Trehalose is not only an antioxidant, it operates as a protein folding stabilizer to help . . .

Maintain Your Youthful Edge
It is important to keep precise folding
“edges” or functionality will be lost

By Will Block

All our words from loose using have lost their edge.
— Ernest Hemingway

S

tress is a natural aspect of aging, but if there is too much stress, aging can become like a speeding vehicle that is out of control. Yet if stress is shock absorbed, that alone can help avoid the inevitability of a serious crash. Not only that . . . adequately cushioned stress can profoundly elevate quality of life and promote youthful aging. When the events of aging get skewed from a healthy pathway, life’s molecular machinery is disrupted and malfunctions. With the passing of time, these events can lead to degenerative disease, of the mind and all the other systems of the body.

If you’ve been reading this publication, you know that stress overdose can have many horrendous consequences, including the problems of protein misfolding (see “The Origami of Aging” in the September 2008 issue, “Youthful Aging Depends on Proper Protein Folding” in the October 2008 issue, and “Chaperoning Your Proteins to Better Health” in the November 2008 issue). Remember that proteins are the principal products of gene expression and consequently determine the shape and function of cells, how they are properly synthesized, assembled, and employed in the service of life.

The Importance of Precise Folding Edges

Bear in mind that proteins have complex three-dimensional structures that establish their functions and stability. As proteins operate within the body, they unfold, and then refold. These properties are necessary to serve their many functions for maintenance of the health of the organism. But if they misfold, they may become unable to unfold properly thereafter. This in turn can render them proteotoxic, and this folding impairment is likely to seed a cascade of harmful molecular events, the end result of which is cellular dysfunction. As is the case with an origami or a folded paper plane, it is important to keep precise folding “edges” or functionality will be lost.

The Neurological Diseases of Misfolding

When these dysfunctions occur in our neurons, the results can be overwhelming, and lead to protein aggregation neurological diseases such as Alzheimer’s, Parkinson’s, Huntington’s, as well as amyotrophic lateral sclerosis (Lou Gehrig’s disease).

While the mechanisms of these diseases are different, the results are similar. For a specific mechanism, consider that the way aggregation occurs in Huntington’s is through what is known as the polyglutamine stretch. This categorizes Huntington’s as a polyQ disease, included within which are various spinocerebellar ataxias. These are genetic disorders characterized by slowly progressive loss of gait coordination and often associated with poor coordination of hands, speech, and eye movements. PolyQ diseases are caused by abnormal expansion of the polyQ stretch, when the number of glutamine molecules within disease-causative proteins increases by as much as twofold. Such stretches trigger misfolding and aggregation, lead to protein aggregation buildup, and eventually result in neurodegeneration.

Fortunately, science is developing support for the idea that that it may be important to offset the consequences of stress, not only for protein aggregation diseases, but for youthful aging. These goals may be achieved through the maintenance of proper protein folding, thus ensuring correct protein function. And recently, the use of supplementation to safely alleviate protein folding damage has made a beachhead.

The Importance of Protein Stability

Alterations in the cellular environment of a protein due to stress, mutation, or change in metabolic activities can result in unstable unfolding, the consequences of which can lead to interactions among other proteins and the ultimate loss or alteration of the protein’s function through misfolding. Not only has this protein deposition been reported for neurological diseases, as mentioned above, but as well for several systemic disorders such as hemolytic anemia, type II diabetes, and cystic fibrosis.

The structural hierarchy of proteins is rigorously defined. Consequently, as little as a single change in any component of this hierarchy—due to chemical or physical alterations—may lead to proper folding failure for a particular protein. In a correctly folded protein, there is usually a center of hydrophobic amino acid residues, which is encircled by components with hydrophilic functionalities. Once misfolded or partially unfolded, the hydrophobic protein core may become exposed to molecules that are similarly misfolded or partially unfolded. A variety of interactions may then result in protein aggregation formation.


Stress overdose can have many
horrendous consequences, including
the problems of protein misfolding.


Depending on the forces engendered, the aggregates fall into either a physical or chemical category, with the undesirable consequences that the aggregated proteins exhibit a range of altered activities from diffused activity to adverse reactions. As previously noted, these protein aggregations are destructive to health and causal for many degenerative diseases. Unfortunately, conventional aggregated protein therapeutics have little efficacy, short shelf life, and have been found to provoke undesirable immune responses.

Osmolytes Specialize in Protein Stability

In the cellular world, certain molecules called chaperones assist the function of proteins in the process of maintaining stability during the processes of unfolding and refolding. There is a class of natural chemical chaperones that specialize in stability for proper folding conformation. These are called osmolytes. They include trehalose, betaine, creatine, glycine, inositol, proline, taurine, and beta-alanine.


Folding impairment is likely to seed a
cascade of harmful molecular events,
the end result of which is
cellular dysfunction.


In our brains, the principal organic osmolytes include amino acids (such as proline, alanine, and glycine), choline, creatine, inositol, and taurine. In our kidneys, high concentrations of osmolytes are maintained that protect tissues exposed to high osmolality, high concentrations of salt and urea salutes. In other tissues, cells there too can also be subjected to hyperosmolality, although to a lesser extent than in the kidneys. Interestingly, plants, fungi, and bacteria also rely on similar osmolytes. Trehalose is particularly concentrated in these kingdoms, especially for protein stability.

Osmolytes Can Also Inhibit Protein Aggregation

By maintaining stability, osmolytes can also help inhibit protein aggregation, which as we now know is the result of protein malfunction caused by misfolding. The role of osmolytes, including trehalose, in stabilizing proteins under stress conditions is now a widely accepted fact. The physical and chemical properties of trehalose are many: low chemical reactivity, nonreducing nature, high glass transition temperature, high affinity for water molecules, existence of a number of polymorphs, and so on.1 Altogether, these properties make trehalose uniquely suitable for stabilizing partially unfolded protein molecules and inhibiting protein aggregation.

Excipients Help Stabilization

Whether inside or outside the cell, stabilization is important for proper function and protein utility. Any strategies to enhance stability must borrow from nature’s mechanisms. Above all, it is necessary to enhance without interfering with protein functionality. Stabilization can involve either avoidance of primary structure alterations by mutagenesis, or modification of the protein’s surroundings through the use of excipients such as sugars, amino acids, polyols, and salts. In this context, an excipient is a substance that is added to a formulation to provide benefits for the processing of the active ingredient, i.e. for increasing critical temperatures or providing protection.

Among the most widely used excipients for the stabilization of proteins are sugars, including sucrose, maltose, and trehalose. As previously noted, trehalose is frequently and widely synthesized in abundance by many living organisms under cellular stress explicitly for stabilization. The typical stress conditions include desiccation and lyophilization (freeze-drying) along with temperature and moisture extremes. Trehalose has also been show to stabilize different disease-related proteins whose instabilities are putative causes of diseases like Huntington’s disease and prion disease.2,3,4 Not so with other sugars.

Possible Trehalose Mechanisms for Protein Stability

Trehalose molecule
The cellular folding machinery has self-correcting mechanisms, and these include heat-shock proteins, chaperones, and regulatory proteins (ubiquitin proteasomes). But when these fail, pathogenesis follows. Life exists in extreme environmental conditions, so Mother Nature has evolved mechanisms to assist in survival. Among these are the mechanisms of osmolytes, which protect proteins and other biomolecules from denaturation under adverse conditions. Trehalose—a disaccharide in which two glucose units are linked together—is a rising evolutionary star found to protect in a variety of situations. It is found ubiquitously in many lower life forms, as well as eukaryotes such as yeast and fungi. It is also reported to be present in animals such as roundworms. Wherever it is found, trehalose serves as a source of energy, and as an osmoregulatory agent, cryoprotectant, and growth regulator. It also plays an important role for desiccation tolerance, among other functions. Among trehalose’s unique properties are inertness and high glass transition temperature (it remains viscous as the heat goes up). Altogether, these properties champion trehalose as a stabilizer of proteins, cells, and liposomes. It even finds use in vaccines, and most importantly as an inhibitor of protein aggregation that can cause the aforementioned diseases.

The Ways Trehalose Works

Trehalose may work in any number of ways including its mechanical entrapment of the native protein conformation in the viscous glassy matrix it forms to restrict protein mobility, thereby inhibiting unfolding under stressful conditions. When temperature is elevated, the glass transition temperature of trehalose remains above the ambient storage temperature and keeps protein in a vitrified (glassy) form, protecting the biomolecule trapped inside.


The use of supplementation to
safely alleviate protein folding
damage is underway.


Water plays a crucial role in the maintenance of the three-dimensional structure of proteins by forming hydrogen bonds with the hydrophilic residues on the protein surface. This stabilizes its structure. However, loss of water through desiccation can compromise the three-dimensional structure and decrease stability. But trehalose has a similar property of forming hydrogen bonds with proteins, so under conditions of water shortage (including desiccation), it can replace water thereby helping to maintain native protein structure.


Conventional aggregated protein
therapeutics have little efficacy,
short shelf life, and have been found
to provoke undesirable
immune responses.


Among its other attributes, trehalose has the ability to structure water molecules around itself under stress conditions, even more so than the water/water interaction. Because trehalose competes with protein for the formation of hydrogen bonds with water, it has a higher hydration volume. Furthermore, the hydrogen bonds it forms with water molecules result in protein compactness that increase rigidity and stability. Also, because of its cryoprotectant property, trehalose prevents the formation of ice crystals, which can denature protein. Any or all of these attributes can explain the stabilizing effect of trehalose under different conditions.

Trehalose as a Rescue Molecule

In ancient Africa during the Middle Stone Age, when much of the world was covered with glaciers, the African deserts were much more extensive than today. Lakes dried up, subtropical lands became more arid, tropical regions became desiccated, and primitive humans lived in a bottleneck caused by the struggle to obtain food (see article on page 4 for another type of rescue). Without sufficient water, plant and animal life tended to become dehydrated more readily. Every living system requires water for existence. Nonetheless, life continued to exist under low-water conditions. So stability facilitation by the presence of rescue molecules such as trehalose may have helped a wide variety of organisms, not only to survive but to thrive throughout the millennia under these harsh and stressful conditions.

It is an interesting aside that lyophilization—a process in which protein solution is frozen, and then dried to remove frozen water by mild heating under high vacuum—has benefited from the use of trehalose. Indeed, trehalose has emerged as one of the very few molecules which can act as a protector against both freezing and drying stress. Summarizing, trehalose facilitates water molecules to surround protein and structures water molecules around itself, thereby preventing the formation of ice crystals when the temperature is lowered during freezing.

Trehalose can also deal with protein water loss and thus can act as both cryoprotectant and lyoprotectant. Its superior physical and chemical properties—high glass transition temperature and chemical inertness—are the reasons for this advantage.

The Effect of Temperature Variation on Proteins and Trehalose

All proteins have temperature ranges within which they optimally operate, but above or below that range they lose function and eventually cease activity. As temperature increases, protein molecule mobility increases and the protein starts to unfold. This leads to greater interaction between partially folded/unfolded proteins, the result of which causes the formation of protein aggregates. In tropical countries where average temperature and humidity levels are very high, this problem is significant for the storage of protein.

However, trehalose has proven to be particularly effective in protecting proteins against deleterious temperature changes. Also, it protects against thermal denaturation through the formation of glassy matrix and by immobilizing the protein against the damage caused by mobility and flexibility under thermal stress.

In many organisms (e.g., yeast), trehalose is synthesized when they are exposed to heat stress. Along with trehalose, heat-shock proteins are produced. And as with trehalose, heat-shock proteins have been found to help stabilize partially folded proteins. And at high temperatures, the thermoprotective effect of trehalose is extended to enzymes that are then able to function under such conditions. Trehalose also has been reported to stabilize and enhance the activity of many enzymes which are used in high heat conditions. Other uses for trehalose involve recombinant DNA technology, reverse transcriptase, and restriction enzymes.

Trehalose for Biomolecules

We have covered a variety of circumstances in which cellular proteins face stress. Among these are oxidative stress, heat shock, and mutations such as the elongation of polyglutamine stretches. Trehalose can negate these stress factors and stabilize biomolecules in vivo. When water stress generates free radicals—more commonly known as reactive oxygen species—endogenous antioxidant enzymes such as superoxide dismutases, ascorbate catalases, and ascorbate peroxidases are normally capable of combating the damage. However, when the increase in free radicals becomes too great, the cellular machinery cannot keep up and the result is oxidative stress, with modifications in amino acid side chains including oxidation and formation of disulfide bridges. Consequently, cellular proteins are damaged and denatured.

Trehalose can offset oxidative stress by arresting free radicals. In yeast, trehalose has been found to offer protection against oxidative stress by inhibiting protein aggregation.5 It has also been found effective against hydrogen peroxide damage, and, in mammalian cells, trehalose has been shown to help prevent the generation of oxidative stress in prion-infected cells and prevent prion-protein aggregation.6 As well, there have been other significant findings for prion diseases. These include reducing the size and modifying the localization of PrPSc (a protease-resistant form of the prion protein, PrPc) protein aggregates.

When mutations lead to expansion of certain stretches of homopolymeric sequences—as mentioned above with polyglutamine stretches—there is an increased tendency to aggregate. And as we have noted, this type of aggregation is the cause of many neurodegenerative diseases like Huntington’s disease and oculopharyngeal muscular dystrophy (OPMD). Cell culture and mouse model studies have found that trehalose inhibits protein aggregation, stabilizes elongated protein sequences, and eradicates the symptoms of the disease. When transgenic mice modeling Huntington’s disease were administered trehalose orally, researchers found it could reduce protein aggregation and alleviate symptoms of the disease.2 In another study with similar design transgenic mice modeling OPMD, the lab animals were fed trehalose. The aggregation of the homopolymeric sequences which are responsible for causing this disease were prevented.4 In the development of Alzheimer’s disease, soluble β-amyloid is converted to aggregated plaque in the brain, and scientists have reported that trehalose can hinder this conversion and reduce aggregation toxicity.3

The Challenge for Supplement Producers

Stabilization of proteins is a challenge for scientists who design biopharmaceutical formulations, work in the protein production industry, and develop therapy regimens for protein-misfolding diseases. Uniting all three task forces are the common themes of preserving protein structure and preventing protein aggregation. Trehalose bridges these arenas of focus and consequently has emerged wearing a heroic mantle. Like a medieval knight—combining all the accoutrements of the perfect war machine—trehalose displays a wide variety of armaments to act as an efficient stabilizer of protein molecules under adverse conditions, thereby inhibiting protein aggregation. Whether developing protein therapeutics formulations, in vitro, or investigating protein-misfolding diseases, in vivo, trehalose is unique. Lest we forget, trehalose is a food and a nutrient, and its rightful home without doubt is the nutritional supplement industry. That’s our challenge.

References

  1. Jain NK, Roy I. Trehalose and protein stability. Curr Protoc Protein Sci 2010 Feb;Chapter 4:Unit4.9.
  2. Tanaka M, Michida Y, Niu S, Ikeda T, Jana NR, Doi H, Kurosawa M, Nekooki M, Nukina N. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington’s disease. Nat Med 2004.10:148-54.
  3. Liu R, Barkhordarian H, Emadi S, Park CB, Sierks MR. Trehalose differentially inhibits aggregation and neurotoxicity of beta amyloid 40 and 42. Neurobiol Dis 2005;20:74-81.
  4. Davies JE, Sarkar S, Rubinsztein DC. Wild-type PABPN1 is anti-apoptotic and reduces toxicity of the oculopharyngeal muscular dystrophy mutation. Hum Mol Genet 2006;15:23-31.
  5. Luo Y, Li W-M, Wang W. Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress? Environ Exper Botan 2008;63:378-84.
  6. Béranger F, Crozet C, Goldsborough A., Lehmann S. Trehalose impairs aggregation of PrPSc molecules and protects prion-infected cells against oxidative damage. Biochem Biophys Res Commun 2008;374:44-8.


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

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