Proper protein folding can protect your brain . . .

Beta-Alanine Fights
Alzheimer’s Amyloid

And other disease aggregations
By Will Block


rotein aggregation is an established signpost of Alzheimer’s and Parkinson’s disease and is also associated with the formation of amyloid plaque. Unfortunately, there are no proven clinical solutions to prevent protein aggregation. Thus, an increasing large number of otherwise healthy individuals are swept toward the abyss of memory loss and ultimately over the edge, with the complete dissolution of their ability to recognize their loved ones and even themselves.

Current conventional therapies are hamstrung and concentrate on the relief of symptoms. (It’s better than nothing!) However, there are new approaches under investigation, and one hopeful course is the targeting of aggregating proteins with small proteins that interrupt the process through which toxic intermediates and mature fibrils are formed. Fibrils are fibrous tissues subject to deterioration. When these take a certain degenerative tilt in the brain, they either become neurofibrillary tangles or amyloid plaque, the two distinguishing physiological characteristics of Alzheimer’s disease.

The Irony of Defeating Self-Recognition in Order to Save It

Curiously, within the aggregating proteins that drive the assembly of fibrils into β-sheet layers* (constituting the fibril core) is something termed a self-recognition element (SRE)—to some extent a misnomer. Nevertheless, by targeting SRE with peptides or synthetic peptides containing subtle modifications—that can pass recognition and bond to the self-recognition site of the parent protein—it is possible to block aggregation. Then—ironically—by inhibiting self-recognition within aggregating tissue, we can help preserve our ability to recognize ourselves!

* A β-sheet (aka β-pleated sheet) is a form of the regular secondary structure found in proteins (see sidebar, "A Protein Primer," below). It consists of beta strands (thus the name) connected laterally to hydrogen bonds, forming a twisted, pleated sheet (the most common form of regular secondary structure in proteins is the alpha helix). The association of β-sheets has been implicated in the formation of protein aggregates and fibrils observed in many human diseases, notably the amyloidoses.

† Peptides (from the Greek for “small digestibles”) are short polymers formed from the linking, in a defined order, of α-amino acids.

Among the strategies to modify SRE and disrupt aggregation include the use of β-sheet-breaking amino acid substitutions (proline is an example1). Other strategies entail the use of N-methylated amino acids to disrupt β-sheets,2 the addition of N- and C-terminal blocking or disrupting groups,2-3 the replacement of amide bonds with ester linkages,4 and introduction of α-disubstituted amino acids, such as α-aminobutyric acid.5 This can get fairly complicated. For our purposes, β-sheet-disruption strategy is the primary focus.

Recovering Cognitive Performance in Alzheimer’s Mice

In vitro, several SRE-targeted modified peptides have been found effective at preventing protein aggregation and reducing cellular toxicity. These β-sheet breakers have also recently been shown to recover cognitive performance in mouse models of Alzheimer’s disease. Not surprisingly, the main component of amyloid plaques in the brains of Alzheimer’s disease patients is called amyloid beta. In one study, researchers reported a significant increase in neuronal survival and a decrease in brain inflammation associated with the reduction of amyloid plaques.6 A much more recent study done in Israel has found similar results.7 In this study, the use of a rationally-designed oligomerization inhibitor enabled modulation of early soluble amyloidal assemblies resulting in cognitive-performance recovery in Alzheimer’s mice.

‡ In chemistry an oligimer is a molecular intermediate between a monomer and a polymer. An agent that inhibits oligimerization, prevents the creation of larger molecules that no longer are able to prevent the formation of early intermediate assemblies of amyloid plaque.

β-Sheet Breakers Disable the Protein Aggregation

Nonetheless, not one of these therapies has yet been embraced for humans in conventional medicine, and the search for new inhibitory agents continues. In a new paper, Dr. Jill Madine of the University of Liverpool and colleagues have identified a new class of modified peptides in which native alanine residues of an SRE are replaced with β-alanine (beta-alanine) or γ-aminobutyric acid (GABA).8 With this type of substitution, the backbone of the SRE is replaced by one or two methylene segments from either beta-alanine or GABA. This causes interruption of the β-sheet pattern of hydrogen bonding to the target protein (either N- or C-terminally**) to the substitution site. Importantly, it does not prevent its other normal functions.

** The N-terminus (also known as the amino-terminus) refers to the start of a protein or polypeptide terminated by an amino acid with a free amine group (-NH2). The C-terminus (also known as the carboxy-terminus) of a protein or polypeptide is the end of the amino acid chain terminated by a free carboxyl group (-COOH).

Thus beta-alanine or GABA substitution acts as a β-sheet breaker, enabling SRE peptides to still interact with the recognition site of the target, but with an important difference: the modifications inhibit further aggregation in the hydrogen-bonding direction and thus inhibit protein aggregation.

The Idea is to Control Protein Misfolding

Unfortunately, as with many peptide inhibitors, the precise mode of action is difficult to determine. What is known is that longer β-peptides composed of multiple β-amino acids readily form helical structures. In the new research, Madine et al. selected as their initial target α-synuclein. In Lewy bodies disease, α-synuclein is the major protein component, a type of neurodegenerative lesion. It is also found in the tissue of those with Parkinson’s disease and some dementias. Futhermore, it resembles fibrillar Abeta, also known as amyloid beta, the primary constituent of amyloid plaques found in the brains of those with Alzheimer’s disease.

Researchers reported a significant
increase in neuronal survival and a
decrease in brain inflammation
associated with the reduction of
amyloid plaques.

In aqueous solution, α-synuclein is predominantly unfolded, but it can misfold and aggregate into amyloid tissue (see “The Origami of Aging” in the September 2008 issue, “Youthful Aging Depends on Proper Protein Folding” in the October 2008 issue, “Chaperoning Your Proteins to Better Health” in the November 2008 issue, and “β-Alanine Fights Fatigue” in the January 2009 issue). Misfolded α-synuclein is prominently involved in Parkinson’s disease.

In a previous study, Dr. Madine and collegues identified a hexapeptide SRE of α-synuclein and demonstrated that N-methylated analogues of this hexameric sequence are effective as inhibitors of α-synuclein aggregation in vitro.9 In their current study, the researchers took these analogues as a starting template sequence, with which to replace alanine with β-alanine and GABA, and tested the peptides for their effectiveness at preventing aggregation of α-synuclein. Of nine peptides tested only beta-alanine and GABA were successful.

In a recent Israeli study,
cognitive performance recovery in
Alzheimer’s mice was reported with
the use of β-sheet breakers.

From a therapeutic standpoint, it is desirable to limit the amount of the inhibitory agents if they are to be utilized as drug candidates. (The bias of Madine et al. is to find a patentable drug, rather than a mere natural material.) Of particular consequence, upon examining tetrapeptide and tripeptide variants of the substituted SRE peptides, β-alanine and GABA, the tetrameric peptides incorporating β-alanine and GABA show no significant effect on amyloid formation compared to α-synuclein. This was also found to be the case with GABA-substituted tripeptides, where the effect was small. By contrast, the β-alanine-substituted tripeptides reduced the amyloid characteristic of α-synuclein by 80% or more relative to control at both day three and day. GABA, the results suggest, acts as a β-sheet breaker when incorporated into the hexameric peptide but is ineffective within shorter peptides. However, β-alanine is moderately effective in the hexamers, but the beta-alanine-substituted tripeptides appear to have the greatest effect in reducing amyloid formation.

A Protein Primer

A protein’s primary structure (amino acid chain, left) folds itself spontaneously into its tertiary structure, or native state (right), which incorporates the two main types of secondary structure: α-helix (coiled segments) and β-pleated sheet (ribbon segments).
Not counting the water, about half of your body weight consists of proteins. These diverse and dazzlingly versatile compounds constitute much of the machinery of life, providing both structural stability and functional capability for the myriad workings of your body. They are the product of life’s master molecule, DNA, whose primary role is to be a genetic “blueprint” for your proteins; it does this by encoding their amino acid sequences in its molecular structure.

All of the biochemical and biomechanical wonders performed by proteins come about as a result of their molecular structures, most of which are exceedingly complex. To help our understanding, we can view these structures at four levels, which develop in sequence when proteins are synthesized in our cells:

  • Primary structure – This is simply the linear sequence of amino acids in the chain, as determined by the gene encoding that protein. A mutation in the gene may alter the amino acid sequence of the protein, resulting in functional changes that can range from inconsequential to fatal.

  • Secondary structure – This is the occurrence, along some segments of the chain, of localized, three-dimensional amino acid structures that are held together by a type of weak chemical bond called a hydrogen bond, whose importance to the existence of life cannot be overstated. The two most common of these protein “scaffold” structures are: (1) the α-helix, a spiral configuration of the amino acid chain; and (2) the β-pleated sheet, a configuration in which the chain loops back and forth upon itself a number of times so that parallel segments of it lie side-by-side in a pattern whose atomic landscape resembles pleats (which run perpendicular to the segments). The prediction and discovery of these structures by Linus Pauling in 1951 was a pivotal event in the history of chemistry.

  • Tertiary structure – This is the protein’s ultimate three-dimensional configuration, the result of the amino acid chain’s intricate twisting and turning and folding in upon itself until it settles into a stable molecular structure, usually more or less globular or ellipsoidal in overall shape (many others, however, are long and fibrous). The process is called intramolecular self-assembly—or simply folding—and it’s enormously complex. Despite the virtually infinite number of ways in which the chain could fold, it almost invariably folds to the one “correct” structure, i.e., the one that we know (from experimental evidence) to be the normal structure of the protein under the physiological conditions in question. This seemingly magical ability to select the one correct structure from an infinity of choices is a consequence of the laws of chemical thermodynamics, which determine the outcomes of all chemical processes and, therefore, of all biological processes.

  • Quaternary structure – This represents the special case in which fully formed protein molecules—sometimes of the same kind, sometimes of different kinds—become bonded to each other to form a supramolecular complex with a definite overall structure. This important process occurs naturally with many proteins, yielding complexes (e.g., hormone and neurotransmitter receptors in our cell membranes) that are essential for life.

    With proteins, structure determines function. In most cases, it’s not so much the overall molecular structure that determines the protein’s function as it is the detailed atomic force fields associated with various features of its irregular surface—the protuberances, depressions, clefts, and channels that give each protein its unique form. All of these features result from the protein’s folding pattern, and all of them influence, in one way or another, the ways in which the protein interacts with the thousands of other kinds of molecules in the chemical “soup” of our cells and body fluids.

    The interactions occur via different kinds of interatomic and intermolecular forces that are governed by the laws of quantum mechanics, and they’re of the same kinds that hold the protein together in its folded configuration in the first place. The mathematical analysis of these interactions in proteins is very difficult because of the large numbers of atoms, their complex geometric arrays, and the varied (and variable) force fields associated with them.

A New Therapy for Misfolding Diseases

The work of Dr. Madine and others demonstrates a new therapeutic strategy: that GABA and beta-alanine-substituted peptides can be used for reducing the aggregation of α-synuclein and other polypeptides associated with misfolding diseases. While there are differences in efficacy depending on the variables outlined above, Madine et al. have nevertheless identified two trimeric β-alanine-substituted peptides that inhibit α-synuclein aggregation.

Of nine peptides tested only
beta-alanine and GABA were

One of the beta-alanine peptides is also effective at reducing aggregations of amyloid beta1–40 and amylin, allowing the conclusion that beta-alanine is a common precursor for designing agents aimed for the treatment of Lewy body disease, as well as Alzheimer’s and type 2 diabetes.†† One of the likely mechanisms entails interrupting the conventional β-sheet pattern of hydrogen bonding N- or C-terminally to the substitution site. This might deter further protein target molecules from bonding to the main-chain of the aggregating species. Another possible mechanism, might involve hydrophobic interactions through valine, ionic bonding through lysine, or hydrogen bonding through glutamine side groups.

†† Of course, the real money is in drugs, so every researcher sees castles in the sky in the form of patentable drugs.

Osmolytes Can Help Prevent Misfolding by Acting as Molecular Chaperones

Apparently, both beta-alanine and GABA can disrupt the formation of protein aggregation in the brains of those with neurodegenerative diseases, by helping to prevent misfolding. But GABA has not been shown to operate as an osmolyte, whereas beta-alanine (an amino acid constituent of the dipeptide carnosine) does act to protect against osmotic stress in lower organisms, various animal models, and plants. (See “The Origami of Aging” in the September 2008 issue). What’s more, beta-alanine has been found to protects neurons from excitotoxicity-induced damage and cell death.

The beta-alanine-substituted
tripeptides appear to have the
greatest effect in reducing
amyloid formation.

Adding to that knowledge, most directly, a recent study has shown that beta-alanine may act as a molecular chaperone to protect the enzyme lactate dehydrogenase against heat-induced inactivation, denaturation and aggregation.10 In this study, the authors explain that the results of similar experiments using other osmolytes suggest that beta-alanine’s suppression of protein denaturation was greater than that of the osmolytes glycerol, trehalose, or proline. Other osmolytes of interest include taurine, inositol, glycine, creatine, and betaine. If you’re interested in preventing amyloid aggregation, osmolytes are important nutrients. Their use can help chaperone you to greater health.


  1. Jiang P, Xu W, Mu Y. Amyloidogenesis abolished by proline substitutions but enhanced by lipid binding. PLoS Comput Biol 2009 Apr;5(4):e1000357. Epub 2009 Apr 10.
  2. Sciarretta KL, Boire A, Gordon DJ, Meredith SC. Spatial separation of beta-sheet domains of beta-amyloid: disruption of each beta-sheet by N-methyl amino acids. Biochemistry 2006 Aug 8;45(31):9485-95.
  3. Kirkitadze MD, Condron MM, Teplow DB. Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis. J Mol Biol 2001 Oct 5;312(5):1103-19.
  4. Gordon DJ, Meredith SC. Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions. Biochemistry 2003 Jan 21;42(2):475-85.
  5. Etienne MA, Aucoin JP, Fu Y, McCarley RL, Hammer RP. Stoichiometric inhibition of amyloid beta-protein aggregation with peptides containing alternating alpha,alpha-disubstituted amino acids. J Am Chem Soc 2006 Mar 22;128(11):3522-3.
  6. Permanne B, Adessi C, Saborio GP, Fraga S, Frossard MJ, Van Dorpe J, Dewachter I, Banks WA, Van Leuven F, Soto C. Reduction of amyloid load and cerebral damage in a transgenic mouse model of Alzheimer’s disease by treatment with a beta-sheet breaker peptide. FASEB J 2002 Jun;16(8):860-2.
  7. Frydman-Marom A, Rechter M, Shefler I, Bram Y, Shalev DE, Gazit E. Cognitive-performance recovery of Alzheimer’s disease model mice by modulation of early soluble amyloidal assemblies. Angew Chem Int Ed Engl 2009;48(11):1981-6.
  8. Madine J, Wang X, Brown DR, Middleton DA. Evaluation of beta-alanine- and GABA-substituted peptides as inhibitors of disease-linked protein aggregation. Chembiochem 2009 Jul 9. [Epub ahead of print]
  9. Madine J, Doig AJ, Middleton DA. Design of an N-methylated peptide inhibitor of alpha-synuclein aggregation guided by solid-state NMR. J Am Chem Soc 2008 Jun 25;130(25):7873-81. Epub 2008 May 30.
  10. Mehta AD, Seidler NW. Beta-alanine suppresses heat inactivation of lactate dehydrogenase. J Enzyme Inhib Med Chem 2005 Apr;20(2):199-203.

Will Block is the publisher and editorial director of Life Enhancement magazine.

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