A Great Leap Forward

The Origami of Aging
How Small Molecules Help Maintain Proper
Protein Folding for Better Health and Longevity

By Durk Pearson & Sandy Shaw

variety of age-related diseases, such as neurodegenerative conditions (including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis), are associated with aggregation of proteins due to improper folding. Other conditions (known as “conformational diseases”) associated with improperly folded proteins include cancer, cystic fibrosis, emphysema, liver disease, prion disease (such as mad cow) and even chronic pain (opioid receptors misfolded in the endoplasmic reticulum).1 As noted below, protein aggregation may also cause cataracts.

“Accumulation of protein aggregates and misfolded moieties is a nearly universal phenomenon during aging. Such aggregates have been specifically referred to as amyloids, lysosomal lipofuscin, ceroid bodies, advanced glycation end-products (AGEs), and cytoplasmic inclusions observed in senescent cells, as well as in affected tissues of age-associated diseases such as diabetes and Alzheimer’s disease.”2 “. . . more general perturbations to protein folding homeostasis, referred to as proteotoxicity, tend to manifest later in life and are closely linked to the time- and use-dependent attrition to cellular and organismal function recognized as aging.”2b Proteins have complex three-dimensional structures that determine their functions and stability. Hence, the quality control of protein folding is very important to health and longevity.

Natural Stabilizers of Protein Structure

An important class of natural chemical chaperones that help stabilize the proper folding conformation of proteins are the osmolytes. These include betaine, inositol, taurine, glycerophosphocholine, choline, and creatine.2 The principal organic osmolytes in the mammalian brain include amino acids (such as proline, alanine, and glycine), choline, creatine, inositol, and taurine.2 The osmolytes are maintained at particularly high concentrations in the kidneys, which are exposed to high osmolality (high concentrations of salt and urea), but cells in other tissues can also be exposed to hyperosmolality, although to a lesser extent than in the kidneys. Plants, bacteria, and yeasts also rely on the same and some other (e.g., trehalose) osmolytes for protein stability. We have been taking a cocktail of osmolytes—including proline, betaine, inositol, taurine, creatine, and glycine—for several months to help maintain our proteins in a properly folded state. We recently added beta-alanine to the mix. Although one probably won’t, in the short term, notice anything as a result of improved protein folding, if one wants to reach the longer term in good condition, maintaining proper protein structure is vital.

How They Work

The interior of cells is comprised of a complex stew of ingredients. Maintaining the stability of proteins involves keeping protein denaturing (structural derangement-inducing) ingredients (such as urea) away from the protein surface by buffering the surface with chaperones such as organic osmolytes that protect against contact with these denaturing agents and do not themselves interfere with protein/solvent (water) interactions.3 As further explained in Reference 3, “. . . the protein surface area exposed to solvent tends toward a minimal value, when these organic osmolytes are present; i.e., proteins fold into their compact native conformations; protein subunits aggregate; and the stability of multiprotein complexes is favored.”

Examples of Osmolyte Protection Against Protein Aggregation

Proline

One paper4 reports that proline protected an aggregation-prone protein, P39A cellular retinoic acid-binding protein, both in vitro and in vivo in an E. coli expression system. Moreover, on the basis of the nonspecific nature of this protection, the authors hypothesize that “the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases, including Huntington’s, Alzheimer’s, and Parkinson’s. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.”

In the commentary5 on the above paper, the author notes that “Naturally occurring osmolytes have been selected by evolution to accumulate in response to intracellular protein aggregation caused by desiccation, heat stress, freezing, high hydrostatic pressure, and the transient or constitutive increase in intracellular denaturants such as urea. . . . Many diverse intracellular osmolyte combinations could certainly be tried. For instance, proline along with other antiaggregation osmolytes such as trehalose may be useful for determining how endogenously synthesized intracellular osmolytes may act synergistically to more effectively prevent general in vivo protein aggregation.”5

Enzyme Inactivation and Aggregation by Heat Prevented by Beta-Alanine

Although beta-alanine (an amino acid constituent of the dipeptide carnosine) has not been included in the lists of osmolytes that we have seen, in fact beta-alanine does act as an osmolyte (protecting against osmotic stress) in lower organisms, in various animal models, and in plants.5a In addition, beta-alanine protects neurons against excitotoxicity-induced damage and death. Most directly, a recent study5a showed that beta-alanine appears to act as a molecular chaperone to protect the enzyme lactate dehydrogenase (LDH) against heat-induced denaturation and aggregation. Beta-alanine was able to disaggregate 27.6% of aggregated LDH at a concentration of 25 mM. It suppressed heat denaturation of LDH by 70% at a molar ratio of approximately 300:1 (beta-alanine/LDH). 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.

Trehalose Decreased Aggregate Formation in Mouse Model of Huntington’s Disease

The osmolyte trehalose was reported to stabilize aggregation-prone proteins.5b,5c A mouse model was used in which mice were genetically engineered to overexpress a truncated huntingtin gene containing a 145 glutamine repeat. (In humans, a polyglutamine repeat of 35 or more in the huntingtin gene results in Huntington’s disease.) Oral administration of trehalose (2% in drinking water of R6/2 mice) decreased polyglutamine-induced protein aggregation, reduced weight loss (due to the disease), ameliorated brain atrophy, and inhibited the formation of truncated huntingtin aggregates in the cerebrum and liver, as well as increasing the treated genetically altered mice’s lifespan by 10% as compared to untreated mice with the identical genetic alteration. Interestingly, the length of CAG (glutamine) repeats in the androgen receptor gene have been found to be associated with androgen-related diseases, such as spinal and bulbar muscular atrophy and prostate cancer; also, longer CAG repeats were associated with lower cognitive functioning in white, older men.5d

Protection Against Prion Protein Misfolding by Protective Osmolytes

Mad cow disease is one of the prion diseases, where a naturally occurring protein, PrPc, normally existing in a soluble globular state, becomes converted into an alternatively folded variant, PrPSc. Trimethylamine N-oxide and other protective osmolytes have been shown to prevent formation of PrPSc in scrapie-infected mouse neuroblastoma cells.6

Osmolytes Prevent Aggregation of Alpha-Synuclein, a Protein Found Misfolded in Parkinson’s Disease

“Recently it has been demonstrated that the naturally occurring osmolyte trimethylamine N-oxide (TMAO) can cause thermodynamically unstable proteins to fold and regain high functional activity. Osmolytes may fold unstructured proteins due to the osmophobic effect, a solvophobic thermodynamic force, arising from the unfavorable interaction between the osmolyte and the peptide backbone.”7 In a recent paper,7 the osmolyte trimethylamine N-oxide induced folding of alpha-synuclein into the native form from an unfolded form. Misfolded alpha-synuclein is importantly involved in Parkinson’s disease.

Counteraction of Urea-Induced Protein Denaturation

Another paper8 reported that the osmolyte trimethylamine N-oxide countered the urea-induced denaturation of the protein chymotrypsin inhibitor 2. “The dominant mode of stabilization of the native state was indirect: TMAO molecules ordered and strengthened water structure, thereby discouraging unfolding of the protein. Furthermore, TMAO decreased urea-protein interactions and strengthened urea-water interactions, thereby mitigating the denaturant action of urea.”

Cataract Risk Reduced by Osmolytes

The osmolytes 4-phenylbutyric acid, trimethylamine N-oxide, or tauroursodeoxycholic acid were used to treat cultured human lens epithelial cells that had been exposed to endoplasmic reticulum (ER) stressors. As the ER is a cellular structure responsible for properly folding newly manufactured proteins, ER stress can result in the release of improperly folded proteins. (In fact, ER stress has been identified as “a central feature of peripheral insulin resistance and type 2 diabetes at the molecular, cellular, and organismal levels.”8b) The osmolytes rescued the cells from the UPR (unfolded protein response), oxidative stress, and apoptosis (programmed cell death) that precede cataract development.9 The osmolytes were also used to treat rats that had been treated with galactose. The lenses of untreated galactosemic rats also undergo similar changes to those of the cells and develop cataracts. As a result of osmolyte treatment, the rats had significantly reduced lens epithelial cell death and partially delayed hypermature cataract formation.9 The osmolyte inositol has also shown protection against cataract-inducing processes, e.g., oxidative stress and glycation.10 In fact, in the human eye lens, the inositol concentration is 5.9 mM, whereas in the eye’s aqueous humor, the inositol concentration is 0.1 mM.


With osmolyte treatment, the rats had
significantly reduced lens epithelial
cell death and partially delayed
hypermature cataract formation.


A recent paper11 described a causative process in the formation of protein aggregates in cataract formation that suggests to us just the type of intracellular milieu that could be ameliorated by osmolytes. “Protein aggregation in the lens increases with age, leading to the accumulation of high molecular weight aggregates that scatter light. . . . Earlier, we showed that in vitro oxidized crystallin peptides enhance the aggregation of beta-crystallin and gamma-crystallin and also exhibit an anti-chaperone-like property. Peptide interactions with lens proteins and the aggregation-facilitating nature of lens peptides may be important in age-related cataract formation . . .”11 Of the results of the new study, they report, “Our data show that peptides generated in the lens interact with crystallins [lens chaperone molecules] and increase their aggregation and precipitation. The results also demonstrate that crystallin peptides generated in vivo exhibit anti-chaperone-like activity. . . . Our study suggests that interaction of crystallin-derived peptides with intact crystallin could be a key event in age-related protein aggregation in lens and cataractogenesis.”11

Why We Chose Particular Osmolytes

We selected osmolytes for inclusion in our cocktail that are naturally found in living tissue in substantial quantities, are readily available, have been used safely in relatively large amounts as supplements or as components of foods for long periods of time, and are reasonably inexpensive. Trehalose and trimethylamine N-oxide, for example, are very expensive.

References

1. Papp and Csermely. Chemical chaperones: mechanisms of action and potential use. Handb Exp Pharmacol 172:405-16 (2006) (Springer-Verlag 2006).
2. Burg and Ferraris. Intracellular organic osmolytes: function and regulation. J Biol Chem 283(12):7309-13 (2008).
2b. Yun et al. Proteasomal adaptation to environmental stress links resistance to proteotoxicity with longevity in Caenorhabditis elegans. Proc Natl Acad Sci USA 105(19):7094-9 (2008).
3. Somero. Protons, osmolytes, and fitness of internal milieu for protein function. Am J Physiol 251 (Regulatory Integrative Comp. Physiol. 20):R197-R213 (1986).
4. Ignatova and Gierasch. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci USA 103(36):13357-61 (2006).
5. Fisher. Proline to the rescue. Proc Natl Acad Sci USA 103(36):13265-6 (2006).
5a. Mehta and Seidler. Beta-alanine suppresses heat inactivation of lactate dehydrogenase. J Enzyme Inhib Med Chem 20(2):199-203 (2005).
5b. Tanaka et al. A novel therapeutic strategy for polyglutamine diseases by stabilizing aggregation-prone proteins with small molecules. J Mol Med 83:343-52 (2005), as cited in Protein Misfolding in Neurodegenerative Diseases, ed. by Smith, Simons, and Sewell; CRC Press, 2008. Surprisingly, the book contained nothing on the osmolytes as a class and only a small amount of data on trehalose and creatine, two osmolytes.
5c. Tanaka et al. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat Med 10(2):148-54 (2004).
5d. Yaffe et al. Androgen receptor CAG repeat polymorphism is associated with cognitive function in older men. Biol Psychiatry 54:943-6 (2003).
6. Bennion et al. Preventing misfolding of the prion protein by trimethylamine N-oxide. Biochemistry 43(41):12955-63 (2004).
7. Uversky et al. Trimethylamine N-oxide-induced folding of alpha-synuclein. FEBS Lett 509:31-5 (2001).
8. Bennion and Daggett. Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: a chemical chaperone at atomic resolution. Proc Natl Acad Sci USA 101(17):6433-8 (2004).
8b. Ozcan et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 306:457-61 (2004).
9. Mulhern et al. Cellular osmolytes reduce lens epithelial cell death and alleviate cataract formation in galactosemic rats. Mol Vis 13:1397-405 (2007).
10. Ramakrishnan et al. Two new functions of inositol in the eye lens: antioxidation and antiglycation and possible mechanisms. Indian J Biochem Biophys 36:129-33 (1999).
11. Santhoshkumar et al. Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation. J Biol Chem 283(13):8477-85 (2008).

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