into a 2-μm internal diameter externally coated nanospray emitter (ES380, Thermo) and desorbed by adjusting the spray voltage to maintain an ion current between 0.1 and 0.2 μA. A hybrid linear ion-trap/FTICR mass spectrometer was used for the analysis (7T, LTQ FT Ultra, Thermo Scientific, Bremen, Germany). Individual charge states of multiply protonated K11V-TR complex ions were selected for isolation and collisional activation in the linear ion trap followed by detection of the resulting product ions in the FTICR cell. Xtract software (Thermo Scientific, Bremen, Germany) was used to compute monoisotopic mass from the measured isotopomer profile.
Briefly, a small aliquot of cylindrin peptide samples, at a concentration of a few mg/mL, were spotted onto a nitrocellulose membrane (Trans-Blot, Bio-Rad, Hercules, Calif.). After blocking with 10% fat free milk in TBST buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween20), the membranes were incubated with polyclonal antibody or monoclonal antibody (˜1:250 dilution in 5% fat free milk, TBST buffer) at room temperature for 1 hour. The membranes were washed three times in TBST buffer before incubating with anti-rabbit HRP-linked antibody (1:5000 dilution in 5% fat free milk, TBST buffer) (Invitrogen, Carlsbad, Calif.) at room temperature for 1 hour. After washing the membranes three times in TBST buffer, the films were developed following the protocol as described in the Kit (Thermo Scientific Pierce ECL Western Blotting Substrate, #32209). Positive controls for A11 and OC were prefibrillar oligomers and fibrils, respectively (14).
Fibrillation assays were initially carried out in fifteen different fibrillation conditions, then narrowed down to four conditions: A—phosphate buffered saline, B—25 mM TRIS pH 8.5, 150 mM sodium chloride, C—10% dimethyl sulfoxide (DMSO), 25 mM TRIS pH 8.5, 150 mM sodium chloride, and D—10 mM CAPS pH 11.0, 150 mM sodium chloride, 1 mM EDTA. Beta cylindrin peptides stock solutions (10 mg/mL in water) were diluted in a fibrillation buffer to a final concentration of 1 mg/mL in a microfuge. Samples were incubated at 50° C. with vigorous shaking (Torrey Pines Scientific, Carlsbad, Calif.) for one week. Most cylindrin peptides grew fibrils in buffer D, and some in buffers B-C. Fibrils did not appear in buffer A, but served as a negative control.
Cell viability was investigated using a CellTiter 96 aqueous non-radioactive cell proliferation assay kit (MTT) (Promega cat. #G4100). SH-SY5Y (ATCC; cat. # CRL-2266), PC-12(ATCC; cat. # CRL-1721), HeLa and HEK293 were used to assess the toxic effect of clyindrin peptides. HeLa and HEK293 cells were cultured in DMEM medium with 10% fetal bovine serum. SH-SY5Y cells were cultured in F12/DMEM 1:1 medium with 10% fetal bovine serum, PC-12 cells were cultured in ATCC-formulated RPMI 1640 medium (ATCC; cat.#30-2001) with 10% heat-inactivated horse serum and 5% fetal bovine serum. Cells were maintained at 37° C. in 5% CO2. For all toxicity experiments, 96-well plates (Costar cat. #3596) were used. HeLa, HEK293 and PC-12 cells were plated out at 10,000 cells per well and SH-SY5Y cells were plated at 25,000 cells per well. Cells were cultured for 20 h at 37° C. in 5% CO2 prior to addition of peptide samples. 10 μl of sample was added to each well containing 90 μL medium, and allowed to incubate for 24 h prior to adding 15 μl Dye solution (Promega. cat. #G4102) into each well, followed by incubation for 4 h at 37° C. in 5% CO2. After incubation, 100 μl solubilization Solution/Stop Mix (Promega cat. #G4101) was added to each well. After 12 h incubation at room temperature, the absorbance was measured at 570 nm. Background absorbance was recorded at 700 nm. Each of the experiments was repeated 3 times with 4 replicates per sample per concentration. The concentration for cylindrin peptides were based on their oligomeric state. That is a trimer for K11V-TR and monomer for K11VV4W-TR. Abeta at 0.5 μM was a positive control. The results were normalized by using the buffer treated cell as 100% viability and cell treated with 0.2% SDS as 0% viability.
Calcein-containing LUVs were prepared as described previously (19), with minor modifications. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn glycero-3-phospho-L-glycerol (POPG) were obtained from Avanti Polar Lipids (Alabaster, Ala.). Mixtures of POPC and POPG in a 7:3 molar ratio were dissolved in 1:1 chloroform:methanol. The solvent was evaporated under dry nitrogen gas to yield a lipid film that was further dried under vacuum for at least 24 hours to remove any residual organic solvent. The film was then hydrated in 70 mM calcein (Sigma Aldrich, St. Louis, Mo.) and 10 mM Tris-HCl (pH 7.4) at a lipid concentration of 5 mM. The suspensions were subjected to 10 freeze-thaw cycles of temperatures of −80 and 50° C., followed by extrusion through two 0.2 μm pore size filters (Whatman, Florham Park, N.J.). Non-encapsulated calcein was separated from calcein-filled LUVs by size exclusion using a Sephadex G-75 (GE Healthcare, Piscataway, N.J.) equilibrated in buffer (10 mM Tris-HCl pH 7.4, 100 mM sodium chloride). The calcein-containing LUVs were concentrated to 3-5 mM and stored at 4° C. Phospholipid content and concentrations of the LUVs were determined by RP-HPLC using 100% Methanol and 100 mM TEAC (tetraethylammonium chloride) pH 7.8 as the solvent and a C18 column. Diameter of LUVs were determined using a Microtrac UPA 150 (York, Pa.). LUV preparations displayed diameters between 170-200 nm for at least 10 days. LUVs were used for experiments within 5 days of preparation.
Pure, lyophilized K11V-TR and K11VV2L-TR peptides were solubilized in water. Three different stock solutions were made at concentrations of 0.2, 0.8, and 2 mM. 5 uL of each peptide stock solution was added to a well containing 195 uL of 7:3 calcein-containing POPC:POPG LUVs in a buffer (10 mM Tris-HCL pH 7.4, 100 mM sodium chloride). Dye leakage was measured using a SpectraMax5 (Molecular Devices, Sunnyvale, Calif.) at 485 nm excitation and a 535 nm emission. Measurements were taken every 10 minutes for 30 hours. Each well contains a final LUV concentration of 100 μM with final K11V-TR and K11VV2L-TR construct concentrations of 5, 20, and 50 μM. Synthetic human Islet Amyloid Polypeptide (hIAPP) residues 8-37, purchased from CS-Bio (Menlo Park, Calif.), was used as a positive control; since hIAPP has been previously shown to interact and disrupt membranes (15, 16). Lyophilized hIAPP was dissolved in 100% hexafluoro-2-propanol (HFIP) and put under vacuum to evaporate the HFIP. hIAPP was then reconstituted in water at 0.2 mM, filtered using a 0.2 μm filter, and added to six wells containing 100 μM Calcein-containing LUVs to a final concentration of 5 μM hIAPP. Fluorescence at a given time point was normalized as described previously (20), using the equation: Fnormalized=(Ft−Fmin)/(Fmax−Fmin) Ft is the measured fluorescence intensity, Fmin the fluorescence of 100 μM calcein-containing LUVs alone, and Fmax is the maximum fluorescence determined by incubation of 100 μM LUVs in the presence of 0.5% Triton X-100.
Considering conformational and geometrical properties of anti-parallel beta-sheets, Salemme and Weatherford attempted to model a six stranded barrel with a minimum of four interchain hydrogen bonds (17). They were unable to present a model, but observed one or more beta-bulges when maintaining beta-sheet twist, and a chain tendency to diverge at the ends of the barrel (17).
A Dali (18) and MATRAS (19) search was performed using the cylindrin as the search model to find similar structures. A cylindrin containing six chains, all with the same chain identification, was used to overcome the minimum chain length limit of 30 amino acids in Dali. The top scoring search result for Dali was alpha-amino acid ester hydrolase (PDB 1RRY) and for MATRAS was cytokine receptor common beta chain (PDB 1EGJ) residues 339-437. The Dali top result has a Z score of 2.7 and rmsd of 3.2, and MATRAS a Z score of 13.74. The MATRAS top scoring structure has the greater similarity, containing seven beta strands in a cylindrical type fashion. The Dali top scoring model has little similarity, and poor alignment to the alpha-amino acid ester hydrolase sequence, which may explain the low Z score of 2.7. Interestingly, a similar structure can be found in the PduU shell protein (PDB 3CGI), but not found using the above search engines. The PduU shell proteins N-termini form a six stranded parallel barrel (20). The parallel beta barrel, containing residues 7-17, has an Ab of 1050 Å2 and Sc of 0.76. Exhibiting similar molecular properties to the anti-parallel cylindrin structure, we classify this as a parallel cylindrin. A similar beta barrel with a shear number of 6 has been observed in the tetrahydrodipicolinate-N-succinyltransferase protein (PDB 1TDT) (21). This barrel is formed from C-terminal hairpins of 3 chains, adopting an antiparallel structure. But unlike the cylindrin, the interior seems to be quite polar, containing H-bonded rings of Asn and Ser sidechains and some waters.
A different crystal packing was observed with the K11V-Br2 peptide, in which valine at position 2 is substituted by (2-bromoallyl)-glycine. In this derivatized structure, the asymmetric unit is two entire neighboring cylindrins. In contrast, cylindrins formed from the wildtype sequence K11V and the derivatized segment K11V-Br8, contain only one crystallographically unique cylindrin. The RMSD of the wildtype structure superimposed on the bromo-derivative structures is 0.76 and 0.37 Å for K11V-Br2 and K11V-Br8, respectively; RMSD of the wild-type backbone on the bromo-derivative backbones is 0.33 and 0.29 Å for K11V-Br2 and K11V-Br8, respectively.
Molecular dynamics (MD) simulations were carried out to examine the structural transition pathway and the energetics associated with the conversion between the native conformation (cylindrin; FIG. S9A) and a cylindrin fibril model (discussed below; FIG. S9B). These two models are the two target structures used in the molecular simulation. NAMD software was used to integrate classical equations of motion of model systems (22), and the Charmm22 all-atom force field with CMAP correction (23) was used. Van der Waals and electrostatic interactions were switched with a 12 Å cutoff distance. The SHAKE algorithm constrained the covalent bond length of a polar hydrogen atom to its donor, which enabled 2 fs integration step. The native model was solvated with TIP3 explicit water molecules in an 80 Å3 cubic box. Initially each target conformation was energy minimized in 1200 steps, heated to 300 K in 100 ps and equilibrated in 500 ps by rescaling temperature periodically. During the equilibration and the production period, Langevin piston algorithm controlled the pressure at 1 atm and the temperature at 300 K. Next, we adopted targeted MD (TMD) simulation (24) to elucidate intermediate conformations in the middle of the transition pathway. After the equilibrating period, cylindrin was gradually transformed to the fibril model in 20 ns by applying a constraining potential on Cα atoms (forward simulation);
U({right arrow over (x)},t)=½k(R({right arrow over (x)})−R*(t))2
where is the coordinate vector of Cα atoms, t is the current simulation time, k is strength of the constraint which was set to 20 kcal/mol/A R(
) is the root-mean-squared deviation (RMSD) of Cα atoms to the β-sheet, and R*(t) is the target RMSD at t which was linearly reduced from the initial RMSD between two end structures (10.04 Å) to zero as the simulation progressed. We also performed a reverse TMD simulation starting from the last snapshot of the forward simulation, and gradually transformed the molecule to cylindrin (backward simulation). The TMD simulations successfully converted the cylindrin to the fibril and vice versa.
TMD simulation is susceptible to hysteresis effect in energy changes which hampers accurate estimation of free energy difference and the transition state energy between two end structures (24). Therefore we employed free energy perturbation (FEP) simulation aimed at an accurate estimation of the free energy change associated with the structural conversion from the cylindrin to the fibril model (25, 26). The relative difference in RMSD (ΔRMSD) of the two end structures was chosen as the reaction coordinate of the transition. The reaction coordinate varied from −10.0 Å to +10.0 Å, which was divided by 40 equally spaced windows. Initial conformers for the FEP simulation were chosen from the previous TMD simulation; for each window iε{n|1δnδ 40}, two of lowest energy conformation from each of the forward and the backward simulation were selected. We applied an umbrella potential to each initial conformation, whose energy minimum was located at the center of the window;
where i is the index of windows, R1() and R2(
) are RMSD of Cα atoms to cylindrin and to the fibril model respectively, k is 20 kcal/mol/Å2, and Ri* is the offset of the umbrella potential aligned with the center of each bin. For each window, the initial conformation was heated to 300 K in 100 ps, while employing a harmonic constraint to Cα atoms to prevent abrupt structural changes. The Langevin piston algorithm was applied to maintain pressure at 1 atm with a temperature of 300 K. During the production period, the offset of the umbrella potential was shifted from i to i+1, i+1 to i+2, i to i−1 and i−1 to i−2 positions respectively. Simulation period at each offset value varied from 0.75 to 1.5 ns depending on convergence of the simulation. The total energy, constraining energy, and reaction coordinate were saved every 0.2 ps, and coordinates were saved every 2.0 ps.
After finishing the FEP simulation we generated an energy histogram of the entire FEP simulation, using weighted histogram analysis method (WHAM) (27). The energy density of state (DOS) information was utilized to compute Gibbs free-energy of each window along the reaction coordinate (ΔRMSD), which was plotted in
where α is index of snapshots, E and dE are discretized energy level and its width (2 kcal/mol), Ω is energy density of state, and Ua is constraining potential of the window. In addition, a hierarchical clustering algorithm was used to define a representative conformation of each reaction coordinate window. We used a Cα RMSD distance of 3.0 Å as the cutoff for defining a structural cluster. The first 3000 snapshots were analyzed for lowest free energy within each window. A representative configuration in the most populated cluster is plotted in
The total free energy of cylindrin and the steric-zipper fibril model were compared within the Molecular Mechanics-Generalized Born/Surface Area approximation method (MM-GB/SA) (30). The Generalized-Born solvation model and the surface area dependent hydrophobic energy were incorporated as functions of the solvation effect. This technique has been successfully applied for comparing the energetic stability of amyloid fiber models (31). The cylindrin steric-zipper fibril model, consisted of a steric-zipper interface wherein the hydrophobic residues (Val 2, Val 4, and Val 8) buried within the bilayer forming the dehydrated interface (FIG. S10). This model was solvated in a tetragonal solvation box (29.15×100×100 Å3). The X dimension of the solvation box was aligned parallel to the fiber axis, as to represent an infinitely long fiber. The model was heated and equilibrated in 600 ps at 300 K, and simulated for 10 ns without structural constraints. The coordinates were saved every 2 ps. The steric-zpper interface was intact during the simulation period. In contrast, the cylindrin was solvated in an 80×80×80 Å3 solvation box, and simulated for 10 ns. After completion of MD simulations, simulated snapshots were analyzed without solvent molecules and analyzed using an implementation of Generalized-Born solvation model in Charmm v31 (32, 33). In the GBSA approximation, the total free energy of a molecule is sum of individual contributions (30);
E
Total
=E
int
+E
vdW
+E
Elec
+E
GB
+E
ASP,
where Eint is summation of covalent bonding energy terms (bond, angle, dihedral, improper dihedral, and CMAP correction), EvdW is Van der Waals energy, EElec is vacuum electrostatic energy, EGB is Generalized-Born solvation energy, EASP is surface area dependent hydrophobic energy, with a surface tension coefficient σ=5 cal/mol/Å2, STrans is translational entropy, and SRot is rotational entropy. The trans-rotational entropy of the steric-zipper fibril was set to zero, since it precipitates in vitro. Unlike the original method, the vibrational entropic contribution was ignored which contributes only a small fraction to the total energy (31). The density of cylindrin was set to 1 mM/L, and any change in this density did not affect our conclusion qualitatively; for example, when the density is set to 1 nM/L resulted in −TSTrans=−4.83 kcal/mol. The MM-GB/SA analysis determined the steric-zipper fibril model has −5.2 kcal/mol/peptide lower free energy than the cylindrin (Table 4).
Because the polyclonal A11 antibody was affinity purified on an AB containing matrix (Kayed et al. Conformation-dependent anti-amyloid oligomer antibodies. Methods Enzymol. 2006; 413:326-44. PubMed PMID: 17046404), cylindrin and AB prefibrillar oligomers presumably share an epitope(s) that is also shared by other toxic oligomers which the A11 antibody recognizes. Several structural features are shared by our current models of cylindrin. They are the radius of the cylindrin, water mediated backbone H-bonds at the ends of the cylindrin, and helical grooves between side chains on the outside surface of the cylindrin. These grooves are akin to the linear grooves between side chains on the outside surface of steric zippers, but are more pronounced because the cylindrin side chains project from a convex surface of the cylindrin.
Fourier transform infrared spectroscopy of cylindrin, K11V-TR dried fibrils display anti-parallel beta sheet characteristics. In the FTIR spectrum (data not shown) we observed peaks at 1628 cm-1 and 1685 cm-1, characteristic of intermolecular and anti-parallel beta sheet (34, 35), respectively. Therefore, an anti-parallel model for cylindrin fibrils was constructed similar to that observed for short steric zippers (36), and subsequently used in targeted molecular dynamics simulation (discussed above).
Results of GBSA calculations for cylindrin and fibril models at 300 K. The energy unit is kcal/mol/peptide.
Because the polyclonal A11 antibody was affinity purified on an AB containing matrix (Kayed et al. Conformation-dependent anti-amyloid oligomer antibodies. Methods Enzymol. 2006; 413:326-44. PubMed PMID: 17046404), cylindrin and AB prefibrillar oligomers presumably share an epitope(s) that is also shared by other toxic oligomers which the A11 antibody recognizes. Several structural features are shared by our current models of cylindrin. They are the radius of the cylindrin, water mediated backbone H-bonds at the ends of the cylindrin, and helical grooves between side chains on the outside surface of the cylindrin. These grooves are akin to the linear grooves between side chains on the outside surface of steric zippers, but are more pronounced because the cylindrin side chains project from a convex surface of the cylindrin.
Fourier transform infrared spectroscopy of cylindrin, K11V-TR dried fibrils display anti-parallel beta sheet characteristics. In the FTIR spectrum (data not shown) we observed peaks at 1628 cm-1 and 1685 cm-1, characteristic of intermolecular and anti-parallel beta sheet (34, 35), respectively. Therefore, an anti-parallel model for cylindrin fibrils was constructed similar to that observed for short steric zippers (36), and subsequently used in targeted molecular dynamics simulation (discussed above).
We identified the oligomer-forming segment of ABC by inspection of its 3D structure (16) and by applying the Rosetta-Profile algorithm to its sequence. This algorithm identifies sequence segments that form the steric-zipper spines of amyloid fibrils (17, 18). We noted that two segments of high amyloidogenic propensity, with sequences KVKVLG (SEQ ID NO:1) and GDVIEV (SEQ ID NO:2), share the same Gly residue 95 at the C-terminus of the first segment and the N-terminus of the second; moreover, the entire 11-residue segment KVKVLGDVIEV (SEQ ID NO:3) forms a hairpin loop in the 3D structure of ABC (
The hairpin, segment KVKVLGDVIEV (SEQ ID NO:3) (termed K11V) formed both amyloid fibrils and oligomers. Upon shaking at elevated temperature, K11V forms fibrils similar to those of the parent protein (ABC) from which the segment is derived (15) and similar to those of a tandem repeat of K11V (K11V-TR) (
Under physiological conditions, the segment K11V, K11V-TR, and a sequence variant with Leu replacing Val at position 2 (K11VV2L), all form stable small oligomers, intermediate in size between monomer and fiber. For each sequence, we determined the number of molecules in the oligomers by size exclusion chromatography (SEC-HPLC) and native mass spectrometry experiments. Purified recombinant K11VV2L, and K11V-TR, a tandem repeat of K11VV2L eluted as oligomeric complexes by SEC (
These ABC K11V oligomers exhibit molecular properties in common with amyloid oligomers from other disease-related proteins. We probed blots of the recombinant segments with the polyclonal A11, amyloid-oligomer-specific conformational antibody (5). Both single and tandem repeat segments are recognized by the A11 antibody (
We next determined the crystal structures of various ABC K11V oligomers. A screen produced X-ray grade crystals of K11V, but structure determination by molecular replacement with fiber-like probes failed, suggesting that the ABC segment oligomers possess a previously unobserved type of amyloid structure. Turning to the SAD method for phase determination, we synthesized K11V derivatives with Br substitutions at positions 2 or 8 of the K11V sequence, K11V-Br2 and K11V-Br8, with the leucine-resembling non-natural amino acid (2-bromoallyl)-glycine. Both derivatives crystallized and led to structure determinations (Table 2) at 1.4 Å resolution. Molecular replacement based on these structures led to the closely related structures of K11V itself, as well as K11V-TR and K11VV2L The structure of K11V, the amyloid-related oligomer, is a six-stranded anti-parallel barrel of cylindrical shape, consistent in mass with our solution studies, which we term a cylindrin. The cylindrin (
a Highest resolution shell shown in parenthesis.
bNumber corresponds to position of (2-Bromoallyl)Glycine residue substitution in eleven amino acid peptide sequence, see Table 1.
c Rsym = Σ | I-<I> | / ΣI.
d Rfree calculated using 5% of the data.
e Rfree calculated using 10% of the data.
To provide adequate cylindrin material for biochemical and toxicological studies, we generated a synthetic gene to express in bacteria a tandem repeat, K11V-TR, of the well diffracting K11VV2L segment, covalently linked through a double glycine linker and containing an additional N-terminal glycine (
For a negative control of cylindrin structure and properties, we generated a variant form of the tandem segment, K11VV4W-TR in which the V4W substitution occurs in both repeats (Table 1). This substitution was predicted on the basis of the K11V crystal structure to disrupt oligomer formation through steric clash of core, buried residues. This variant peptide eluted in the mass range of a dimeric/monomeric species by SEC-HPLC and displayed dramatically reduced cell toxicity (
To compare cylindrins to fibers, we consider a cylindrin to be a toxic, amyloid-related, oligomeric, cylindrically shaped beta-barrel formed from anti-parallel, extended protein strands and having the cylinder filled with packed sidechains. A cylindrin resembles a steric zipper in having a packed interior, but differs from a steric zipper in an important respect which may illuminate the reaction pathway from oligomers to fibrils. When unrolled into a beta-sheet, each anti-parallel pair of strands in the cylindrin sheet (
The atomic coordinates of ABC cylindrin are shown in Table 5.
An important question is whether the ABC cylindrin is a possible model for amyloid oligomers formed by well-studied toxic proteins, such as Abeta and hIAPP. There is evidence that amyloid oligomers share common structural features. For example, studies have suggested oligomers are beta-sheet rich (38-40), and several toxic oligomers are recognized by the A11 conformational antibody (41), which also recognizes the cylindrin. A11 also recognizes alpha-hemolysin, a soluble beta barrel protein (42). Thus the cylindrin structure may represent the common structural core of amyloid oligomers. To investigate this possibility, we used the Rosetta-Profile method (18) to ask if other toxic sequences, or segments of them, are compatible with the cylindrin structure. Some of these cylindrin-forming sequences are shown in Table 7. We found that the C-terminal segment of Abeta is reasonably compatible with the cylindrin structure, and with a two-residue registration shift between pairs of anti-parallel strands, a very good fit with the cylindrin structure is obtained. (
Cylindrins of Abeta have also been generated. For example, the tandem 3 (GG) appears to dimerize immediately as it comes off of an MBP trap column, eluted at 200 mM NaCl, 20 mM Tris 7.5 (room temp) as seen by SDS-PAGE. The product, as measured by the size of the oligomer, appears to contain a mixture of dimer/monomer after Q˜400 mM NaCl, 10% glycerol, 20 mM Tris 7.5. On SEC (150 mM NaCl, 20 mM tris 7.5 room temp), it appears to be a mix of monomer and a broad peak of dimer and higher-mers. Abeta cylindrins made by a method of the invention will be tested for toxicity. It is expected that they will be cytotoxic.
SOD1 binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. This amyloid protein is involved in several pathological conditions or diseases. For example, mutations (over 150 identified to date) in this gene have been linked to familial amyotrophic lateral sclerosis. Several pieces of evidence also show that wild-type SOD1, under conditions of cellular stress, is implicated in a significant fraction of sporadic ALS cases, which represent 90% of ALS patients.
The SOD1 cylindrin-forming sequences were identified by methods as described herein, using the ABC cylindrin structure obtained in Example I as the profiled structure. Some of the cylindrin-forming segments which were identified, as well as variants thereof, are shown in Table 7.
Copies of the cylindrin-forming segments, KVKVWGSIKGL (SEQ ID NO:61) and PVKVWGSIKGL (SEQ ID NO:60), were chemically synthesized and purified by HPLC. The peptides were allowed to form aggregates as follows. It is presumed that condition 1 forms primarily amyloid fibrils, and condition 2 forms the cylindrin.
Buffer: 50 mM Tris Base
Condition 1: 37° C., shaking, overnight (produces mildly toxic species)
Condition 2: 37° C. incubation, non-agitated, overnight (produces toxic species)
The cylindrins were assayed for toxicity as follows:
Protein concentrations: 50 uM to 800 uM
Cell line: 1) Hela 2) ES derived motor neurons (GFP+)
Toxicity assay: MTT assay, and visual inspection of the GFP+ neuron morphologies
Sample incubation time: 24 hours before MTT reagents were added.
The SOD1-derived peptide KVKVWGSIKGL (SEQ ID NO:61) was crystallized and data collection was performed as follows: The peptide was crystallized via hanging-drop vapor diffusion at 18° C. The peptide was dissolved at 50 mg/mL in 50 mM Tris, then was mixed 1:1 with the reservoir solution: 0.2M Na Citrate pH 5, 13% PEG 6000. The peptide grew into needle-shaped crystals in 2-3 days. For data collection, hundreds of crystals were mounted because of their quick decay. However, two larger crystals were able to be used to collect complete data sets, by diffracting from several points along the length of the crystals. Diffraction from iodine-soaked crystals was used to obtain phases by SIRAS.
The structure of the SOD1-derived peptide KVKVWGSIGKL (SEQ ID NO:61) is an open-ended cylindrin. Each peptide adopts a beta-strand structure, with a hydrogen-bonded turn at the C-terminus. Pairs of strands hydrogen bond in an antiparallel fashion with their C-terminal turns pointing at the N-terminus of the paired strand (red strands of panel a). These pairs of strands H-bond out-of-register with additional pairs of strands, combining to form an open-ended, curved, antiparallel beta-sheet “corkscrew.” 16 strands form one turn of the corkscrew (panel b), and hydrophobic, ‘inward’-pointing side chains mostly fill up the bore of the corkscrew (panel c). This structure and its component peptide have many features in common with the ABC cylindrin: (1) each peptide forms a beta-strand; (2) beta-strands H-bond out-of-register; (3) beta-strands form an antiparallel beta-sheet; (4) the beta-sheet is curved, with hydrophobic residues filling the inner space; (5) an essential glycine points inward, allowing packing of other side chains and thereby supporting the curvature (panel d); (6) the peptide is toxic to cultured cells; (7) the peptide additionally forms amyloid-like fibrils; and (8) the peptide amyloid-like fibrils are non-toxic to cultured cells. The biggest difference between this structure and that of the ABC cylindrin is that the ABC cylindrin is a closed oligomer (that is, of fixed size), whereas this SOD1 cylindrin is an open oligomer, and can contain any number of peptide units.
The atomic coordinates of the SOD1 cylindrin structure are shown in Table 6.
Using the ABC cylindrin as the profiled structure, and following methods disclosed herein, the inventors have determined cylindrin-forming sequences of a variety of representative amyloid or amyloid-related proteins. Table 7 shows some of these sequences, as well as mutant forms of the peptides which have been used to characterize the cylindrins. Shown are sequences from alphaB crystallin (ABC), Amyloid beta peptide (Abeta, or Aβ) of Alzheimer's disease, Islet amyloid polypeptide (IAPP, associated with diabetes type 2), Prion protein (PrP), Superoxide dismutase1 (SOD1), α-Synuclein (associated with Parkinson's disease), Tau and TDP43. In this table, TR means tandem repeat; Arctic, E22del, and Iowa are various mutant Abeta sequences; Capped means an acetyl group (CH3—CO—) is on the N-terminus, and an amino group (—NH2) on the C-terminus to make them more protein-like (no terminal charges). The upper case letters (for example, in the SOD1 segments) represent sequences that are important for the formation of a cylindrin. The lower case letters represent looped out regions (intervening sequences) between the sequences involved in the formation of the cylindrin. The loops are important for the ability of the strands of the cylindrin to fold back upon one another to form the antiparallel strands of the cylindrin.
Skilled workers, using the ABC cylindrin structure, the SOD1 cylindrin structure, or others, can readily identify cylindrin-forming sequences from any amyloid or amyloid-related protein of interest, using the methods described herein. Using conventional methods, such as those described herein, cylindrin-forming segments are synthesized, allowed to aggregate to form cylindrins, shown to be toxic, crystallized and their 3D structures determined, and/or used to identify agents which inhibit or reduce cylindrin-mediated activities, such as cytotoxicity.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications cited above, including U.S. Provisional Applications 61/588,478, filed Jan. 19, 2012, and 61/590,085, filed Jan. 24, 2012, and in the figures are hereby incorporated in their entirety by reference, particularly with regard to the information for which they are cited.
This application claims the benefit of the filing date of U.S. Provisional Applications 61/588,478, filed Jan. 19, 2012, and 61/590,085, filed Jan. 24, 2012, each of which is incorporated by reference herein in its entirety.
This invention was made with Government support under Grant No. AG029430 awarded by the National Institutes of Health, Grant No. 0445429, awarded by the National Science Foundation, and grant No. DE-ACO2-06CH11357, awarded by United States Department of Energy. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/22574 | 1/22/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61588478 | Jan 2012 | US | |
| 61590085 | Jan 2012 | US |
