Environmental Factors can Modify Genotype Risks by Slight Changes in Protein Conformation: The Role of Water

Shahabi et al, Graphical Abstract

Abstract
In biological systems, water is not simply arranged, but is highly structured. Interactions between structured water and proteins have an important role in the preservation of protein conformation, which is vital for their proper function. Any agent that can exert a direct effect on an organism might also change the structure of water molecules, resulting in slight changes in protein conformation. In disease states, these changes could efficiently target the proteins that are products of mutated genes, but display normal, or approximately normal, functions in a normal environment. Physiological compensatory agents such as stress proteins can correct abnormal protein conformation. However, if slightly altered conformations of proteins (due to altered water structure) persist, they may not be able to be restructured by stress proteins, resulting in abnormal protein function. The purpose of this paper is to provide a hypothesis for a mechanism through which protein structure and function is modulated by altering the surrounding water microenvironment.


Introduction
A multifactorial disease is a disorder caused by the interaction of genetic and non-genetic, environmental factors. Therefore, the development of a multifactorial disease requires both a specific genotype factor and a suitable environment. The routes by which the environment affects the onset or course of a disease vary depending on environmental factors (1).

Proteins are often a favoured target of these factors. At the molecular scale, proteins carry out the essential physiological processes, and are thus fascinating molecular devices. They play a variety of roles in life processes; there are structural proteins, catalytic proteins, transport and storage proteins, regulatory proteins and proteins of the immune system and the immunoglobulin superfamily (2). Proteins have a three-dimensional structure and their function is completely dependent on this structure (2,3). The most important target of stressors is the conformation of proteins, while the primary aim of the stress response of biological systems is to inhibit or to reverse this conformational change (2,3). However, the question remains: how do these environmental factors affect proteins? The effects may be exerted in a direct manner. For example, the concentration of an important ion in the synthesis or function of a protein will naturally affect the functions of that protein (4). Yet, their direct effects cannot explain all consequences of environmental stressors. The purpose of this paper is to provide a hypothesis for a mechanism through which environment can affect protein function.

Water is the most abundant molecule in living cells, and scientific evidence indicates that this simple molecule has an important role in more than merely inter-molecular interactions. Water is the only solvent in biological systems, and all biological activities are performed in water (5). Therefore, water may be a very good medium through which the effects of environmental pressures are transferred to proteins in a biological system.

Hypothesis
Upon acknowledging the important role of water and proteins in biological systems, we ask: Can environmental factors influence the behaviour of biological systems by targeting proteins through the alteration of the structure and nature of water?

Research has indicated that if certain physical and chemical conditions of water, as a protein solvent, such as the pH, changes far from normal, the conformation of proteins will change and their function will be lost (2,3). However, such abrupt changes in water conditions rarely occur in a biological system. If they do happen, they do not persist because they are inconsistent with the vital functions of the biological system. Here, we intend to consider environmental factors that do not interfere with vital functions. Therefore, if the water in a biological system transfers the effects of environmental factors to proteins, it must affect proteins in a way that would lead to functional disorder, not loss.

It has been shown that water in biological systems is not simple, but is distinctly structured. According to our current understanding, water is a mixture of randomly hydrogen-bonded molecules and larger structures comprised of tetrahedral oxygen centres which, when hydrogen-bonded to each other, lead to five-member and other rings that can aggregate to form three-dimensional structures or clusters (6-9). Yet, many ionic and non-ionic solutions have been divided into two groups according to their effects on structured water: structure makers, which stabilize structured water, and structure breakers, which destabilize it (6,10). Solute molecules that possess hydrogen-bonding groups will provoke the formation of further hydrogen-bonding chains of water molecules and change the structure of solvent water, giving the solute a secondary identity of associated water which may play a role in molecular recognition. Solutes that do not have hydrogen-bonding capability or regions of solutes that are non-polar may also produce partial cage-like water structures that are characteristic of the solute (6). Interactions between structured water and proteins play an important role in the preservation of protein conformation (6). Conformation of proteins can be in turn changed in the presence of different solutions since solute molecules can alter the structure of water by forming or destabilizing hydrogen-bonded constructs. As a result, the structure of water is changed. Since structured water affects protein conformation, this can also result in a new protein conformation.

Most proteins are found in a distinct three-dimensional structure in vivo and in vitro, and this native structure is necessary for integral function (2,3). Some solutes effect structured water so severely that the proteins lose their tertiary and/or secondary structures, resulting in complete function loss (6). Other solutes, however, may have very few effects on structured water and subsequent protein conformation, with only slight effects on protein function. In this paper, we will discuss the effects of these protein conformation changes.

It is presumed that slight changes in protein conformation do not affect the function of all proteins to an equal extent. The function of proteins with mutations resulting in slightly altered conformations may be more susceptible to further slight conformational changes due to altered water structure. In normal structured water, this minute conformational abnormality may still allow normal protein function, or may be compensated by redundant physiological agents such as stress proteins, which could enable these proteins to return to their native conformation. When structured water changes slightly, the conformation of these genetically susceptible proteins might change to a conformation with an abnormal function, while changes in the conformation of normal proteins would still be tolerated.

According to our conjectures, it can be deduced that protein conformation is representative of the structured water of that biological system. The structure of water can be altered by any factor that can affect hydrogen bonds (6), including increased body temperature or a structure breaker and/or changes in equilibrium of ionic or non-ionic milieu. In every internal environment imbalance, there is an altered protein conformation that reflects that imbalance. Thus, any environmental factor that alters the internal environmental balance of a biological system can confer a new conformation to the proteins of the biological system. Accordingly, two individuals with different genetic backgrounds, when exposed to the same environment, may develop two different disorders (Figure 1). For example, one person may develop major depression, another person may develop rheumatoid arthritis and others may develop no disorder, according to their genetic predisposition, when exposed to a chronic stressful condition.

Figure 1: The Effect of Environment on Protein Structure In Person 1, gene A produces protein A, which is conformationally slightly abnormal, butfunctions normally in a normal environment. In Person 2, gene B produces protein B, which isconformationally slightly abnormal, although it functions normally in a normal environment.When these two individuals are exposed to the same environmental factors, they will havesimilar changes in water structure. However, the protein A in Person 1, and protein B inPerson 2 will have abnormal conformations. Ultimately in Person 1, the functions attributed toprotein A will be affected, whereas in Person 2, the effects of protein B will be affected. As aresult, these individuals can develop different disorders in the same environment.

As discussed previously, if alterations of protein conformation due to changes in structured water are slight, protein function may not be lost, but the function of genetically susceptible proteins might be diminished. A specific change in protein conformation may affect the function of some proteins more than others. During an internal environmental imbalance, the functions of certain genetically susceptible proteins can be more affected than others. In another type of imbalance, the affected proteins may also vary. This is in agreement with the diverse effects of varying environmental conditions on different diseases (11). For example, geographic locations may lead to dissimilar risks for two different diseases (12). Some reasons have been suggested for this difference (1,11), although it may also be due to the effect of a specific geographic region on specific internal environments. The water structure due to this internal environment may induce a protein conformation that is slightly different from native. This conformational change may not affect the function of all genetically susceptible proteins evenly.

Proteins such as enzymes, structural proteins, motor proteins, receptors and antibodies may be functionally altered by conformational changes (2). These proteins have important roles in maintaining homeostasis. Here, we will attempt to explain the changes in protein function as a result of slight changes in the conformation of receptors, antibodies and tumour suppressor proteins. However, it is obvious that slight changes in protein conformation can also affect the function of other proteins as well.

Slight conformational changes in a receptor and/or its ligand may be not so severe as to inhibit binding but may change the binding affinity. The affinity between a receptor and its ligand has an important role in the subsequent events after binding; reducing affinity may change the quality of the intracellular signalling (3). In addition to the role of structured water in determining the conformation of a receptor and its ligand, water molecules surrounding the receptors and their ligands may have an important role in the primary ligand recognition process. A ligand does not need to come into direct contact with the receptor to affect the recognition process (6). An agonist is recognized by the receptor and provokes a conformational change in the macromolecule. The change in conformation, and possibly ligand recognition, are likely mediated by the water that surrounds both partners (6). Thus, any changes in an internal environment can affect the recognition process and its subsequent events by changing structured water. This is consistent with the finding that the affinity of a receptor to its specific ligand may differ in various tissues (13).

Similarly, altering the structure of water causes slight conformational changes in antibody and/or an antigenic proteins, the binding affinity may be affected. Studies have shown that changes in pH, temperature and other conditions pertaining to hydrogen bonds in water, the primary solvent of antibodies, may result in changes of the affinity of an antibody to its antigen (14). The affinity of the antigen-antibody binding site has an important role in the subsequent immune reaction and direction of the immune response. For example, tolerance to some self-antigens may be affected due to changes in the affinity of autoantibodies to these antigens (15-18). In addition, the affinity of T cell receptors (TCRs) for MHC-peptide complexes may be altered due to the slight conformational changes in the TCR and/or MHC. Alteration of the affinity between TCR and MHC-peptide complexes may result in shifts in cytokine patterns as well as certain immune-mediated disorders (19).

The function of tumour suppressor proteins also depends on their conformation. For example, zinc and cadmium increase and decrease respectively, the function of p53 by affecting its conformation (20). It can accordingly be hypothesized that any slight conformational change induced by alterations in water structure may affect the function of a genetically susceptible tumour suppressor protein, and consequently tumorigenesis.

Stress Response
In a broad sense, the word “stress” often refers to some entity that when applied to another, upsets or disturbs equilibrium. These alterations or changes in stasis usually impair or alter normal function and if left unchecked, may eventually have lethal consequences. From a physiological point of view, a stressor may disrupt homeostasis. This disruption may be at the cellular, tissue, organ, organ system, or organism level. The extent of the homeostatic disruption depends on both the intensity and duration of the stress (21). Regardless of the source of the stress, the physiological response is similar for each stress response. After organisms have been exposed to a stressor, they respond by synthesizing stress proteins. If an organism can synthesize sufficient quantities of stress proteins to restore homeostasis, stress resistance may be conferred. However, if the stress is too large and/or long that the quantity of synthesized stress proteins is not sufficient to cope with cellular perturbations, homeostasis may not be restored (21).

Protein denaturation is one of the stressors that may induce the stress response in living organisms (22). Stress proteins, named heat shock proteins (HSPs), are chaperones, and one of their functions is to correct the distorted conformation of proteins (22). Current research has shown that conformational changes associated with stress response activators are severe enough to lead proteins toward denaturation (21, 22) but it is possible that slight conformational changes of genetically susceptible proteins due to changes in water structure could also induce the production of stress proteins given sufficient time. If these slightly altered conformations of genetically susceptible proteins persist, stress proteins may become unable to correct this distortion, and the abnormal functions of altered conformation proteins will appear.

Discussion
Environmental factors that directly disrupt the equilibrium of the internal environment of a living organism may also indirectly exert their impact by altering structured water, thereby inducing changes in protein conformation. We will attempt to explain the above hypothesis through the use of an example.

There is indisputable evidence that particularly in the high-risk populations such as the elderly, the diabetic and patients with renal disease, long term salt intake impacts blood pressure and may lead to cardiovascular and renal end-organ damage (23). The adverse effects of salt cannot be fully explained by increases in osmotic pressure and other mechanisms that have been suggested in literature (23). According to our discussion, long-term salt consumption could alter protein conformation by altering water structure. Therefore, osmotic pressure-independent side effects of long-term salt consumption may be due to changes in the conformation of genetically susceptible proteins that may decrease their function. Furthermore, some of the effects contributing to increases in osmotic pressure may be due to effects of increased osmotic pressure on protein conformation.

According to this hypothesis, every agent that can directly alter the internal environment of the body, such as nutrients, air and water pollutants, microbes, social stresses, metabolic disorders and fluctuations in hormone levels, may also indirectly affect homeostasis by changing the structure of water and consequently protein conformation. For example, the effects of certain pathogens on the onset and progression of autoimmune diseases (24) can, at least partially, be attributed to the altered conformation of proteins. As a result, this matter may explain the effects of nutrients, air and water pollution, long-term social stresses, metabolic disorders and changes in hormone levels on increased incidences of morbidity and mortality of certain diseases.

As previously mentioned, following slight conformational alterations after changes in the internal environment, the function of proteins with a slightly non-ideal conformation may be more seriously affected than normal proteins. This concept is in agreement with other evidence that emphasizes that as potential genotype-risk modifiers, environmental factors may have major public health implications.

Multifactorial diseases are caused by both genetic and environmental factors. The inheritance of ‘defective’ genes will vary from individual to individual, and any single mutation is likely to contribute only a small risk. Context dependency, i.e. the importance of environmental factors in influencing genetic risk, is now becoming evident. Thus, a mutation may have a modest risk effect in individuals who maintain a low environmental risk, but a major effect in a high-risk environment (1,11). Altered water structure can be considered an important mechanism by which environmental factors can modify genotype risks. Consequently, according to one’s genetic susceptibility, a person may or may not develop a disorder as a result of stressors that created altered internal environments.

Again according to genetic susceptibility, two different individuals may develop two different disorders because of altered internal environments (Figure 1). This is in agreement with the observation that one type of environmental stress may have varying effects on different people. A stressful condition can affect the internal environment of the body and result in specific water structure, but two people who have different genetic susceptibilities will develop different disorders according to the proposed mechanisms.

Phenotypic differences in certain diseases due to variations in sex, age and geography emphasize that environmental exposures in combination with genetic susceptibility plays a role in the development of diseases (12,25-28). Among other suggested mechanisms, the conformational changes of proteins can be considered as a mechanism through which the occurrence, onset or course of certain diseases can be influenced by the above-mentioned factors. Each of these factors can provide an internal environment and water structure that may impose a particular conformation on a genetically susceptible protein that is suitable for development of a specific disease. This appropriate conformation may not be available in another sex, age or geographical category.

The increased occurrence of some diseases such as cancer, autoimmune diseases, neurodegenerative diseases and infections during the aging process (29,30) may be attributed to changes in protein conformation among many others reasons. Changes in the expression of stress proteins could be an important factor in organisms as they age because senescence is physiologically characterized by a decrease in an organism’s ability to respond to environmental stress/stimuli (31). Induced expression of stress proteins is dramatically reduced with age (31,32). Stress proteins have an important role in maintaining the naïve conformation of proteins; decreased expression of stress proteins results in the body’s reduced ability to prevent conformational changes in proteins (33). As a result, the function of genetically susceptible proteins will be abnormal, and the propensity to certain diseases may be enhanced.

This process may lead to diminished efficacy of immune responses against pathogens, reduced tolerance to self-antigens, diminished activity of tumour suppressor proteins, or disorders that may predispose the individual to neurodegenerative diseases such as Alzheimer’s disease (AD). AD is a neurodegenerative disorder characterized pathologically by senile plaques, neurofibrillary tangles and cerebral amyloid angiopathy (CAA). The major component of senile plaques and CAA is the amyloid-β protein (Aβ), a 4 kDa polypeptide (34) that is proteolytically cleaved from the amyloid-β precursor protein (35,36). Conformational changes of Aβ may lead to its aggregation into oligomers, protofibrils and mature fibrils (37). Aβ is particularly neurotoxic when it is in an aggregated state (38,39). It has been shown that HSPs inhibit Aβ aggregation and cerebrovascular Aβ toxicity (40,41). The diminished expression of HSPs associated with aging may therefore be one of causes of the increased incidence of AD in the aged. However, the conformational changes of Aβ in AD are severe, but it may be that HSPs play such a role in correcting slight conformational changes of genetically susceptible proteins. If slight conformational changes of genetically susceptible proteins remain unchecked, the function of these proteins will be diminished. Decreased expression of HSPs resulting in lower levels of stress compensation can result in downregulated functions of genetically susceptible proteins.

On the other hand, it has been shown that increased physical exercise can decrease the occurrence of certain diseases and increase stress tolerance (42,43) by increasing stress protein expression (21). Thus, exercise may prevent the slight conformational changes of genetically susceptible proteins. As a result, agents that could previously affect protein conformation directly or through changes to the internal environment could be less effective than before and homeostasis may be maintained more efficiently.

More studies and research in this field are clearly required to explore the unknown properties of water. This new perspective on water and protein conformation may transform many of the current viewpoints and theories related to biochemical processes. While it is necessary to pursue the theoretical aspects of this hypothesis, developing an appropriate model that would enable researchers to explore the diverse dimensions of this hypothesis is also essential. Empirical evidence on the changes in water structure and how it may affect protein conformation are new areas of study for future interdisciplinary research.

Experimental Model
This hypothesis can be tested with concrete studies examining the relationship between environmental factors, water structure in the organisms and slight conformational changes of proteins using techniques such as X-ray crystallography and Nuclear Magnetic Resonance.

We outline a way to study the effect of a structure-breaker and a structure-maker macromolecule on the affinity of an antibody to its antigen or the conformation of antigen-antibody complexes without direct contact between the macromolecules.

There are three containers. In container A, there is the structure-breaker macromolecule, antigen X and its antibody (antigen X and its antibody must be suitable for immunoprecipitation assays). In container B, there is the structure-maker macromolecule, antigen X and its antibody. In container C, there is only antigen X and its antibody. The macromolecules are separated from the antibody and antigen by a semi-permeable membrane that inhibits the passage of macromolecules, but is permeable to water. For this purpose, the pores of the semi-permeable membrane must be as small as possible. There is no direct contact between the macromolecules and antigen or antibody in containers A and B, but the water structure that is affected by the macromolecules may also affect the antigen and/or the antibody conformation and their affinities. The affinity between the antigen and its antibody can be measured by immunoprecipitation assays (44) and the conformation of the combining site of the antigen-antibody complexes can be studied by X-ray crystallography (45). Because there is no direct contact between macromolecules and the antigen and antibody in containers A and B, any differences between the conformation of the combining site of the antigen-antibody complexes in containers A and B and that of the antigen-antibody complexes in container C can be attributed to the changes in water structure by the structure-breaker or structure-maker macromolecules. This conclusion may be drawn if the affinity between antigen and antibody in containers A and B are different from that in container C.

Conclusion
In brief, environmental factors may affect water by affecting its structure. The structure of water as a protein solvent has specific traits that if changed, may slightly change the conformation of proteins. This change in protein conformation may decrease functional efficacy of genetically susceptible proteins. Therefore, the water structure of biological systems is the proper avenue through which environmental factors can affect protein conformation and subsequently, function. It is clear that other important mechanisms also have a role in transferring the effects of environmental factors to a biological system.

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