דור מהיר של עמילואיד מחלבונים שפת אם<em> במבחנה</em
Summary
Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.
Abstract
חלבונים לבצע משימות קריטיות באורגניזמים באמצעות הפעלת פונקציות שהושרו משלושה קפלי ממדים הספציפיים שלהם. למרות שמבני יליד פוליפפטידים להגשים מטרות רבות, עכשיו זה הכיר בכך שרוב החלבונים יכולים לאמץ הרכבה חלופית של עמילואיד ביתא העשיר גיליון. סיבי עמילואיד מסיסים בתחילה קשורים למחלות אנושיות מרובות, אבל הם מוצגים יותר ויותר כשחקנים פונקציונליים משתתפים בתהליכים תאיים חשובים שונים. בנוסף, עמילואיד הופקד ברקמות מטופל מכיל רכיבי nonproteinaceous, כגון חומצות וglycosaminoglycans (בדיחות) גרעין. cofactors אלה יכול להקל על היווצרות של עמילואיד, וכתוצאה מדור סוגים שונים של משקעים בלתי מסיסים של. על ידי ניצול של ההבנה שלנו כיצד חלבוני misfold באמצעות שלב ביניים של מקדים עמילואיד מסיס, שפתחנו שיטה להמיר חלבוני יליד סיבי עמילואיד במבחנה </em>. גישה זו מאפשרת לאדם להכין עמילואיד בכמויות גדולות, בדוק את המאפיינים של עמילואיד נוצרו מחלבונים ספציפיים, ולהעריך את השינויים המבניים המלווים את ההמרה.
Introduction
Proteins are the most abundant biological macromolecules present in all types of cells. They occur in a great variety of sizes, structures, and post-translational modifications, and fulfill an enormous range of important biological functions when in their native forms. More than two dozens of aberrant polypeptides have been implicated in numerous human pathological conditions, such as Alzheimer's disease, Parkinson disease, and Type 2 Diabetes1-4. The terminal misfolded proteins accumulate as amyloid fibrils the insoluble stable aggregates that occur extracellularly or intracellularly.
Despite the implication of specific proteins in certain diseases, increasing evidence supports the notion that all polypeptides have intrinsic properties that enable amyloid transformation. A genome-wide sequence survey identified the “amylome”, by which amyloid-prone fragments constitute roughly 15% of all coding peptide segments from E. coli to humans5. Accordingly, an increasing number of functional amyloids, which participate in various important cellular processes, have been discovered in recent years. For example, bacteria assemble amyloids to form biofilm and spore structures that are critical for their survival and pathogenesis6-9. Peptide hormones form amyloid deposits during storage within mammalian secretory granules before being released, further implying that the protein amyloid form can serve beneficial biological functions6,10,11. Moreover, proteins can also assemble into amyloid fibrils to relay critical cellular signals12,13.
For many years, the most studied amyloids are prepared from individual peptides that are associated with human diseases, such as amyloid beta or prion peptide. These peptides usually lack well defined structure and spontaneously form amyloid in solution over time, a process mediated by a partially misfolded intermediate that serves as the precursor of insoluble amyloid. The precursor of amyloid is also called soluble protein oligomer, which is rare and unstable when generated from natural peptides14,15. However, as described earlier, almost any protein in principle can adopt amyloid conformation under the appropriate conditions. We recently established a method to prepare stabilized soluble oligomers of native proteins, which readily form amyloid in the presence of various cofactors16. The resulting amyloid fibrils reflect the complex nature of the terminal protein misfolding aggregates17,18. Here we use the term protein-only amyloid to refer the protein fibrils without cofactors and hybrid amyloid for the fibrils containing nonproteinaceous components.
Previously, various methods have been developed to generate amyloid in vitro by applying conditions known to favor protein misfolding, such as high temperature, hydrophobic environment, and pH variations19-23. However, they associate with various problems, such as low efficiency, reversible folding, large batch variations or slow kinetics. The approach described here is reproducible, easy to scale up, and allows one to precisely control the timing and condition of amyloid conversion.
Protocol
Representative Results
Discussion
The method described here offers a rapid and flexible means to prepare amyloid fibrils in vitro from virtually any protein or peptide of choice. Several different types of amyloid can be reliably prepared – protein-only fibrils or hybrid aggregates containing DNA, RNA, or glycosaminoglycans. The procedures involved are simple, straight forward, and do not require advanced technical training. The amyloid prepared by this method is quite stable and can be safely stored at 4 ºC or frozen at -80 ºC f…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by grants to W.C. from National Institutes of Health Grant AI074809 and The University of Texas M. D. Anderson Cancer Center Institutional Research Grant Program.
Materials
| 2-(N-morpholino)ethanesulfonic acid (MES) | Sigma-Aldrich | M8250 |
| NaCl | Fisher | BP358-10 |
| 1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC) | Thermo/Pierce | 22980 |
| DNA from salmon sperm | Sigma | D1626 |
| RNA from torula yeast | Sigma | R6625 |
| Heparin from porcine intestinal mucosa | Sigma | H3149 |
| Tris base | Fisher | BP152-1 |
| Equipment: | ||
| Material Name | Company | Catalogue Number |
| Water bath | Fisher Science | Isotemp 210 |
| Slide-A-Lyzer dialysis cassette | Thermo/Pierce | 66380 |
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