Biochimica et Biophysica Acta 1850 (2015) 1822–1831

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Review

Macromolecular crowding: Macromolecules friend or foe Shruti Mittal, Rimpy Kaur Chowhan, Laishram Rajendrakumar Singh ⁎ Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi - 110 007, India

a r t i c l e

i n f o

Article history: Received 13 March 2015 Received in revised form 27 April 2015 Accepted 4 May 2015 Available online 8 May 2015 Keywords: Protein folding Protein aggregation Enzyme activity Protein stability Excluded volume effect

a b s t r a c t Background: Cellular interior is known to be densely crowded due to the presence of soluble and insoluble macromolecules, which altogether occupy ~40% of the total cellular volume. This results in altered biological properties of macromolecules. Scope of Review: Macromolecular crowding is observed to have both positive and negative effects on protein folding, structure, stability and function. Significant data has been accumulated so far on both the aspects. However, most of the review articles so far have focused on the positive aspect of macromolecular crowding and not much attention has been paid on the deleterious aspect of crowding on macromolecules. In order to have a complete knowledge of the effect of macromolecular crowding on proteins and enzymes, it is important to look into both the aspects of crowding to determine its precise role under physiological conditions. To fill the gap in the understanding of the effect of macromolecular crowding on proteins and enzymes, this review article focuses on the deleterious influence of crowding on macromolecules. Major Conclusions: Macromolecular crowding is not always good but also has several deleterious effects on various macromolecular properties. Taken together, the properties of biological macromolecules in vivo appears to be finely regulated by the nature and level of the intracellular crowdedness in order to perform their biological functions appropriately. General Significance: The information provided here gives an understanding of the role played by the nature and level of cellular crowdedness in intensifying and/or alleviating the burden of various proteopathies. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The present understanding of various biological processes has been acquired through investigations largely made under dilute experimental conditions where the total macromolecular concentration never exceeds 10 g/l. However, biological macromolecules are known to evolve and function under crowded intracellular environments consisting of a plethora of both soluble and insoluble macromolecules like proteins, nucleic acids, ribosomes and carbohydrates with their sum concentration reaching around several hundred g/l. For example, the total concentration of protein and RNA inside the bacterium, Escherichia coli is in the range of 300–400 g/l [1]. Altogether, these macromolecules occupy a significant fraction (~40%) of the total cellular volume [2], making it virtually unavailable to the other macromolecules present. Such media are termed ‘crowded’ or ‘volume-occupied’ rather than ‘concentrated’, because no single species of macromolecule is necessarily present at a high concentration. In fact, the level of crowdedness varies among different cell types and cellular compartments. Human lens contains approximately 340 g/l protein [3]; the red blood cells contain about 350 g/l hemoglobin [4]; while the total protein content in the mitochondrial ⁎ Corresponding author. Tel.: +91 9811630757 (mobile). E-mail addresses: [email protected] (S. Mittal), [email protected] (R.K. Chowhan), [email protected] (L.R. Singh).

http://dx.doi.org/10.1016/j.bbagen.2015.05.002 0304-4165/© 2015 Elsevier B.V. All rights reserved.

matrix may reach up to 500 g/l [5]. Macromolecular crowding is observed not only in the cellular interior but also in the extracellular matrix of tissues. For example, blood plasma contains ~ 80 g/l protein, a concentration high enough to cause significant crowding effects [2]. The degree of volume occupancy by these macromolecules is expected to have major thermodynamic and kinetic consequences on the properties of macromolecules present in the cell [6–9]. The term ‘macromolecular crowding’ connotes the non-specific influence of steric repulsions on specific reactions, and processes that occur in highly volumeoccupied media [10]. It was Minton and Wilf [11] who brought the influence of crowding on macromolecules to the forefront in terms of theory and experiment and coined the term “macromolecular crowding” in 1981. The influence of macromolecular crowding on various properties of macromolecules has been examined in depth by adding high concentrations of inert synthetic or natural macromolecules, termed crowding agents or crowders, to the system in vitro to create an in vivo like scenario [2]. It is generally believed that macromolecular crowding (i) enhances protein stabilization against denaturation by heat, cold or denaturant. It has been argued that macromolecular crowding stabilizes globular proteins due to excluded volume effect because the native state occupies less space than the denatured state [12–15]; (ii) alters the reaction rates depending on the nature of reactions (diffusion-limited or transition-state-limited). Since, macromolecular crowding decreases

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the diffusion of macromolecules, the rate of diffusion-limited reactions is decreased with the increasing crowder concentration [16,17]. However, the rate of transition-state-limited reactions is increased as crowding is expected to enhance the relative abundance of the transition state complex [18]; (iii) increases the catalytic activity of enzymes, either due to alteration in the conformation of the enzyme to a higher activity state [19–22] or due to an increase in the effective concentration of the enzyme resulting from decrease in the amount of the available free water [20]; (iv) increases protein–protein association leading to oligomer formation depending on the conformation of monomer [18,23–26]; and (v) inhibits aggregation of β-rich proteins [27,28]. All these effects of crowding on macromolecules have been described as “positive effect” in this article. In contrast to the facts described above, there exists large volume of data that oppose the findings (Fig. 1). All these opposing effects are

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described as “hostile or negative effect” in the entire manuscript. For instance, macromolecular crowding has been demonstrated to have a destabilizing influence on the stability of Myoglobin [29]. Recent investigations made by Fan et al. suggested a decrease in the activity of recombinant human brain-type creatine kinase under crowded conditions [30]. Furthermore, in a systematic study by Dobson and coworkers, crowding was found to disrupt the refolding of reduced lysozyme and caused aggregation [31,32]. All these evidences suggest that the stabilizing or positive effect of crowding on macromolecules is not universally true. Therefore, in addition to the understanding of the positive effects of crowding on macromolecular properties, it is important to have a knowledge of the hostile effects of macromolecular crowding as well so as to have a complete picture of the effect macromolecular crowding has on macromolecular properties. This review article is therefore, designed to give a collective knowledge on almost all the

Fig. 1. Consequences of macromolecular crowding on proteins. Illustration of both the positive and negative effects of macromolecular crowding on proteins.

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progresses made so far towards the understanding of hostile influence of crowding on macromolecular properties. We have also provided new future insights that might be useful for the scientists working in the avenue. 2. Deleterious effects of crowding on macromolecules Besides the positive effects of crowding on macromolecular properties, several hostile effects of crowding on macromolecular properties and biological processes have been showcased by several researchers worldwide (Fig. 1). The hostile effects of macromolecular crowding on the properties of macromolecules that might lead to altered biological processes are outlined below: 2.1. Macromolecular crowding destabilizes macromolecules and increases conformational fluctuation Macromolecular crowding, in general, has been predicted and well documented by several experimentalists to increase the thermodynamic stability of macromolecules [33,34]. Increase in the macromolecular structure and stability under crowded conditions is mainly accounted by the excluded volume effect which predicts that any reaction that increases the available volume (or decreases the excluded volume) is stimulated under crowded conditions [7,35]. Furthermore, the randomness of a solution decreases with an increase in the number of solute particles present. As a consequence, the entropy of the solute is less than it would be in an ideal solution in which the solute molecules occupied no space. The decrease in entropy results in an increase in the free energy of the solute [36] and also the chemical potential of each species of macromolecules present in that solution. For a heteropolymer like an amino acid chain, a total collapse is possible under crowded conditions. For such chains, the short range interactions (hydrogen bonds, hydrophobic interactions, etc.) bring a sufficient enthalpic gain to compensate for the entropic loss caused by the collapse, and hence significantly stabilize the native state of the protein [37]. Therefore, crowding will confer a stabilizing influence on the folded states of proteins, thereby indirectly destabilizing the more expanded unfolded state [38,39]. In contrast, the destabilizing influence of crowding in terms of structure and stability of certain macromolecules has also been reported through different approaches (see Table 1). Dextran 70 has been demonstrated to decrease the stability of rPrPC (properly folded prion protein) by 8 °C and rPrP80R by 12.5 °C [40]. Similar to Dextran 70, Ficoll 70 was also shown to enhance unfolding of myoglobin by urea [29]. Furthermore, in a recent development by our laboratory [41], holo α-lactalbumin was observed to be destabilized by several degrees (in terms of Tm, melting temperature) in the presence of polymer

crowders due to reduction in its calcium-binding affinity. Interestingly, a recent study by Pielak and co-workers using nuclear magnetic resonance (NMR) underlines the role of natural protein crowders (bovine serum albumin and lysozyme) in destabilizing chymotrypsin inhibitor 2 as a consequence of weak nonspecific protein–protein interactions [42]. In another study, molecular dynamics simulation and NMR experiments also suggested reduced native state stability of chymotrypsin inhibitor 2 in the presence of protein crowders due to increased protein–protein interactions [43]. Protein crowders (as opposed to polymer crowders) are suggested to result in a competition between stabilizing volume exclusion effects and destabilizing nonspecific interactions by the authors. Furthermore, the destabilizing influence of the reconstituted E. coli cytosol on chymotrypsin inhibitor 2 stability was observed to be stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions [44]. In another development [45], PEG 2000 was found to decrease the thermal stability of apo human α-lactalbumin thereby accelerating its degradation by trypsin. Isothermal titration calorimetry (ITC) measurements revealed a weak, nonspecific interaction between the apo form of the protein and PEG 2000 suggesting that the lack of crowding-induced stabilizing influence on protein stability is due to the amelioration of the stabilizing excluded volume effects of crowding agents by nonspecific interactions between protein and crowder. In accordance with the above in vitro observations, in vivo investigations also suggest a large conformational fluctuation of macromolecules under crowded conditions relative to that under dilute conditions. For instance, the stability (in terms of Tm) of the VlsE protein (without the N-terminal lipidation signal) was observed to be 3 °C lower in U2OS cells relative to that in aqueous buffer and 6 °C lower than in the presence of 150 g/l Ficoll [46]. Furthermore, Ubiquitin (Ub) and Ub-3A (mutant of Ub) in HeLa cells [47]; and carbonic anhydrase I in human RBCs [48] through exchange rates have been shown to have a more dynamic structure relative to dilute in vitro conditions. It has been argued that the nonspecific interactions of the test protein with other macromolecules might be responsible for the observed increase in the exchange rates. In another interesting observation, it was found that the stability of Cellular retinoic acid binding protein I (mutant of CRABP I) within E. coli cells was quite similar to that under dilute in vitro conditions. However, the unfolding transition was more co-operative under the intracellular crowded conditions [49,50]. Crowding-induced stabilization is mainly explained based on the excluded volume effect. However, the above observations on the destabilizing nature of crowders indicate the role of some destabilizing factor that overwhelms the stabilizing effect conferred by excluded volume effect resulting in a net destabilizing effect. This destabilizing factor has been suggested to be the nonspecific interaction of crowders with

Table 1 Macromolecular crowding destabilizes macromolecules. Protein

Crowding agents

Experimental conditions

Observations

References

Recombinant prion proteins (rPrPC and rPrP80R) Dextran 70 Holo α-lactalbumin Ficoll 70; Dextran 70 Equine skeletal muscle Myoglobin Ficoll 70

pH 4.0–6.0 pH 4.5 Presence of urea; pH

[40] [41] [29]

Chymotrypsin inhibitor 2

7.0 pH 7.0

Change in protein conformation Reduction in calcium-binding affinity Change in protein conformation due to nonspecific interactions Weak nonspecific protein–protein interactions Increased protein–protein interactions

[42,44,54]

Weak nonspecific interactions between the test protein and crowder Excluded volume effect Nonspecific interactions between the test protein and crowder Nonspecific interactions between the test protein and crowder Molecular chaperone activity changes the thermodynamic equilibrium of the protein

[45]

The headpiece domain of chicken villin and segment B1 of streptococcal protein G Apo human α-lactalbumin

BSA; lysozyme; reconstituted E. coli cytosol; anionic protein lysate from E. coli The headpiece domain of chicken villin and segment B1 of streptococcal protein G PEG 2000

Simulation

VlsE (without the N-terminal lipidation signal) Ubiquitin (Ub) and Ub-3A (mutant of Ub)

In vivo (U2OS cells) In vivo (HeLa cells)

pH 7.6 pH 7.4

Human carbonic anhydrase I (hCAI)

In vivo (RBCs)

tetra-Cys CRABP I (mutant of CRABP I)

Cytoplasm of E. coli cells

pH 7.0

Presence of urea

[43]

[46] [47] [48] [49,50]

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the test molecules by several investigators [51–53]. Therefore, not only the excluded volume effect, but also the nonspecific interactions between the test and background molecules must also be taken into account while interpreting the effect of macromolecular crowding on the stability of macromolecules. Therefore, it appears that the effect of crowding on the stability of proteins depends on both the properties of the test protein and background molecules and the solvent conditions used thereby, highlighting the relevance of studying various factors responsible for the altered macromolecular properties within living cells. At molecular level, all the above reports on the destabilizing influence of macromolecular crowding on protein stability indicate the involvement of attractive chemical interactions that counteract the effect of hard-core repulsions (steric repulsions), thereby decreasing the thermodynamic stability. To test this idea, Pielak and co-workers [54] employed anionic proteins from E. coli as crowding agents to assess the stability of the anionic test protein chymotrypsin inhibitor 2. It was speculated that the repulsive interactions between the similarly charged crowders and test protein should strengthen the effect of hard-core repulsions, resulting in an increase in the thermodynamic stability of the test protein. Surprisingly, anionic protein crowders destabilized chymotrypsin inhibitor 2. The authors, therefore, conclude that proteins possess an inherently favorable and ubiquitous interaction with other proteins. Although weak, these interactions can overcome the stabilizing effect of hard-core repulsions associated with macromolecular crowding. On thermodynamic grounds, Zhou [55] recently proposed that all the macromolecular crowders (polymers or proteins) act similarly, with an entropic component favoring the folded state and an enthalpic component favoring the unfolded state. The net effect of the two is suggested to be destabilizing below a crossover temperature but stabilizing above it. Destabilizing influence of crowding on macromolecules might play an important role under cellular conditions. Several processes including reversible binding of a signaling molecule involve protein–protein recognition and their interaction, which are mediated by local unfolding of the interacting partners. Thus, destabilization of such molecules to capture the other interacting partner is biologically important for several signaling cascades. Because, it would be hard for the interacting molecules to bind to a stable compact conformation. Therefore, destabilization of macromolecules might play an important role in processes like reversible binding of a signaling molecule to its target. Furthermore, cells might alter the extent of crowding by change in cell volume as a strategy to regulate the stability of macromolecules contained within [56] and hence regulation of a signaling process. The heterogeneity of the crowded physiological environment is thus suggested to create regions where cellular macromolecules are stabilized or destabilized, depending on the local degree of volume exclusion and the local extent of nonspecific interactions [42].

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2.2. Macromolecular crowding reduces enzyme activity From the investigations made so far, high solute concentrations resulting from macromolecular crowding is known to have at least three different components: an excluded volume effect [18]; an increased viscosity [57]; and a hydration effect [58–60] (see Fig. 2). All these components have been demonstrated to affect the activity of enzymes. It has been shown that macromolecular crowding results in an enhanced enzymatic activity due to a change in the conformation of the enzyme brought about by crowding as a consequence of excluded volume effect. Change in the conformation of enzymes to a high activity state involves either the formation of a compact and enzymatically more active structure [19], or the self-association of monomeric enzymes to an oligomer with high enzymatic activity [20,22]. In addition to conformational change, macromolecular crowding also increases enzyme activity if the association of enzyme and substrate leads to a more compact enzyme-substrate complex or if there is a decrease in volume of the transition state during catalysis. In contrast to the increase in enzyme activity under crowded conditions, a list of enzymes have also been demonstrated to display reduced catalytic efficiency in the presence of macromolecular crowding which are given in Table 2. We have discussed the role played by macromolecular crowding in the reduction of enzymatic activity in terms of the above mentioned individual forces acting on the enzymes. 2.2.1. Excluded volume Excluded volume theory predicts that crowding favors the compaction of an unfolded polypeptide, but it does not necessarily predict that an unfolded polypeptide will be induced to fold exclusively to its functional native state, as the compaction force will make little or no distinction between the unique functional native state and other compact nonnative states. Interestingly, macromolecular crowding due to increase in the excluded volume effect was observed to significantly decrease the refolding yield of recombinant Human brain-type creatine kinase (rHBCK), by favoring aggregation over correct folding [30]. In another development, Monterroso and Minton [61] demonstrated that macromolecular crowding induces the formation of a compact near-native (non-functional) bovine carbonic anhydrase b. A recent work by Sugimoto and group [62] suggested that DNA hydrolysis by exonuclease I was strongly inhibited by PEG 8000 by decreasing Vmax. In another study, change in the solvent polarity of the active site under crowded conditions was found to be responsible for the decrease in the trypsin enzymatic activity [63]. In addition, high concentrations of polysaccharide crowders (Ficoll 70 and Dextran 70) were observed to have an inhibitory effect on the enzymatic activity of S. cerevisiae multi-copper oxidase, Fet3p by decreasing Km due to an increase in the effective concentration of the enzyme and the substrate [64]. Similar to the effect of

Macromolecular crowding

Excluded volume effect

Increased viscosity

Reduction in the diffusional mobility of reactants

Hydration effect

Competition between protein folding and aggregation

Protein aggregation

Thinning of hydration layer

Non-native conformations

Activity decreases Fig. 2. Different components of macromolecular crowding leading to decrease in the catalytic activity of enzymes. Macromolecular crowding decreases enzyme activity by reducing diffusional mobility of reactants; thinning of hydration layer; favoring protein aggregation and formation of non-native conformations.

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Table 2 Macromolecular crowding inhibits enzyme activity. Protein

Crowding agents

Experimental conditions

Observations

References

Alkaline phosphatase

25 °C

Reduction in the rate of enzyme-substrate encounter

[67]

Urease Pyruvate decarboxylase Glutamate decarboxylase EcoRV mitochondrial creatine kinase α-chymotrypsin rHBCK GAPDH

Ficoll 70 and 400; Dextran 15, 40, 70, 200 and 500 Dextran 70 and 120; PEG; Glycerol Dextran 70 Dextran 70 Ficoll 70 Dextran 20, 40, 70 and 500 Dextran 5, 50, 150, 275 and 410 Dextran 70; Ficoll 70; PEG 2000 PEG 20000; Dextran 70; BSA

pH 7.0 pH 6.5 pH 5.0 pH 7.5 pH 7.4 25 °C; pH 8.0 25 °C; pH 9.0 pH 7.5

[20] [20] [20] [68] [69] [70] [71] [72]

rHBCK

PEG 2000

Bovine carbonic anhydrase b

Ficoll 70

Refolded from 3.0 M GdmCl; pH 8.0 Refolded from 5.0 M GdmCl

Exonuclease I S. cerevisiae multi copper oxidase, Fet3p Trypsin Hexokinase B. burgdorferi VlsE

PEG 8000 Ficoll 70; Dextran 70

37 °C; pH 9.5 pH 5.0

PEG 8000; PEG 1500 BSA Ficoll 70

25 °C; pH 7.7 25 °C; pH 7.0 25 °C; pH 7.0

α-chymotrypsin

PEG 400

–

Telomerase

PEG 200 and 8000

25 °C; pH 7.0

Reduction in the rate of enzyme-substrate encounter Reduction in the rate of enzyme-substrate encounter Reduction in the rate of enzyme-substrate encounter Increased diffusion resistance Increased diffusion resistance Increased diffusion resistance Reduced diffusional mobility of the molecules Decreased association of GAPDH dimers into enzymatically active tetramers due to reduced diffusion Protein aggregation favored over the correct folding due to excluded volume effect Formation of compact non-functional near-native conformation over the functional native conformation Decrease in Vmax of the reaction Restricted internal dynamics due to volume exclusion making the protein less structurally strained Change in solvent polarity of the enzyme active site Enzyme catalysis involves rehydration Native state of the protein deformed as a consequence of volume exclusion Loss of critical water residues from the hydration shell of the enzyme Uptake of water molecules for the formation of G-quadruplex/ligand complex disfavored under crowded conditions

polysaccharide crowders, the enzyme activity of hexokinase has also been reported to decrease with the increasing concentration of protein crowder, BSA [65] due to BSA-induced conformational change/ rehydration that accompanies catalysis and/or diffusion of product from the enzyme-product complex. Interestingly, by combining computational and experimental studies of B. burgdorferi VlsE, WittungStafshede and group [66] recently discovered that crowding can deform the native state of a distinctly aspherical globular protein, because a non-native state takes up less space than its native state. 2.2.2. Viscosity Macromolecular crowding is known to increase the viscosity of the solvent which is one of the important parameters affecting enzyme catalysis. As a result of increased viscosity, crowding is expected to decrease the rate of a diffusion-limited reaction, where the overall rate of the reaction is limited by the encounter of the reactants. For instance, the enzymatic activity of alkaline phosphatase [67], urease [20], pyruvate decarboxylase [20] and glutamate decarboxylase [20] has been reported to decrease as a function of polymeric crowding agent concentration, due to reduction in the rate of enzyme-substrate encounter. Furthermore, an increase in Km of EcoRV [68], mitochondrial creatine kinase [69] and α-chymotrypsin [70] in the presence of increasing polymeric crowder concentration has been suggested to result from an increased diffusion resistance in the sample. In contrast to these observations, a recent study by Wang and co-workers [71] demonstrated that Dextran 70 (up to 100 g/l) had no significant effect on the enzymatic activity of recombinant Human Brain Creatine Kinase (rHBCK), thereby excluding the possibility of any effect on the enzymesubstrate encounter and any specific interaction of the crowder molecules with the enzyme. However, at higher Dextran 70 concentration (200 g/l), ~32% reduction in activity was observed, which was indeed attributed to increased viscosity. In another systematic study, refolding of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated under crowded conditions. Upon dilution, the denatured GAPDH monomer is known to fold and associate into dimers which then undergo a conformational adjustment before further associating into tetramers (a prerequisite for reactivation). Crowding was observed to decrease the reactivation rate of GAPDH, due to decrease in the

[30] [61]

[62] [64] [63] [65] [66] [77] [78]

diffusional mobility of dimers thereby affecting their association into enzymatically active tetramers [72]. 2.2.3. Hydration effect Cells are known to contain two phases of water: water of hydration and bulk water. Water of hydration is strongly adsorbed to all the macromolecules present in the cell, and the remaining water represents the bulk water. Thus, the volume of water of hydration depends on the total concentration of macromolecules present, i.e., with increasing solute concentration, the volume of water of hydration surrounding any macromolecule decreases. In particular, it is the water of hydration rather than the bulk water that is required for enzyme activity [73,74]. For instance, Clegg and Mansell reported that the rate of glycolysis and respiration of mouse L cells remains unaffected when the cell volume is reduced to 40% by sorbitol dehydration [75]. Therefore, the actual activity of a given enzyme in the cytoplasm is a function of the concentration of all the macromolecules present. Majority of the studies aimed at investigating the effect of macromolecular crowding on enzyme activity focused more on excluded volume and viscosity than on hydration. However, recently using explicit solvent molecular dynamics simulation, Feig and his group examined the structure and dynamics of the hydration shell of protein G and protein G/villin system under crowded conditions [76]. The results suggest that the structure of water within the hydration shell is modestly affected at low levels of crowding (b30% volume), but is significantly altered beyond the first hydration shell at high levels of crowding (N30% volume). Furthermore, analysis of self-diffusion rates and dielectric constants revealed a linear reduction in hydration dynamics with increasing crowder concentration. In yet another development, the role of hydration on the functionality of α-chymotrypsin was established in the presence of a polymer crowder, PEG. The enzyme activity was observed to be reduced in the presence of PEG due to decrease in kcat, which is attributed to the loss of critical water residues from the hydration shell of the enzyme [77]. Furthermore, Sugimoto and group investigated the potential of 2 anionic (phthalocyanine and Hemin) and 2 cationic (TMPyP4 and PIPER) G-quadruplex-ligands in binding to the telomere G-quadruplex and inhibiting telomerase activity under crowded conditions (using PEG) [78]. It was found that the

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binding affinity of the anionic ligands to G-quadruplex DNA remained unaffected while the binding affinity of the cationic ligands reduced under crowded conditions relative to that observed under dilute conditions. The difference in the binding behavior of the ligands to Gquadruplex DNA under crowded conditions is explained on the basis of water molecules that are released and taken up, respectively for the formation of G-quadruplex/ligand complex. As a consequence of differential binding affinities, the anionic ligands were found effective in inhibiting telomerase activity. In contrast, the telomerase-inhibiting capacity of cationic ligands was found to be significantly reduced under crowded conditions. Despite these elegant studies, more studies are required to establish the relationship between water of hydration of particular enzymes and thus their activity with increasing crowder concentration. 2.3. Crowding alters protein folding leading to aggregation: a cause to the progression of many neurodegenerative and other genetic diseases The primary sequence of a globular protein is important in making the unfolded proteins en-route to its native, functional threedimensional structure via intramolecular interactions [79]. However, proteins (if not properly folded) often have a tendency to interact intermolecularly in the course of protein unfolding, refolding, or de novo folding, leading to the formation of aggregates/amyloids. Presently, the formation of aggregates is of intense medical interest since the

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deposition of aggregates in vivo is related to the pathogenesis of many human genetic diseases including neurodegenerative diseases, metabolic disorders, cardiovascular disorders, etc. [80–85]. Aggregation is a problem frequently encountered during the folding of polypeptide chains both in vitro and in vivo. Considerable effort has so far been made to achieve a fundamental understanding of the basic cause and factors affecting protein folding leading to disease development via aggregate formation [86]. However, most solvent environments used traditionally to study protein folding leading to aggregate formation were dilute as compared to the highly crowded intracellular environment wherein proteins perform their biological functions [87,88]. Thus, consideration of the protein folding behavior under conditions mimicking the intracellular milieu has become a necessity due to the demands of advances in medical therapy. In this spirit, the effects of macromolecular crowding on the folding of several proteins have been extensively studied during the past 15 years. One primary discovery is that it enhances aggregation propensity of several proteins (see Table 3) during many kinetic studies of protein folding that are initiated by rapidly transferring a polypeptide from a fully denaturing medium to a buffer favoring renaturation. However, factors responsible for the aggregation appear to depend on the type and/or physico-chemical properties of the protein under study. For instance, polymer crowders have been recently shown by our laboratory [41] to induce aggregation of holo α-lactalbumin under acidic conditions by reducing its calcium-binding affinity. In addition, aggregation during refolding of rHBCK [30],

Table 3 Macromolecular crowding enhances protein aggregation and amyloidosis. Protein

Crowding agents

Experimental conditions

Holo α-lactalbumin

Ficoll 70; Dextran 70

pH 4.5

rHBCK

PEG 2000; Dextran 70; Calf Thymus DNA Dextran 20

Azotobacter vinelandii flavodoxin Reduced, denatured lysozyme GAPDH

Dextran 70; Ficoll 70; Ovalbumin; BSA PEG 20000; Dextran 70; BSA

GroEL

Ficoll 70; Dextran 70; BSA

Rabbit muscle creatine kinase

PEG 2000; Ficoll 70; Dextran 70; Calf Thymus DNA Dextran 70; Ficoll 70; BSA Cytoplasm of E. coli cells

Reduced, denatured lysozyme P39A tetra-Cys CRABP I

Human PrP (wild type; E196K; Ficoll 70; Ficoll 400 D178N) E. coli preMaltose binding protein Ficoll 70 Human apolipoprotein C-II Dextran 10 Apo α-lactalbumin

Dextran 68

Human Tau fragment, tau-(244–441) Bovine core histones

Ficoll 70; Dextran 70

Human Superoxide Dismutase 1 mutant A4V β-lactoglobulin

Dextran 70; PEG 20000

PEG 3500

β-synuclein

Dextran 70; PEG 400, 8000 and 20000 PEG 10000

Insulin

Ficoll 70; PEG 3500

α-Synuclein α-Synuclein

Ficoll 70; PEG 3500 Ficoll 70 and 400; Dextran 138000; PEG 200, 400, 600, 3350 and 10000; Lysozyme; BSA Ficoll 70; PEG 3500; BSA PEG 2000

S-carboxymethyl α-lactalbumin FG Nucleoporins

Observations

Protein destabilization due to reduced calcium-binding affinity Refolded from 3.0 M GdmCl; pH 8.0 Intermediates aggregate before having enough time to fold to a state resistant to aggregation 2.0 M GdmCl Intermediates aggregate before having enough time to fold to a state resistant to aggregation Refolded from 8.0 M urea; pH 8.5 Intermediates aggregate before having enough time to fold to a state resistant to aggregation Refolding from 4.0 M GdmCl Intermediates aggregate before having enough time to fold to a state resistant to aggregation Refolded from 5.0 M urea; pH 7.5 Increased tendency of non-assembly competent monomeric GroEL to undergo self-association Refolded from 3.0 M GdmCl; pH 7.5 Increase in volume exclusion and possibly weak and nonspecific crowder–protein interactions pH 8.5 Increase in volume exclusion 37 °C Accumulation of intermediates with an extended non-native helix pH 7.5 Production of more fragmented fibrils increases the apparent rate constant for fibrillation refolded from 2.0 M GdmCl; pH 7.2 Intermediates due to slow-folding of the protein 20 °C; pH 7.4 Destabilization of the monomeric form of the protein relative to the aggregated form 20 mM DTT; 37 °C; pH 7.4 Destabilization of the protein tertiary structure leading to the intermolecular association between protein molecules Hyperphosphorylated by GSK-3β; Acceleration of the nucleation step of pH 7.5 phosphorylated human Tau misfolding 37 °C; pH 2.0 Stabilization of partially folded protein conformation leads to accelerated fibrillation pH 7.5 Reduction in protein stability enhances aggregation rate more than folding 2.0 M GdmCl; pH 7.0 Decrease in the nucleation time and accelerated protein amyloid formation Zn2+ Stabilization of the amyloidogenic intermediate by Zn2+ 20% acetic acid; pH 2.0 Stabilization of the partially folded protein conformation 1.0 M TMAO Accumulation of partially folded intermediates pH 7.5 Metal ions minimize the coulombic charge–charge repulsion between the charged protein molecules 37 °C Crowding modulated partial folding of protein pH 7.4 Space restriction or sequestering of water

References [41] [30] [89] [31] [72] [90] [91] [92] [49] [94,95] [97] [98] [99]

[94,95] [100] [95] [28] [101] [100] [100] [93,102–104]

[100] [105]

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Azotobacter vinelandii flavodoxin [89], reduced, denatured lysozyme [31] and GAPDH [72] was found to be due to the accumulation of aggregation-prone intermediates that aggregate before having enough time to fold to a state resistant to aggregation. In another interesting study, both polysaccharide and protein crowders were demonstrated to favor aggregation during GroEL refolding [90]. It is suggested that crowding increases the tendency of monomeric GroEL, either assembly-competent or not, to undergo self-association at equilibrium, and therefore promotes its productive oligomerization or unproductive aggregation [90]. Not only single crowding agents, but mixed crowded conditions have also been observed to enhance aggregation of rabbit muscle creatine kinase [91] and reduced, denatured lysozyme [92]. However, aggregation in the presence of mixed crowding agents was found to be less serious than that in the presence of a single protein crowder (BSA) which is suggested to be due to more effective excluded volume of BSA compared to the polysaccharide crowding agents. Crowding within the cytoplasm of E. coli cells has also been demonstrated to favor the formation of insoluble aggregates of P39A tetra-Cys CRABP I (a mutant of tetra-Cys CRABP I) in the course of refolding after incubation at 37 °C in contrast to the soluble tetra-Cys CRABP I [49]. Enhanced aggregation is argued to be a result of the slow-folding of P39A and stabilization of an intermediate with an extended non-native helix as a consequence of amino acid substitution (proline to alanine) [49]. Taken together, increase in aggregation under crowded conditions stems from the following reasons (Fig. 3): (a) favored accumulation and association of aggregation-prone intermediates; (b) crowding slows down the refolding rate of proteins as a consequence of increased viscosity; (c) volume excluded to aggregates is less than that of the

Crowding promotes partial unfolding

(1)

Native protein

Accumulation of aggregationprone intermediates

Aggregate

Accumulation of aggregationprone intermediates

Aggregate

Crowding slows folding

(2)

Denatured protein

(3)

polypeptide chains; (d) reduction in the activity of water as a consequence of increased cosolute concentration resulting in decreased protein solubility and thus, increased aggregation [93]. In addition to enhanced aggregation propensity of proteins under macromolecularly crowded conditions, crowding has been reported to accelerate amyloid fibril formation. A mild concentration of Ficoll (upto 200 g/l) was found to dramatically accelerate the fibrillation of human prion protein, PrP and its mutants (E196K, D178N, E196K/ E219K, Q217R/E219K and D178N/M129V) by its effect on the nucleation phase and increasing the apparent rate constant for the growth of fibrils [94–96]. In addition, crowding has been implicated in prion replication due to the production of more fragmented fibrils upon incubation. These fragmented fibrils are generally considered more infectious than larger species [94,95]. Furthermore, E. coli preMaltose binding protein was observed to form amyloids during refolding in the presence of Ficoll 70 in contrast to the formation of amorphous aggregates under dilute conditions [97]. Similar to Ficoll, another polymer crowding agent, Dextran has also been reported to accelerate the rate of fibril formation of human apolipoprotein C-II [98] and reduced apo α-LA [99]. Intriguingly, Ficoll and Dextran have also been evidenced to induce fibrillation of phosphorylated human Tau (244–441) which was otherwise lost under crowded conditions by accelerating the nucleation step of its misfolding [94,95]. In addition to Ficoll and Dextran, fibrillation of bovine core histones [100] and β-synuclein (in the presence of Zn2+) [101] has been shown to be accelerated upon addition of PEG. Accelerated fibrillation of insulin [100], α-synuclein [100], human Superoxide Dismutase1 mutant A4V [95] and β-lactoglobulin [28] has also been demonstrated upon addition of polymer crowders. Not only polymer

Monomer Aggregate Crowding favors

Excluded volume

Available volume

More excluded volume

Less excluded volume

Fig. 3. Crowding favors protein aggregation. Crowding induces protein aggregation by at least three ways: (1) by promoting partial unfolding of native protein molecules leading to the accumulation of aggregation-prone intermediates; (2) by reducing the rate of protein folding due to increased diffusion resistance; and (3) by favoring formation of protein aggregates that exclude less volume than that of the monomeric polypeptide chains.

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crowders, but protein crowders have also been shown to be efficient in accelerating the fibrillation of several proteins. For example, both polymer and protein crowders accelerate fibrillation of α-synuclein [93, 102–104] and S-carboxymethyl-α-lactalbumin [100]. In addition, amyloid formation by FG Nucleoporins was demonstrated to be dramatically accelerated under crowding conditions, thereby providing an insight into how the intrinsically disordered FG Nups form amyloid fibrils that can grow into networks to adapt the macroscopic phenotype of a hydrogel [105]. All these evidences suggest that the increased incidence of amyloid disease in the aged persons may be a direct consequence of increased total intracellular protein concentration (or, equivalently, a decrease in cell-water content) in the cells of aging tissues. Interestingly, the water content in brain and liver cells are significantly decreased with advancing age [106–108] ultimately increasing the level of crowdedness in cells. We therefore, propose that with advancing age the pathogenesis of neurodegenerative disorders increases as a result of increase in the effective concentration of amyloidogenic proteins. In addition, it has earlier been reported that the intracellular levels of calcium and other metal ions help to accelerate the aggregation of Aβ protein [109,110]. Under intracellular crowded conditions, the effective concentration of metal ions also increases. Taken together, it appears that crowding may significantly affect the aggregation behavior of the amyloidogenic proteins. Therefore, we propose that in crowded physiological environments, different macromolecules have different effects on the aggregation propensity of nearby proteins. 3. Are cells stressed due to macromolecular crowding? Besides the positive effects of crowding on macromolecules, all the above discussed hostile effects of macromolecular crowding on the biological processes suggest that the high concentration of macromolecules in vivo might lead to many damaging consequences, which might be a major challenge for the normal function of the cell. Indeed, the extent of such hostile effects might be much more in vivo than expected. During translation on polysomes, several unfolded polypeptides with exposed hydrophobic residues might be at a high risk of interacting with each other leading to aggregation [111]. In addition, the densely populated intracellular environment could intensify protein aggregation to a much greater extent due to an increase in the effective concentration of macromolecules. It, therefore, appears that there must be existence of some strategy in vivo to cope up with such deleterious consequences of macromolecular crowding on proteins. One such strategy is the existence of chaperones. It might be possible that cells are all the time stressed due to highly crowded nature of the intracellular environment leading to the constitutive expression of housekeeping chaperones and basal expression of inducible chaperones. In support of this speculation, it has been known that different levels of intracellular crowdedness are responsible for various cellular signaling strategies [112,113]. Therefore, different cell types and/or variation in the magnitude of cellular crowdedness due to certain factors might lead to the expression of specific chaperones or chaperone complexes. Indeed, intracellular environment is known to have at least three classes of chaperones: (i) molecular chaperones that have the ability to catalyze refolding of misfolded proteins (e.g., Hsp60, Hsp70, Hsp90) [114–117]; (ii) small heat shock chaperones that sequester the unstable or unfolded proteins so as to prevent their aggregation thereby maintaining them in a folding competent state (e.g., Hsp33, Hsp26, Hsp27) [118–120]; and (iii) chemical chaperones that help to stabilize unstable proteins (e.g., glycine, sarcosine, trimethylamine N-oxide) [121,122]. In addition, there is existence of many chaperones to offer housekeeping assistance against the misfolded/unfolded macromolecules in the crowded intracellular environment. Large volume of data are available in the literature on how these different chaperones assist in protein folding in vitro under dilute conditions. However, there is existence of very few data on how specific chaperones help to retard such macromolecular

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crowding-induced harmful situations on macromolecules [99,123]. Therefore, in the future, it is important to investigate the influence of macromolecular crowding on the efficiency of chaperones. More work on this aspect is required to understand how macromolecular crowding and chaperones work in association to get a complete picture of how biological processes operate in the living cells. It has been known that macromolecular crowding induces/ enhances the pathogenicity of many human diseases caused due to proteopathic conditions (such as Alzheimer's, Parkinson's, transmissible spongiform encephalopathy). Investigating specific chaperone systems or refoldasome complexes that work against each of the deleterious consequences induced due to macromolecular crowding will yield several insights to fix diseases caused by protein malfunctioning or proteopathies. Nevertheless, therapeutic agents aimed at tackling proteins with misfolded/aggregated conformations in vitro, a major pathological condition in a number of genetic diseases should be developed under molecularly crowded conditions. This will resolve inconsistencies faced during clinical trials of therapeutic agents developed under dilute in vitro conditions. 4. Summary and perspectives It is well known that solvent conditions and level of crowding can easily alter various biological processes. Depending on concentrations and solvent conditions, crowding can have various deleterious consequences on macromolecules. Thus, our long standing belief that macromolecular crowding stabilizes macromolecules, enhances enzyme catalytic efficiency, etc. appears not to be universally true. In fact, the properties of biological macromolecules seem sensitive to the degree of crowdedness of the medium wherein they are dissolved. Thus, the properties of all the macromolecules in vivo appear to be finely regulated under different physiological conditions in order to perform their biological functions appropriately. This suggests that cells might alter the extent of crowding by changes in cell volume as a strategy to regulate the properties of different macromolecules contained within [56]. The heterogeneity of the crowded physiological environment is thus suggested to create regions where cellular macromolecules are stabilized or destabilized, depending on the local degree of volume exclusion and the local extent of non-specific interactions [42]. Transparency Document The Transparency document associated with this article can be found, in online version. Acknowledgment This work is supported by a grant from the Department of Science and Technology [ref no.: SR/SO/BB-0003/2011]. SM, RKC and LRS acknowledge the Council of Scientific and Industrial Research for the financial assistance provided in the form of research fellowship [file no.: 09/045(1047)/2011-EMR-1 and 09/045(1245)/2012-EMR-1]. References [1] S.B. Zimmerman, S.O. Trach, Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli, J. Mol. Biol. 222 (1991) 599–620. [2] R.J. Ellis, Macromolecular crowding: obvious but underappreciated, Trends Biochem. Sci. 26 (2001) 597–604. [3] J.J. Harding, Cataract: Biochemistry, Epidemiology and Pharmacology, Chapman and Hall, London, 1991. 1–333. [4] R.J. Ellis, Macromolecular crowding: an important but neglected aspect of the intracellular environment, Curr. Opin. Struct. Biol. 11 (2001) 114–119. [5] P. Srere, The infrastructure of the mitochondrial matrix, Trends Biochem. Sci. 5 (1980) 120–122. [6] T.C. Laurent, The interaction between polysaccharides and other macromolecules. 5. The solubility of proteins in the presence of dextran, Biochem. J. 89 (1963) 253–257.

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