Dendrimers destabilize proteins in a generation-dependent manner involving electrostatic interactions

Dendrimers are well-defined chemical polymers with a characteristic branching pattern that gives rise to attractive features such as antibacterial and antitumor activities as well as drug delivery properties. In addition, dendrimers can solubilize prion protein aggregates at very low concentrations, but their mode of action is unclear. We show that poly(propylene imine) dendrimers based on di-aminobutane (DAB) and modified with guanidinium surface groups reduce insulin thermostability and solubility considerably at microgram per microliter concentrations, while urea-modified groups have hardly any effect. Destabilization is markedly generation-dependent and is most pronounced for generation 3, which is also the most efficient at precipitating insulin. This suggests that proteins can interact with both dendrimer surface and interior. The pH-dependence reveals that interactions are mainly mediated by electrostatics, confirmed by studies on four other proteins. Ability to precipitate and destabilize are positively correlated, in contrast to conventional small-molecule denaturants and stabilizers, indicating that surface immobilization of denaturing groups profoundly affects its interactions with proteins.     

Post-aggregation Oxidation of Mutant Huntingtin Controls the Interactions between Aggregates

Aggregation of protein molecules is a pathological hallmark of many neurodegenerative diseases. Abnormal modifications have often been observed in the aggregated proteins, supporting the aggregation mechanism regulated by post-translational modifications on proteins. Modifications are in general assumed to occur in soluble proteins before aggregation, but actually it remains quite obscure when proteins are modified in the course of the aggregation. Here we focus upon aggregation of huntingtin (HTT), which causes a neurodegenerative disorder, Huntington disease, and we show that oxidation of a methionine residue in HTT occurs in vitro and also in vivo. Copper ions as well as added hydrogen peroxide are found to oxidize the methionine residue, but notably, this oxidative modification occurs only in the aggregated HTT but not in the soluble state. Furthermore, the methionine oxidation creates additional interactions among HTT aggregates and alters overall morphologies of the aggregates. We thus reveal that protein aggregates can be a target of oxidative modifications and propose that such a “post-aggregation” modification is a relevant factor to regulate properties of protein aggregates.     

Influence of dendrimers on red blood cells

Dendrimers, highly branched macromolecules with a specific size and shape, provide many exciting opportunities for biomedical applications. However, most dendrimers demonstrate toxic and haemolytic activity because of their positively charged surface. Masking the peripheral cationic groups by coating them with biocompatible molecules is a method to reduce it. It was proven that modified dendrimers can even diminish haemolytic activity of encapsulated drugs. Experiments confirmed that anionic dendrimers are less haemotoxic than cationic ones. Due to the high affinity of dendrimers for serum proteins, presence of these components in an incubation buffer might also influence red blood cell (RBC)-dendrimer interactions and decrease the haemolysis level. Generally, haemotoxicity of dendrimers is concentration-, generation-, and time-dependent. Various changes in the RBCs’ shape in response to interactions with dendrimers have been observed, from echinocytic transformations through cell aggregation to cluster formation, depending on the dendrimer’s type and concentration. Understanding the physical and chemical origins of dendrimers’ influences on RBCs might advance scientists’ ability to construct dendrimers more suitable for medical applications.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Selective molecular recognition in amyloid growth and transmission and cross-species barriers

Mutual conformational selection and population shift followed by minor induced-fit optimization is the key mechanism in biomolecular recognition, and monomers and small oligomers binding to amyloid seeds in fibril growth is a molecular recognition event. Here, we describe amyloid aggregation, preferred species, cross-species barriers and transmission within the broad framework of molecular recognition. Cross-seeding of amyloid species is governed by conformational selection of compatible (complementary) states. If the dominant conformations of two species are similar, they can cross-seed each other; on the other hand, if they are sufficiently different, they will grow into different fibrils, reflecting species barriers. Such a scenario has recently been observed for the tau protein, which has four repeats. While a construct consisting of repeats 1, 3 and 4 can serve as a seed for the entire four-repeat tau segment, the inverse does not hold. On the other hand, the tau protein repeats with the characteristic U-turn shape can cross-seed Alzheimer's amyloid β and, similarly, the islet amyloid polypeptide. Within this framework, we suggest that the so-called "central dogma" of amyloid formation, where aggregation takes place through nonspecific backbone hydrogen bonding interactions, which are common to all peptides and proteins, is a simple reflection of the heterogeneous, polymorphic free-energy landscape of amyloid species. Here, we review available data and make some propositions addressing this key problem. In particular, we argue that recent theoretical and experimental observations support the key role of selective molecular recognition in amyloidosis and in determining cross-species barriers and transmission.     

Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects

Arginine has been used to suppress protein aggregation and protein-protein or protein-surface interactions during protein refolding and purification. While its biotechnology applications are gradually expanding, the mechanism of these effects of arginine has not been fully elucidated. Arginine is more effective at higher concentrations, an indication of weak interactions with the proteins. The effects of weakly interacting additives, such as arginine, on protein solubility, stability and aggregation have been explained from three different approaches: i.e., (1) the effects of additives on the structure of water, (2) the interactions of additives with the amino acid side chains and peptide bonds and (3) the preferential interactions of additives with the proteins. Here we have examined these properties of arginine and compared with those of other additives, e.g., guanidine hydrochloride (GdnHCl) and certain amino acids and amines. GdnHCl is a strong salting-in agent and denatures proteins, while betaine is a protein stabilizer. Several amino acids and amine compounds, including betaine, which stabilize the proteins, are strongly excluded; i.e., the proteins are preferentially hydrated in these solutions. On the other hand, GdnHCl preferentially binds to the proteins. Arginine is intermediate between these two extreme cases and shows a more complicated pattern of interactions with the proteins. The effects of additives on water structure, e.g., the surface tension of aqueous solution of the additives and the solubility of amino acids in the presence of additives also shed light on the mechanism of the effects of the additives on protein aggregation. While arginine increases the surface tension of water, it favorably interacts with most amino acid side chains and the peptide bonds, a property shared with GdnHCl. Thus, we propose that while arginine is similar to GdnHCl in the amino acid level, arginine interacts with the proteins differently from GdnHCl.     

The crowd you're in with: Effects of different types of crowding agents on protein aggregation

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein–polymer interactions influence the structure and dynamics of proteins in crowded environments.     

Inhibiting protein aggregation with nanomaterials: The underlying mechanisms and impact factors

Protein aggregation is correlated with the onset and progression of protein misfolding diseases (PMDs). Inhibiting the generation of toxic aggregates of misfolded proteins has been proposed as a therapeutic approach for PMDs. Due to their unique properties, nanomaterials have been extensively investigated for their ability to inhibit protein aggregation and have shown great potential in the diagnosis and treatment of PMDs. However, the precise mechanisms by which nanomaterials interact with amyloidogenic proteins and the factors influencing these interactions remain poorly understood. Consequently, developing a rational design strategy for nanomaterials that target specific proteins in PMDs has been challenging. In this review, we elucidate the effects of nanomaterials on protein aggregation and describe the mechanisms through which nanomaterials interfere with protein aggregation. The major factors impacting protein-nanomaterial interaction such as size, charge, concentration, surface modification and morphology that can be rationally addressed to achieve the desired effects of nanomaterials on protein aggregation are summarized. The prospects and challenges to the clinical application of nanomaterials for the treatment of PMDs are also discussed.     

Dendrimer-protein interactions studied by tryptophan room temperature phosphorescence

Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer generation and surface charge

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.     

Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis.     

Polyamidoamine dendrimers with different surface charge as carriers in anticancer drug delivery

Second-generation (G2) polyamidoamine (PAMAM) dendrimers are branched polymers containing 16 surface primary amine groups. Due to their structural properties, these polymers can be used as universal carriers in various drug delivery systems. Amine-terminated PAMAM dendrimers are characterized by a high positive surface charge, leading to effective but nonspecific interactions with negatively charged cell plasmatic membranes. To reduce the nonspecific internalization of PAMAM dendrimers, their primary amine groups are often modified by acetic or succinic anhydrides, polyethylene glycol derivatives and other compounds. In this work, the role of primary amine groups, which are localized on the surface of doxorubicin-conjugated (Dox) dendrimers, was studied with regard to their intracellular distribution and internalization rates using SKOV3 human ovarian adenocarcinoma cells. It was demonstrated that all Dox-labeled G2-derivatives containing different numbers of acetamide groups synthesized in this work show high rates of cellular uptake at 37°С. As expected, the conjugate carrying the maximum number of primary amine groups demonstrated the highest rates of binding and endocytosis. At the same time, the G2-Dox conjugate containing the maximum number of acetamide groups showed colocalization with LAMP2, a marker of lysosomes and late endosomes, as well as the highest level of cytotoxic activity against SKOV3 cells. We conclude that second-generation PAMAM dendrimers are characterized by varied pathways of internalization and intracellular distribution due to the number of primary amine groups on their surface and, as a consequence, a different surface charge.     

Amyloid fibril formation by native and modified bovine β-lactoglobulins proceeds through unfolded form of proteins: a comparative study

The misfolding and extracellular amyloid deposition of specific proteins are associated with a large family of human pathologies, often called protein conformational diseases. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Recently, β-lactoglobulin (β-lg) was driven toward amyloid aggregation under specific extreme conditions. In the present study, citraconylation was employed to neutralize the charges on accessible lysine residues of β-lg and different approaches such as turbidimetry, thermodynamic analysis, extrinsic fluorimetry and theoretical studies have been successfully used to compare the different behaviors of the native and modified proteins. Kinetic analyses of native β-lg aggregation showed a gradual development of turbidity, whereas the modified β-lg displayed an increased propensity toward aggregation. Our results clearly demonstrated that the stability of modified β-lg is markedly reduced, compared to the native one. Using of TANGO and WALTZ algorithms (as well as modelling softwares) which describe aggregation tendencies of different parts of a protein structure, we suggested critical importance of some of the lysine residues in the aggregation process. The results highlighted the critical role of protein stability and elucidated the underlying role of hydrophobic/electrostatic interactions in lactoglobulin-based experimental system.     

Solvation thermodynamics of biopolymers. I. Separation of the volume and surface interactions with estimates for proteins

The present paper is a systematic first approach to the problem of solvation thermodynamics of biomolecules. Most previous approaches have been only crude estimates of solvent contributions, and have simply assessed solvation free energy as proportional to surface areas. Here we estimate the various contributions and divide them into (a) hard-core interactions dependent upon the entire volume of solute and (b) the remainder of interactions manifested through surfaces, such as van der Waals, charge-charge, or hydrogen bonds. We have estimated the work to create a cavity with scaled-particle theory (SPT), the van der Waals interactions on the surface, and hydrogen bonds between the surface and the solvent. The conclusion here is that this latter term is the largest component of the solvation free energy of proteins. From estimates on nine diverse proteins, it is clear that the larger the protein, the more dominant is the hydrogen-bond term. In the next paper, we indicate that correlations between hydrogen-bonding groups on the surfaces could increase the magnitude of the hydrogen-bond contribution.     

Review: Why is arginine effective in suppressing aggregation?

Arginine is finding a wide range of applications in production of proteins. Arginine has been used for many years to assist protein refolding. This effect was ascribed to aggregation suppression by arginine of folding intermediates during protein refolding. Recently, we have observed that arginine facilitates elution of antibodies during Protein-A chromatography and solubilizes insoluble proteins from inclusion bodies, which both can be ascribed to weakening of protein-protein interactions. In order to gain understanding on why arginine is effective in reducing protein-protein interactions and suppressing aggregation, the effects of arginine on stability and solubility of pure proteins have been examined, which showed that arginine is not a protein-stabilizer, but is an aggregation suppressor. However, there is no explanation proposed so far on why arginine suppresses aggregation of proteins. This review addresses such question and then attempts to show differences between arginine and strong denaturants, which are also known as an aggregation suppressor.     

Photophysical properties of Newkome-type dendrimers in aqueous medium.

Newkome-type first, second and third generation dendrimers, having t-butyl (GB), ethyl (GE) and carboxylic (GA) end groups, were synthesized. A pyrene group, which can act as fluorescent sensor, was attached to the core of the dendrimers and their photophysical properties in aqueous solution were studied. These dendrimers were found to aggregate in aqueous solution, which manifested as an excimer peak in the pyrene emission spectra for the first and second generation dendrimers with ethyl and t-butyl end groups. The excimer peak however was not seen in case of the third generation dendrimer. Dendrimers with carboxylic end groups, did not show the excimer peak in water, which implies the hydrophobic nature of the aggregation. It is observed that the intensity of the excimer peak decreases with the increase in the size of the dendrimer. Lifetime studies carried out on the first and second generation dendrimers showed the formation of excimer species as a risetime in the decay curve. The aggregation of the third generation dendrimer was proposed from the quenching studies using silver ions and CCl(4) as quenchers.     

Oxidative stress-enhanced SUMOylation and aggregation of ataxin-1: Implication of JNK pathway

Although the polyglutamine protein ataxin-1 is modified by SUMO at multiple sites, the functions of such modification or how it is regulated are still unknown. Here we report that SUMO-1 or Ubc9 over-expression stimulated the aggregation of ataxin-1 and that oxidative stress, such as hydrogen peroxide treatment, further enhanced SUMO conjugation and aggregation of ataxin-1. Accordingly, co-treatment with antioxidant N-acetyl-cysteine attenuated the effect of oxidative stress. Ataxin-1, which can activate c-Jun N-terminal kinase (JNK) pathway by itself, strongly associated with apoptosis signal-regulating kinase 1 (ASK1) while not interacting with JNK. Finally, treatment of JNK-specific inhibitor caused a reduction in the oxidant-enhanced SUMOylation and aggregation of ataxin-1. Together these results indicate that SUMO modification of ataxin-1 promotes the aggregation of ataxin-1 and that oxidative stress and JNK pathway play roles in this process.     

Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration

The barrier functions of the stratum corneum and the epidermal layers present a tremendous challenge in achieving effective transdermal delivery of drug molecules. Although a few reports have shown that poly(amidoamine) (PAMAM) dendrimers are effective skin-penetration enhancers, little is known regarding the fundamental mechanisms behind the dendrimer-skin interactions. In this Article, we have performed a systematic study to better elucidate how dendrimers interact with skin layers depending on their size and surface groups. Franz diffusion cells and confocal microscopy were employed to observe dendrimer interactions with full-thickness porcine skin samples. We have found that smaller PAMAM dendrimers (generation 2 (G2)) penetrate the skin layers more efficiently than the larger ones (G4). We have also found that G2 PAMAM dendrimers that are surface-modified by either acetylation or carboxylation exhibit increased skin permeation and likely diffuse through an extracellular pathway. In contrast, amine-terminated dendrimers show enhanced cell internalization and skin retention but reduced skin permeation. In addition, conjugation of oleic acid to G2 dendrimers increases their 1-octanol/PBS partition coefficient, resulting in increased skin absorption and retention. Here we report that size, surface charge, and hydrophobicity directly dictate the permeation route and efficiency of dendrimer translocation across the skin layers, providing a design guideline for engineering PAMAM dendrimers as a potential transdermal delivery vector.