BIOCHEMISTRY - DR. JAKUBOWSKI
Last Update: 03/10/16
Learning Goals/Objectives for Chapter 2H: After class and this reading, students will be able to
H1. Protein Aggregation
We have seen that protein aggregates complicate the lives of people who study protein folding in vitro and who try to express human proteins in prokaryotes like E. Coli in vivo. Instead of viewing these aggregates as junk, some now study them avidly. It turns out that these aggregates are not as non-specific as earlier believed. In addition, an understanding of how and when they form will give us clues into the etiology and treatment of some of the most debilitating and feared diseases. Much of this review is based on the following reference: Taubes, G. Misfolding the Way to Disease, G. Science, 271, 1493-1495 (1996)
Clues Showing the Specificity of Aggregate Formation
Protein Aggregates and Disease
This knowledge of protein folding and aggregation was soon turn toward understanding several diseases in which protein aggregates were observed which either initiated or were associated with disease. These protein aggregates were termed "amyloid deposits" and seemed to be associated and perhaps causative of several neurodegenerative diseases. The name amyloid was first used by a German pathologist, Rudolf Virchow, who in 1853 described waxy tissue deposits associated with eosinophils (a type of immune cell). These deposits seemed to resemble starch (made of amylose and amylopectin) so he termed them amyloid. All known amyloid deposits are, however, composed of protein, not starch. It now appears that these disease may be caused by improper protein folding and subsequent aggregation. Except in certain rare inherited diseases, the amyloid deposits are composed of normal proteins (often called wild-type proteins) which seems to polymerize into fibrils. Sometimes, in inherited conditions, or when mutations appear in a specific protein, the amyloid protein deposits consist of the mutant protein. The proteins in these deposited fibers are composed predominantly of beta sheets which are perpendicular to the fiber axis, while the polymerized protein usually has little beta sheet structure. Examples are given below:
H2. Prions and Disease
The normal cellular form of this protein, PrPc is highly conserved in mammals, and is widely expressed in embryogenesis. Techniques exist to delete or make ineffectual genes in mice. When a mice knockout of the PrPc (i.e. the gene for the protein was deleted in all cells) was made, the mice appeared normal. More recent data suggests however, that these mice had altered circadian rhythms and sleep patterns, which suggest a possible link to Fatal Familial Insomnia. The PrPc is a normal membrane protein in neurons. It is anchored to the membrane through a glycosyl-phosphatidyl inositol link, with the protein chain on the outside of the neuronal plasma membrane. The PrPc (without the PI link) is water soluble, protease sensitive, and consists of 42% alpha helix and 3% beta sheet.
The problem in the transmissible spongiform encephalopathies (TSE's) is that amyloid-like protein aggregates form which appear to be neurotoxic. The protein found in the plaques (in cases other that those that are inherited) has the same primary sequence as the PrPc but a different secondary and presumably tertiary structure. The protein found in the plaques, called the PrPsc (the scrapie form of the the normal protein) is insoluble in aqueuous solution, protease resistant, and has a high beta sheet content (43%) and lower alpha helix content (30%) than the normal version of the protein PrPc.
Figure: Cartoon Models of PrPc and PrPsc
A genetic, inheritable form of disease also exists, in which a mutant form of the PrPc occurs, whose normal structure is destabilized by the mutation. The aggregates caused by the mutant form of the disease are understandable in light of the other diseases which we discussed above. The question is how does the normal PrPc form PrPsc . Evidence shows that if radiolabeled PrP*c from scrapie free cells is added to unlabeled PrPsc from scapie infected cells, the PrP*c is converted to PrP*sc! It appears that the PrPc protein has two forms not that much different in energy, one composed of mostly alpha helix and the other of beta sheet. A dimer of PrPc.PrPsc might form, which destabilizes the PrP*c causing a conformational shift to the PrPsc form, which would then aggregate. Exposure to the PrPsc form would then catalyze the conversion of normal PrPc to PrPsc . Hence, it would be transmissible by contact with just the PrPsc form of the protein. Likewise species specificity could be explained if only dimers of PrPc.PrPsc formed from proteins of the same species could occur. The inherited form of the disease would be explained since the mutant form of the normal protein would more easily form the beta structure found in the aggregate.
It has recently been found that the very same mutation in PrPc, Asp178Asn can cause two different diseases - CJD and FFI. Which disease you get depends on if you have 1 of two naturally occurring, nonharmful variants at amino acid 129 of the normal PrPc gene. If you have a Met at that position, and acquire the Asp178Asn mutation, you get CJD. If, on the other hand, you have a Val at amino acid 129 and acquire the Asp178Asn mutation, you get FFI. This disease was first observed in 1986 and has been reported in five families in the world. It occurs in the late 50's, equally in men and women. It is characterized by a progressive loss of the ability to sleep and disrupted circadian rhythms. The brain shows neuronal loss. It is known that amino acids 129 and 178 occur at the start of alpha helices, as predicted from propensity calculations. Chronic exposure to micromolar levels of synthetic fragment 106-126 of PrPc kills hippocampal neurons. This peptide also has the greatest tendency to aggregate synthetic PrPc peptides.
A series of recent studies have expanded on our knowledge of prion structure. Nelson et al. have obtained the crystal structure of a fibril aggregate made of a short peptide (7 amino acids) from the yeast prion protein Sup35. As presumably occurs in amyloid fibers, these crystals show beta-sheet structures stacked vertically to produce fibril structures. The unit of stacking appears to be pairs of beta-sheets, with the inner side amino acids of one member of the pair interacting with the inner amino acid side chains of the other member of the pair, in a process which excludes water. Similar studies by Ritter et al, using NMR and fluorescence, found pair of beta sheets to be the motif of the fibril. Using fluorescence, they identified two regions, each 15 amino acids, important in collapse to molten-globule like state for nucleation of fibril formation.
Kuru killed many members of the Fore tribe in New Guinea until the cannibalistic practice of eating dead relatives was stopped. Analysis of the genes for the prion protein in the Fore tribe and other ethnic groups in the world show two version differing by just one amino acid in all people (remember that a single gene is represented in both maternal and paternal chromosomes. That these two forms exist through the world suggest that they have been selected for by evolution and confer some biological advantage. People who have just one form of the protein are more susceptible to the development of prion diseases. Mead and Collinge have shown that about 75% of older Fore women (who had lived through cannibalistic practices) had two different prion genes, compared to about 15% of women from other ethnic groups. This high percentage suggests that these women were protected from the disease, leading through natural selection to a high percentage of heterozygotes in this defined population. The general presence of two forms of the prion gene (which probably offers protection from prion disease) suggests that cannibalism might have been widespread in our early ancestors.
There appears to be one main difference between the formation of amyloid fibers from prion proteins and others such as mutant lysozymes. If you add mutant lysozyme to normal lysozyme, the amyloid fibers contain only the mutant protein. However, if you incubate mutant prion proteins with normal prions, the normal proteins become pathological.
QED - Protein Aggregates are not just test tube artifacts, but rather matters of life and death.
CDC: Prion Disease
Jsmol: Protopedia - Prions
H3. Misfolding and Aggregation - Summary
Recent work has shown the proteins considered to be completely harmless can generate misfolded intermediates that aggregate to produce pre-fibril structures that are toxic to cells. This process is usually prevented in the cell by interaction of nascent forms of the proteins with chaperones, which sequester exposed hydrophobic patches and prevent aggregation. (Obviously prion proteins and the others mentioned above are exceptions). Amyloid fibers (characterized by subunits with an abnormal amount of beta-structure) can be made from many different types of proteins as noted above. Is this property specific to just a handful of proteins, or is it more common than expected from the limited examples noted so far? The new studies show that when a bacterial protein HypF is incubated at pH 5.5 in the presence of trifluoroethanol, aggregates (but not fibrils) form with enhanced beta structure. These aggregates slowly form into fibrils characteristic of amyloid protein fibers. The early aggregates (before fibril formation) proved cytotoxic. Similar results were seen with dimers and trimers (prefibril states) of the amyloid-b peptide released from cultured neurons.
A diverse group of proteins that do not share significant secondary or tertiary structure can form amyloid-like protein aggregates. Even though their monomer forms share little in common, the insoluble amyloid aggregates have a common structure in which the monomer in the aggregates has significant b-structure whose strands run perpendicular to the aggregate axis. Since it has recently been shown that almost any protein, under the "right" set of conditions can form such aggregates, the stabilizing feature of protein aggregates must be potentially found in any protein. Evidence suggests that is the presence of a polypeptide backbone, which can form stable interstrand H-bonds in beta secondary structure, and not the side-chains, that is the source of the common amyloid structure. In contrast, native, nonamyloid forms of normal proteins must arise through specific interactions of unique side chain sequence and structure, which out competes nonspecific interactions among backbone atoms found in amyloid structures. Nonspecific aggregation becomes more prevalent when buried hydrophobic side chains and buried main chain atoms become more solvent exposed. Such exposure occurs when native proteins form intermediate molten globule states when subjected to altered solvent conditions or when destabilizing mutants of the wild-type protein arise. Some mutations may alter the cooperativity of folding which would increases the fraction of nonnative proteins states. Other mutations that decrease the charge on the protein or increasing their hydrophobicity might enhance aggregation. In addition, chemical modifications to proteins (such as oxidation or deamination) might destabilize the native state, leading to the formation of the molten globule state. Once formed, this state may aggregate through sequestering exposed side chain hydrophobes or through inter-main chain H bond formation. Aggregate formation appears to proceed through the initial formation of soluble units (which may or not be more toxic to cells than the final aggregate). Aggregates are kinetically stable species. Since amyloid aggregates are cytotoxic and almost any protein can form them, albeit with different propensities, nature, through evolutionary selection, has presumably disfavored proteins with high tendencies to form such aggregates.
Wasmer et al have determined a solid state NMR structure of an amyloid fiber state for a prion forming domain of the HET-s protein from the fungus P. anserina. It consists of a left-handed b solenoid.
Clearly accurate protein folding is required for cell viability. Aberrant protein folding clearly can be the cause of serious illness. Given the extraordinary nature of the task and its failure, the process governing protein folding must be highly regulated. The diagram below shows the steps that determine intracellular concentrations and locations of normal and aberrant protein structures.
Potential therapies of diseases of proteostasis include replacing aberrant proteins, shifting the equilibria toward active forms with small ligands, or modulating the pathways with agents that influence pathways such as signal transduction, transcription, translation, degradation, and translocation using molecules like siRNAs to modulate concentrations of chaperons, disaggregases, and signal pathways.
H4. Recent Links and References
Wasmer, C. Amyloid Fibrils of the HET-s(218-289) Prion Form a b Solenoid with a Triangular Hydrophobic Core. Science. 319, 1523 (2008)
Balch, W. et al. Adapting Proteostasis for Disease Intervention. Science. 319, 916 (2008)
Krishnan and Linquist, Structural insights into a yeast prion illuminate nucleation and strain diversity. Nature, 435, 765 (2005)
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Archived version of full Chapter 2H: Protein Aggregates and Disease