Formation Of Insoluble Aggregates Research Articles

Effect of Oxidative Damage on the Stability and Dimerization of Superoxide Dismutase 1

During their life cycle, proteins are subject to different modifications involving reactive oxygen species. Such oxidative damage to proteins may lead to the formation of insoluble aggregates and cytotoxicity and is associated with age-related disorders including neurodegenerative diseases, cancer, and diabetes. Superoxide dismutase 1 (SOD1), a key antioxidant enzyme in human cells, is particularly susceptible to such modifications. Moreover, this homodimeric metalloenzyme has been directly linked to both familial and sporadic amyotrophic lateral sclerosis (ALS), a devastating, late-onset motor neuronal disease, with more than 150 ALS-related mutations in the SOD1 gene. Importantly, oxidatively damaged SOD1 aggregates have been observed in both familial and sporadic forms of the disease. However, the molecular mechanisms as well as potential implications of oxidative stress in SOD1-induced cytotoxicity remain elusive. In this study, we examine the effects of oxidative modification on SOD1 monomer and homodimer stability, the key molecular properties related to SOD1 aggregation. We use molecular dynamics simulations in combination with thermodynamic integration to study microscopic-level site-specific effects of oxidative “mutations” at the dimer interface, including lysine, arginine, proline and threonine carbonylation, and cysteine oxidation. Our results show that oxidative damage of even single residues at the interface may drastically destabilize the SOD1 homodimer, with several modifications exhibiting a comparable effect to that of the most drastic ALS-causing mutations known. Additionally, we show that the SOD1 monomer stability decreases upon oxidative stress, which may lead to partial local unfolding and consequently to increased aggregation propensity. Importantly, these results suggest that oxidative stress may play a key role in development of ALS, with the mutations in the SOD1 gene being an additional factor.

Global kinetic analysis of seeded BSA aggregation

Accelerated aggregation studies were conducted around the melting temperature (Tm) to elucidate the kinetics of seeded BSA aggregation. Aggregation was tracked by SEC-HPLC and intrinsic fluorescence spectroscopy. Time evolution of monomer, dimer and soluble aggregate concentrations were globally analysed to reliably deduce mechanistic details pertinent to the process. Results showed that BSA aggregated irreversibly through both sequential monomer addition and aggregate–aggregate interactions. Sequential monomer addition proceeded only via non-native monomers, starting to occur only by 1–2°C below the Tm. Aggregate–aggregate interactions were the dominant mechanism below the Tm due to an initial presence of small aggregates that acted as seeds. Aggregate–aggregate interactions were significant also above the Tm, particularly at later stages of aggregation when sequential monomer addition seemed to cease, leading in some cases to insoluble aggregate formation. The adherence (or non-thereof) of the mechanisms to Arrhenius kinetics were discussed alongside possible implications of seeding for biopharmaceutical shelf-life and spectroscopic data interpretation, the latter of which was found to often be overlooked in BSA aggregation studies.

Using proteomics to characterise storage-induced aggregates in acidic whey protein isolate drinks

Formation of insoluble aggregates in acidic whey protein isolate (WPI) beverages during storage is a quality concern. Earlier studies showed that changes in processing methods from 95 °C for 180 s–120 °C for 20 s could prevent it. In the present study, WPI drinks were stored for up to 12 months and insoluble aggregates formed in re-solubilised WPI and its liquid phase analysed by proteomics. Insoluble aggregates were found to be enriched in caseinomacropeptides (CMP). Increased peptide formation by weak acid hydrolysis, especially of CMP, was found at 120 °C. Further enhanced hydrolysis of CMP was obtained by deamidation as well as by the presence of the genetic variant A of CMP. Batch variations may therefore be assigned to variations in CMP as well as variations in the distribution of genetic variants of A and B in different WPI batches used for the production of the WPI products.

Development of a Transnasal Delivery System for Recombinant Human Growth Hormone (rhGH): Effects of the Concentration and Molecular Weight of Poly-L-arginine on the Nasal Absorption of rhGH in Rats.

A novel system for delivering recombinant human growth hormone (rhGH) that is noninvasive and has a simple method of administration is strongly desired to improve the compliance of children. The aim of this study was to investigate the potential for the intranasal (i.n.) co-administration of rhGH with poly-L-arginine (PLA) as a novel delivery system by evaluating the effects of the concentration and molecular weight of PLA on the nasal absorption of rhGH. The influence of the formation of insoluble aggregates and a soluble complex in the dosage formulation on nasal rhGH absorption was also evaluated by size-exclusion chromatography and ultrafiltration. PLA enhanced the nasal absorption of rhGH at each concentration and molecular weight examined. Nasal rhGH absorption increased dramatically when the PLA concentration was 1.0 % (w/v) due to the improved solubility of rhGH in the formulation. A delay in rhGH absorption was observed when the molecular weight of PLA was increased. This appeared to be because the increase in molecular weight caused the formation of a soluble complex. It seems that the PLA concentration affects the absorption-enhancing effect on rhGH, while the molecular weight of PLA affects the time when the maximum plasma rhGH concentration was reached (Tmax) of rhGH after i.n. administration, mainly because of the interactions among rhGH, PLA, and additives. Therefore, the transnasal rhGH delivery system using PLA is considered to be a promising alternative to subcutaneous (s.c.) injection if these interactions are sufficiently controlled.

Factors to Govern Soluble and Insoluble Aggregate-formation in Monoclonal Antibodies.

The aggregation formation of monoclonal antibodies as biopharmaceuticals induced by heat stress was evaluated by size-exclusion chromatography, and the formation rate was correlated with several physicochemical parameters of the antibodies to clarify the factors to govern the aggregate formation. The parameters we studied were: the melting temperature (Tm) and the standard enthalpy of the melting point (ΔmH°) evaluated by differential scanning calorimetry under given and common conditions; the wavelength (λmax) and the intensity (Fint) of the maximum fluorescence peak of 1-anilinonaphthalene-8-sulfonate as a probe dye; the z-average diameter (D) evaluated by dynamic light scattering; and the isoelectric point (pI) and the hydrophobic index (Hpho) of the complementarity determining region calculated from the amino acid sequence. Multivariate statistical analysis with these explanatory variables based on Akaike's information criterion indicates that the soluble aggregate formation is negatively correlated with Tm and pI, while the insoluble aggregate formation is positively correlated with Fint and pI. Based on these results, the mechanisms of the aggregate formation and methods to prevent the formation are discussed.

A fast and specific method to screen for intracellular amyloid inhibitors using bacterial model systems

The aggregation of a large variety of amyloidogenic proteins is linked to the onset of devastating human disorders. Therefore, there is an urgent need for effective molecules able to modulate the aggregative properties of these polypeptides in their natural environment, in order to prevent, delay or halt the progression of such diseases. On the one hand, the complexity and cost of animal models make them inefficient at early stages of drug discovery, where large chemical libraries are usually screened. On the other hand, in vitro aggregation assays in aqueous solutions hardly reproduce (patho)physiological conditions. In this context, because the formation of insoluble aggregates in bacteria shares mechanistic and functional properties with amyloid self-assembly in higher organisms, they have emerged as a promising system to model aggregation in the cell. Here we show that bacteria provide a powerful and cost-effective system to screen for amyloid inhibitors using fluorescence spectroscopy and flow cytometry, thanks to the ability of the novel red fluorescent ProteoStat dye to detect specifically intracellular amyloid-like aggregates. We validated the approach using the Alzheimer's linked Aβ40 and Aβ42 peptides and tacrine- and huprine-based aggregation inhibitors. Overall, the present method bears the potential to replace classical in vitro anti-aggregation assays.

324 - Oxidative Signature of Cerebrospinal Fluid from Mild Cognitive Impairment and Alzheimer Disease Patients

All key biological macromolecules are susceptible to carbonylation – an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage.

Protein aggregation in foam fractionation of bovine serum albumin: Effect of protein concentration

In foam fractionation of proteins, a clear understanding of their aggregation induced by the gas-liquid interface has great importance to reducing the loss of their bioactivity. In this work, the effects of the concentration of bovine serum albumin (BSA) on its aggregation induced by the gas-liquid interface were investigated at the molecular level to clearly explain how this protein aggregated in foam fractionation. The results show that reducing the BSA concentration made the protein structure more unfolded so that it enhanced the protein aggregation induced by the gas-liquid interface. In foam fractionation, the BSA aggregates were mainly formed in the desorption of the BSA molecules from the gas-liquid interface. Their formation was closely related to the BSA enrichment ratio. In the concentration from 0.05g/L to 0.20g/L, the highly unfolded BSA structure and the high enrichment jointly resulted in the formation of insoluble aggregates.

Towards the design of hypolipidaemic peptides: Deoxycholate binding affinity of hydrophobic peptide aggregates of casein plastein

Plastein is a thixotropic product of protease-induced peptide aggregation. This study evaluated the effects of the modified peptide structure of plastein on its affinity for a secondary bile acid. Casein plastein formation resulted in decreased free amino nitrogen and increased mean particle size, surface hydrophobicity and hydrophobic-to-hydrophilic amino acid ratio of the peptides. The net negative surface charge of casein hydrolysate (CH) was decreased (P < 0.05) in crude plastein (CPc) and completely lost in the isolated plastein (CPi) resulting in the formation of insoluble aggregates. The surface and structural changes in CPi were associated with 38.5 ± 0.0% binding of physiological level of sodium deoxycholate, higher (P < 0.05) than the activities of CPc (23.1 ± 0.0%) and CH (12.8 ± 4.4%). Maximum specific ligand binding (Bmax) was higher (P < 0.05) for CPc (0.391 ± 0.021 mM/mg protein) than CH (0.246 ± 0.043 mM/mg protein) and CPi (0.234 ± 0.005 mM/mg protein). However, CPi had the strongest affinity for sodium deoxycholate with the lowest dissociation constant (Kd) of 2.719 ± 0.146 mM, which was 2.5- and 3-folds lower (P < 0.05) than those of CH and CPc, respectively. Understanding deoxycholate-binding affinity of peptides can facilitate the design of potent food-based BA sequestrants for managing hypercholesterolaemia (dyslipidaemia) and associated health conditions.

Dual-Stimuli-Responsive l-Serine-Based Zwitterionic UCST-Type Polymer with Tunable Thermosensitivity

The synthesis of l-serine-based zwitterionic polymers, poly(l-serinyl acrylate)s (PSAs), of controllable molecular weights and low polydispersities via reversible addition–fragmentation chain transfer (RAFT) polymerization in water at 70 °C is described. The obtained homopolymer PSA exhibits dual responsiveness toward pH and temperature in aqueous solution. The PSA exhibits an isoelectric point near pH 2.85 where the PSA molecules exist in its zwitterionic form. In the pH range of 2.3–3.5, the aqueous PSA solution appears as a two-phase system due to the formation of insoluble aggregates through the intra- and intermolecular electrostatic interaction between the pendent ammonium and carboxylate groups of the neighboring zwitterionic PSA molecules. Furthermore, in this pH range, the two-phase PSA solution becomes one-phase upon heating, exhibiting distinct reversible upper critical solution temperature (UCST)-type phase transition. The cloud point (Tp) is found to increase with increasing molecular weights...

Unrelated solubility-enhancing fusion partners MBP and NusA utilize a similar mode of action.

The tendency of recombinant proteins to accumulate in the form of insoluble aggregates in Escherichia coli is a major hindrance to their overproduction. One of the more effective approaches to circumvent this problem is to use translation fusion partners {solubility-enhancers (SEs)}. E. coli maltose-binding protein (MBP) and N-utilization substance A (NusA) are arguably the most effective solubilizing agents that have been discovered so far. Here, we show that although these two proteins are structurally, functionally, and physicochemically distinct, they influence the solubility and folding of their fusion partners in a very similar manner. These SEs act as "holdases" that prevent the aggregation of their fusion partners. Subsequent folding of the passenger proteins, when it occurs, is either spontaneous or chaperone-mediated.

How protein aggregations and lens fiber disorder result in nuclear and cortical cataracts

AbstractCommonly, lens opacities and cataract are considered protein aggregation disorders. Increased light scattering is attributed to larger high‐molecular‐weight fractions of proteins. The impact on transparency of fiber membranes and their age‐ and disease‐related changes is addressed to a lesser extent in the literature. In the lens cortex of transparent human lenses, the lens fiber membranes have high refractive indexes compared with the cytoplasm, whereas there is almost no difference in the refractive indexes of membranes and cytoplasm in the lens nucleus. Therefore, in the lens cortex, transparency is obtained by a high spatial order of the lens fiber lattice to compensate for light scattering caused by differences of the refractive indexes. Any disturbance of the ordered lens fiber architecture in the cortex results in light scattering. In typical nuclear cataract, there is no major fiber disruption or extracellular debris to explain the opacity. The strong implication is that local fluctuations in crystallin density, due to the formation of insoluble aggregates, are the major cause of nuclear opacification. These fluctuations in density and refractive index, are considered to produce significant light scattering.

Functional screening in Drosophila reveals the conserved role of REEP1 in promoting stress resistance and preventing the formation of Tau aggregates.

Pathological modifications in the microtubule-associated protein Tau is a common characteristic observed in different neurological diseases, suggesting that analogous metabolic pathways might be similarly affected during neurodegeneration. To identify these molecules and mechanisms, we utilized Drosophila models of human Tau-mediated neurodegeneration to perform an RNA interference functional screening against genes considered to be implicated in the pathogenesis of different neurodegenerative disorders. We found that the downregulation of the Drosophila REEP1 homolog protein enhanced Tau toxicity with increased formation of insoluble aggregates. On the contrary, the overexpression of either the Drosophila or the human REEP1 protein was able to revert these phenotypes and promote neuronal resistance to ER stress. These studies identify a new function for the REEP1 protein in vivo and a novel cellular mechanism to prevent Tau toxicity.

Facile preparation of aqueous fullerene C60 nanodispersions

Aqueous solutions of the fullerene C60 (nC60) were prepared by simple mixing of the solution of C60 in N-methylpyrrolidone (MP) with deionized water or an aqueous solution of a low-molecular-weight natural substance (L-amino acids, monosaccharides, peptides, or glycerol) used as stabilizing agents (SAs) followed by exhaustive dialysis against distilled water. During dialysis, all low-molecular-weight compounds are removed through the pores and the fullerene clusters remain in the solution. The efficiency of conversion of C60 from the crystalline state to the solution approaches the quantitative value, and solutions with a C60 concentration of up to 250 mg/L can be obtained; moreover, these solutions are stable for at least 10–12 months. The formation of insoluble aggregates has been observed when basic and acidic organic compounds were used as SA. The UV-VIS spectra of solutions have a profile characteristic of nC60 solutions obtained by other well-known procedures (maxima at 220, 265, 340, and 450 nm). Mass spectra of aqueous solutions and FTIR spectra of dried nC60 samples were indicative of the possible partial hydroxylation of the fullerene. A measurement of the sizes and ξ potential of the C60 particles in solutions by the dynamic light scattering method showed that their average diameter is about 100 nm and the charge is −30 mV, whereas the electron microscopy data demonstrated that the particles have a typical size of approximately 20 nm and contain both crystalline and amorphous phases. The proposed method is promising for the preparation of solutions of endofullerenes and, probably, higher fullerenes.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its "codon-preferred" DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ~2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10(-8) m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.

Dyrk1A-mediated phosphorylation of RCAN1 promotes the formation of insoluble RCAN1 aggregates

The mechanisms underlying aggregate formation in age-related neurodegenerative diseases remain not well understood. Here we investigated whether dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is involved in the formation of regulator of calcineurin 1 (RCAN1) aggregates. We show that RCAN1 self-associates and forms multimers, and that this process is promoted by the Dyrk1A-mediated phosphorylation of RCAN1 at the Thr192 residue. Transgenic mice that overexpress the Dyrk1A exhibited lower levels of phospho-Thr192-RCAN1 in 10-month-old-group compared to littermate controls, when analyzed with soluble hippocampus lysates. These results suggest that the phosphorylation of RCAN1 by Dyrk1A stimulates the formation of insoluble aggregates upon aging.

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T(m) values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C(H)2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C(H)2 domain (notably heavy chain 241-251) and the mAb's overall physical stability.

191st ENMC International Workshop: Recent advances in oculopharyngeal muscular dystrophy research: From bench to bedside 8-10 June 2012, Naarden, The Netherlands

This workshop hosted a group of 19 clinicians and basic scientist from 5 countries (France, USA, Canada, Denmark and The Netherlands). Recent research developments and possible collaboration in advancing therapies for oculopharyngeal muscular dystrophy (OPMD), a late onset, rare and progressive neurodegeneration disorder were discussed in this meeting. The disease is caused by alanine expansion mutations in the gene encoding for poly(A)-binding protein nuclear 1 (PABPN1), resulting in accumulation of mutant PABPN1 in the cell nucleus and the formation of insoluble aggregates. Therefore, OPMD is categorized together with neurodegenerative disorders that are caused by single amino acid repeats. While the genetic cause for those disorders is often recognized, the molecular basis for symptoms is still obscure. PABPN1 is ubiquitously expressed and it is not known why symptoms develop only after midlife and initially only in subsets of skeletal muscles [1,2]. While the genetic cause for OPMD was reported in 1998 and good surgical treatments exist for major early symptoms, there are currently no medical options for the more general skeletal muscle involvement, no validated biomarkers and no clinical severity scale.

Efficient expression systems for cysteine proteases of malaria parasites

Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes.