Chemical chaperones are a class of
small molecule
Within the fields of molecular biology and pharmacology, a small molecule or micromolecule is a low molecular weight (≤ 1000 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Many drugs ar ...
s that function to enhance the folding and/or stability of proteins. Chemical chaperones are a broad and diverse group of molecules, and they can influence protein stability and polypeptide organization through a variety of mechanisms. Chemical chaperones are used for a range of applications, from production of recombinant proteins to treatment of protein misfolding ''in vivo''.
Classes of chemical chaperones
There are many different small molecules that can function to enhance protein stability and folding, many of them can be broadly grouped into large classes based both on their structure and their proposed mechanism of action. The parameters that define these groups are not strictly defined, and many small molecules that exert a chemical chaperoning effect do not readily fall into one of these categories. For example, the free amino acid
arginine
Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
is not classically defined as a chemical chaperone, but it has a well-documented anti-aggregation effect.
Osmolytes
Cellular
osmolyte
Osmolytes are low-molecular weight organic compounds that influence the properties of biological fluids. Their primary role is to maintain the integrity of cells by affecting the viscosity, melting point, and ionic strength of the aqueous solution. ...
s are polar small molecules that are synthesized or taken up by cells to maintain the integrity of cellular components during periods of osmotic or other forms of stress.
Osmolytes are diverse in chemical structure, and include polyols, sugars, methylamines, and free amino acids and their derivatives. Examples of these include
glycerol
Glycerol (), also called glycerine in British English and glycerin in American English, is a simple triol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known ...
,
trehalose
Trehalose (from Turkish '' tıgala'' – a sugar derived from insect cocoons + -ose) is a sugar consisting of two molecules of glucose. It is also known as mycose or tremalose. Some bacteria, fungi, plants and invertebrate animals synthesize it ...
,
trimethylamine n-oxide
Trimethylamine ''N''-oxide (TMAO) is an organic compound with the formula (CH3)3NO. It is in the class of amine oxides. Although the anhydrous compound is known, trimethylamine ''N''-oxide is usually encountered as the dihydrate. Both the anhydro ...
(TMAO), and
glycine
Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid (carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinogeni ...
.
Despite being most active at relatively high concentrations, osmolytes don’t display any effects on normal cellular processes – for this reason, they are also commonly referred to as “compatible solutes”.
Osmolytes exert their chaperoning effects indirectly by changing the interaction of the protein with solvent, rather than through any direct interaction with the protein. Unfavorable interactions between proteins and osmolytes increases the solvation of the protein with water. This increased hydration favors more compact polypeptide conformations, in which hydrophobic residues are more tightly sequestered from polar solvent. Thus, osmolytes are thought to work by structuring partially folded intermediates and thermodynamically stabilizing folded conformations to a greater extent than unfolded conformations.
Hydrophobic compounds
Chemical compounds that have varying degrees of hydrophobicity that still are soluble in aqueous environments can act as chemical chaperones as well. These compounds are thought to act by binding to solvent-exposed hydrophobic segments of unfolded or improperly folded proteins, thereby “protecting” them from aggregation.
4-phenylbutyrate
Sodium phenylbutyrate, sold under the brand name Buphenyl among others, is a salt of an aromatic fatty acid, 4-phenylbutyrate (4-PBA) or 4-phenylbutyric acid. The compound is used to treat urea cycle disorders, because its metabolites offer an ...
(PBA) is a prominent example of this group of compounds, along with
lysophosphatidic acid
Lysophosphatidic acid (LPA) is a phospholipid derivative that can act as a signaling molecule.
Function
LPA acts as a potent mitogen due to its activation of three high-affinity G-protein-coupled receptors called LPAR1, LPAR2, and LPAR3 (also kn ...
s and other lipids and detergents.
Pharmacological chaperones
Another class of chaperones is composed of protein ligands, cofactors, competitive inhibitors, and other small molecules that bind specifically to certain proteins. Because these molecules are active only on a specific protein, they are referred to as pharmacological chaperones. These molecules can induce stability in a specific region of a protein through favorable binding interactions, which reduce the inherent conformational flexibility of the polypeptide chain.
Another important property of pharmacological chaperones is that they are able to bind to the unfolded or improperly folded protein, and then dissociate once the protein is properly folded, leaving a functional protein.
Applications
Recombinant protein expression
Beside clinical applications, chemical chaperones have proved useful in ''in vitro'' production of recombinant proteins.
Re-folding of insoluble proteins from inclusion bodies
Recombinant expression of protein in ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' often results in the formation of insoluble protein aggregates called
inclusion bodies
Inclusion bodies are aggregates of specific types of protein found in neurons, a number of tissue cells including red blood cells, bacteria, viruses, and plants. Inclusion bodies of aggregations of multiple proteins are also found in muscle cells ...
. These protein bodies require refolding ''in vitro'' once extracted from ''E.coli'' cells by strong detergent. Proteins are thought to unfold during the solubilization process, and subsequent removal of detergent by dilution of analysis allows their refolding.
Both folding enhancers and aggregation suppressors are often employed during the removal of denaturant to facilitate folding to the native structure and to prevent aggregation. Folding enhancers assist protein to assume the native structure as soon as possible when the concentration of detergent is drastically decreased at once as in the dilution process. On the other hand, aggregation suppressors prevent protein folding intermediates from aggregating even after a long exposure to intermediate level of detergent as seen in dialysis. For example, it has been reported that
Taurine
Taurine (), or 2-aminoethanesulfonic acid, is an organic compound that is widely distributed in animal tissues. It is a major constituent of bile and can be found in the large intestine, and accounts for up to 0.1% of total human body weight. It ...
significantly increases the yield of in vitro refolding for Fab fragment
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
.
Periplasmic expression
The discovery of chemical chaperones’ effect on protein folding led to periplasmic protein expression, especially for ones that require an oxidative environment to form disulfide bonds for proper folding. Folding of proteins that are difficult to do in the cytoplasm can be enhanced in the periplasm where the osmotic pressure can be readily controlled. The osmotic pressure of the periplasmic space can be simply altered by changing that of the medium as osmolytes freely penetrate the outer membrane. Proteins is secreted to this space when an appropriate signal sequence is attached to its terminal. A good example of folding enhancement by periplasmic expression is the disulfide bond-containing plasminogen activator variant (rPA). Folding of rPA is shown to increase when folding enhancers or arginine is added to the culture medium.
Use of halophiles in protein production
Halophile
The halophiles, named after the Greek word for "salt-loving", are extremophiles that thrive in high salt concentrations. While most halophiles are classified into the domain Archaea, there are also bacterial halophiles and some eukaryotic species, ...
s are a type of extremophiles that have adapted to extreme salt solutions. Halophiles are classified into two categories: 1) extremely halophilic
archaea
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
, and 2) moderately halophilic
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
. The extremely halophilic archaea have adapted to require high salt concentrations (2.5M) in the living environment by incorporating the high salt concentration into the cell. On the other hand, the moderately halophilic bacteria achieve living in a wide range of salt concentrations by synthesis or incorporation of organic compounds. Many halophilic bacteria and archaea are easy to maintain, and their high cellular osmotic pressure has been exploited in recombinant protein production. The cellular environments of halophiles can be fine-tuned to accommodate folding of protein of interest by adjusting the concentration of osmolytes in the culture medium. Successful expression and folding of Ice nucleation protein, GFP, α-amylase, nucleotide diphosphate kinase, and serine racemase have been reported in halophiles.
Protein folding diseases
Since chemical chaperones promote the conservation of the native structure of proteins, the possibilities of developing chemical chaperones for clinical applications have been explored for various protein folding diseases.
Cystic fibrosis
Cystic fibrosis
Cystic fibrosis (CF) is a rare genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. Long-term issues include difficulty breathing and coughing up mucus as a result of frequent lung infections. O ...
(CF) is a disease resulting from a failure to maintain the level of cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a chloride channel in pulmonary tissues. ΔF508 point mutation in CFTR protein interferes with maturation of the protein has been found in a number of CF patients. It is found that the mutant CFTR mostly fails to transport to the cell membrane and is degraded in the ER; however, ones that successfully make it to the cell membrane are fully functional. As a result, a number of chemical chaperones have been shown to promote the trafficking of ΔF508 CFTR to the plasma membrane.
Transthyretin Amyloidoses
Partially denatured
transthyretin
Transthyretin (TTR or TBPA) is a transport protein in the plasma and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol to the liver. This is how transthyretin gained its name: ''transports thyroxine and retinol'' ...
(TTR) can promote the formation of
amyloid
Amyloids are aggregates of proteins characterised by a Fibril, fibrillar morphology of 7–13 Nanometer, nm in diameter, a beta sheet (β-sheet) Secondary structure of proteins, secondary structure (known as cross-β) and ability to be Staining, ...
fibrils in cells, and this aggregation can lead to cellular toxicity and a variety of human disease pathologies. Many small molecule inhibitors of TTR amyloid formation have been discovered that act by kinetically stabilizing the TTR tetramer. This prevents monomer misfolding events by disfavoring the dissociation of the TTR tetramer.
Tafamidis
Tafamidis, sold under the brand names Vyndaqel and Vyndamax, is a medication used to delay disease progression in adults with certain forms of transthyretin amyloidosis. It can be used to treat both hereditary forms, familial amyloid cardiomyopa ...
is one such small molecule that has been approved by several international regulatory agencies for the treatment of Transthyretin Familial Amyloid Polyneuropathy.
See also
*
Chaperone (protein)
In molecular biology, molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are a number of classes of molecular chaperones, all of which function to assi ...
, proteins that perform the same function
References
{{reflist
Protein biosynthesis
Protein folding
Molecular chaperones