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PubMed2005

Spray freezing into liquid versus spray-freeze drying: influence of atomization on protein aggregation and biological activity.

Protein aggregation and enzyme activity were compared for reconstituted lysozyme particles produced by two cryogenic technologies, spray freezing into liquid (SFL) and spray-freeze drying (SFD). The particles were characterized by enzyme activity measurements, scanning electron microscopy (SEM), light scattering, X-ray photoelectron spectroscopy (XPS) and BET specific surface area analysis. Highly porous microparticle aggregates of protein nanoparticles, observed by SEM, were produced by both processes. The smaller degree of protein aggregation and smaller losses in enzyme activity for the SFL process relative to the SFD process were due primarily to the spraying step. The higher stability of the SFL versus SFD powders was consistent with the smaller surface excess of lysozyme measured by XPS in SFL, resulting from the reduced time of exposure to the air-water interface during atomization. For pure lysozyme, the degree of aggregation and enzyme activity were comparable for lyophilization and SFL, despite the much larger particle surface area for SFL.

Enzyme Stability↗

PubMed2005

Protein aggregation and its inhibition in biopharmaceutics.

Protein aggregation is arguably the most common and troubling manifestation of protein instability, encountered in almost all stages of protein drug development. Protein aggregation, along with other physical and/or chemical instabilities of proteins, remains to be one of the major road barriers hindering rapid commercialization of potential protein drug candidates. Although a variety of methods have been used/designed to prevent/inhibit protein aggregation, the end results are often unsatisfactory for many proteins. The limited success is partly due to our lack of a clear understanding of the protein aggregation process. This article intends to discuss protein aggregation and its related mechanisms, methods characterizing protein aggregation, factors affecting protein aggregation, and possible venues in aggregation prevention/inhibition in various stages of protein drug development.

Biopharmaceutics↗

PubMed2001

Impairment of the ubiquitin-proteasome system by protein aggregation.

Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.

Acetylcysteine↗

PubMed2004

Reversion of protein aggregation mediated by Sso7d in cell extracts of Sulfolobus solfataricus.

In eukaryotic cells and in Escherichia coli, reversion of protein aggregation is mediated by the network of chaperones belonging to Hsp70 and Hsp100 families [Weibezahn, Bukau and Mogk (2004) Microb. Cell Fact. 3, 1-12]. The thermophilic prokaryotes of the archaea domain lack homologues of these chaperone families, and the mechanisms they use to rescue aggregated proteins are unknown [Macario, Malz and Conway de Macario (2004) Front. Biosci. 9, 1318-1332]. In the present study, we show that stable protein aggregates can be detected in extracts of starved cells of the thermophilic archaeon Sulfolobus solfataricus, and that the protein Sso7d interacts with the aggregates and mediates the disassembly of the aggregates and the re-activation of insolubilized beta-glycosidase in the presence of ATP hydrolysis. Furthermore, we report that heat-induced protein aggregates in extracts of exponential cells of S. solfataricus contain Sso7d that rescues insolubilized proteins in the presence of ATP hydrolysis. Results of these experiments performed in cell extracts are consistent with an in vivo role of Sso7d in reverting protein aggregation.

Adenosine Triphosphate↗

PubMed2005

Bistability explains threshold phenomena in protein aggregation both in vitro and in vivo.

Neurodegenerative disease can originate from the misfolding and aggregation of proteins, such as Amyloid-beta, SOD1, or Huntingtin. Fortunately, all cells possess protein quality control machinery that sequesters misfolded proteins, either refolding or degrading them, before they can self-associate into proteotoxic oligomers and aggregates. This activity is largely performed by the stress response chaperones (i.e., Hsp70). However, the expression level of molecular chaperones varies widely among cell types. To understand the potential consequence of this variation, we studied the process of protein aggregation in the presence of molecular chaperones using mathematical modeling. We demonstrate that protein aggregation, in the presence of molecular chaperones, is a bistable process. Bistability in protein aggregation offers an explanation for threshold transitions to high aggregate concentration, which are observed both in vitro and in vivo. Additionally, we show that slight variations in chaperone concentration, due to natural fluctuations, have important consequences in a bistable system for the onset of protein aggregation. Therefore, our results offer a possible theoretical explanation for neuronal vulnerability observed in vivo and the onset of neurodegenerative phenotypes in neurons lacking an effective heat-shock response.

Animals↗

PubMed1983

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein.

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.

Amino Acids↗

PubMed2006

Insufficient expression of cyclooxygenase-2 protein is associated with retarded degradation of aggregated protein in diabetic glomeruli.

To elucidate the involvement of cyclooxygenase (COX) in degradation of aggregated protein in diabetic glomeruli, we used streptozotocin (STZ)-induced diabetic mice and aggregated bovine serum albumin (a-BSA) as a model protein. There was a higher deposition of a-BSA in diabetic glomeruli compared to normal glomeruli 18 h after a-BSA injection at 4 and 8 weeks after STZ. Degradation of a-BSA was confirmed using isolated glomeruli. Diabetic glomeruli produced prostaglandin E(2) (PGE(2)) more than normal glomeruli in the basal level at 8 weeks. a-BSA caused further increase of PGE(2) production in normal glomeruli, but not in diabetic glomeruli. Niflimic acid, a selective COX-2 inhibitor, reduced PGE(2) production of normal glomeruli in the a-BSA loading group, but not that in the control group. In diabetic glomeruli, niflimic acid reduced PGE(2) production in both the control group and a-BSA loading group. In normal glomeruli, a-BSA increased expressions of both COX-2 mRNA and protein. However, in diabetic glomeruli, a-BSA increased COX-2 mRNA expression but not COX-2 protein expression. These results suggest that retarded degradation of aggregated protein in diabetic glomeruli is associated with lack of further expression of COX-2 protein and further production of PGE(2) in response to aggregated protein.

Animals↗

PubMed2025

Prediction and Evaluation of Protein Aggregation with Computational Methods.

Protein and peptide aggregation has recently become one of the most studied biomedical problems due to its central role in several neurodegenerative disorders and of biotechnological importance. Multiple in silico methods, databases, tools, and algorithms have been developed to predict aggregation of proteins and peptides to better understand fundamental mechanisms of various aggregation diseases. Here, we attempt to provide a brief overview of bioinformatic methods and tools to better understand molecular mechanisms of aggregation disorders. Furthermore, through a better understanding of protein aggregation mechanisms, it might be possible to design novel therapeutic agents to treat and hopefully prevent protein aggregation diseases.

Computational Biology↗

PubMed2006

Protein aggregation and bioprocessing.

Protein aggregation is a common issue encountered during manufacture of biotherapeutics. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment (eg, media components) and implementing appropriate strategies to minimize the extent of aggregation. Steps to remove aggregates have been successfully used at a manufacturing scale. Care should be taken when developing a process to monitor the compatibility of the equipment and process with the protein to ensure that potential aggregation is minimized.

Cell Culture Techniques↗

PubMed2003

Protein aggregation in motor neurone disorders.

Toxicity associated with abnormal protein folding and protein aggregation are major hypotheses for neurodegeneration. This article comparatively reviews the experimental and human tissue-based evidence for the involvement of such mechanisms in neuronal death associated with the motor system disorders of X-linked spinobulbar muscular atrophy (SBMA; Kennedy's disease) and amyotrophic lateral sclerosis (ALS), especially disease related to mutations in the superoxide dismutase (SOD1) gene. Evidence from transgenic mouse, Drosophila and cell culture models of SBMA, in common with other trinucleotide repeat expansion disorders, show protein aggregation of the mutated androgen receptor, and intraneuronal accumulation of aggregated protein, to be obligate mechanisms. Strong experimental data link these phenomena with downstream biochemical events involving gene transcription pathways (CREB-binding protein) and interactions with protein chaperone systems. Manipulations of these pathways are already established in experimental systems of trinucleotide repeat disorders as potential beneficial targets for therapeutic activity. In contrast, the evidence for the role of protein aggregation in models of SOD1-linked familial ALS is less clear-cut. Several classes of intraneuronal inclusion body have been described, some of which are invariably present. However, the lack of understanding of the biochemical basis of the most frequent inclusion in sporadic ALS, the ubiquitinated inclusion, has hampered research. The toxicity associated with expression of mutant SOD1 has been intensively studied however. Abnormal protein aggregation and folding is the only one of the four major hypotheses for the mechanism of neuronal degeneration in this disorder currently under investigation (the others comprise oxidative stress, axonal transport and cytoskeletal dysfunctions, and glutamatergic excitotoxicity). Whilst hyaline inclusions, which are strongly immunoreactive to SOD1, are linked to degeneration in SOD1 mutant mouse models, the evidence from human tissue is less consistent and convincing. A role for mutant SOD1 aggregation in the mitochondrial dysfunction associated with ALS, and in potentially toxic interactions with heat shock proteins, both leading to apoptosis, are supported by some experimental data. Direct in vitro data on mutant SOD1 show evidence for spontaneous oligomerization, but the role of such oligomers remains to be elucidated, and therapeutic strategies are less well developed for this familial variant of ALS.

Animals↗

PubMed2001

Specificity in intracellular protein aggregation and inclusion body formation.

Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels.

Cell Line↗

PubMed2002

p62 Is a common component of cytoplasmic inclusions in protein aggregation diseases.

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network.

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.

Bacterial Proteins↗

PubMed2004

Xenobiotic-induced autoimmunity and protein aggregation diseases share a common subnuclear pathology.

The cell nucleus constitutes a prime target of idiopathic and xenobiotic-induced autoimmunity. Despite of the high prevalence of rheumatic autoimmune diseases, the molecular mechanisms inducing systemic autoimmunity are largely unknown. In appreciation of Rudolf Virchow's cellularpathology, this review introduces the new concept of subnuclear pathology to autoimmune responses against the cell nucleus. Aberrant nucleoplasmic clusters consisting of nuclear autoantigens and proteasomes are observed in xenobiotic-treated cell lines, splenic cells from animal models of xenobiotic-induced autoimmunity, and dendritic cells of scleroderma patients. Aggregation of nuclear proteins in clusters inhibits nuclear functions such as replication and transcription, and induces altered proteasomal degradation of nuclear autoantigens and cellular senescence. Since these modifications of nuclear structure, function and proteolysis resemble the pathology of neurodegenerative protein aggregation diseases, the hypothesis is put forward that xenobiotic-induced autoimmunity may also be a consequence of protein aggregation.

Autoantigens↗

PubMed2005

The amino-terminal domain of ClpB supports binding to strongly aggregated proteins.

Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.

Amino Acid Motifs↗

PubMed2002

Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates.

We have studied at pH 4.2 and three protein (Pr):polysaccharide (Pol) weight ratios (8:1, 2:1 and 1:1) the structure and stability of beta-lactoglobulin/acacia gum/water dispersions containing protein aggregates (BLG/AG/W) or free from aggregates (AF-BLG/AG/W). Phase diagrams were characteristic of complex coacervation. BLG/AG/W dispersions displayed a larger biphasic area than AF-BLG/AG/W dispersions, that moved towards the protein axis. It was concluded that protein aggregates affected complex coacervation both by entropic (size and molecular masses of aggregates) and enthalpic (surface properties of aggregates) effects. Laser light scattering measurements revealed that the particles diameter (d(43)) induced by demixing was controlled by protein aggregates in AF-BLG/AG/W dispersions. At 1 wt.% biopolymer concentration, particles were 15-20 times larger in AF-BLG/AG/W dispersions than in BLG/AG/W dispersions at (Pr:Pol) ratios of 2:1 or 1:1. Confocal scanning laser microscopy showed that AF-BLG/AG/W dispersions only contained spherical coacervates. BLG/AG/W dispersions contained both coacervates and aggregates coated with AG or/and BLG/AG coacervates. At a (Pr:Pol) ratio of 2:1 and 1:1, coacervates were vesicular or multivesicular. Coacervates were smaller in BLG/AG/W dispersions than in AF-BLG/AG/W dispersions. It was concluded that protein aggregates have the intrinsic ability to stabilize complex coacervates and could be used to design multifunctional delivery systems. This study showed that composite dispersions containing both protein aggregates embedded in protein-polysaccharide coacervates and free coacervates may be performed. In this respect, the design of protein aggregates with controlled size distribution and surface properties could open new possibilities both in the non-chemical control of coacervates stability and in the development of multifunctional delivery systems.

Drug Compounding↗

PubMed2005

Reduced transglutaminase-catalyzed protein aggregation is observed in the presence of creatine using sedimentation velocity.

Transglutaminases (TGases) are enzymes that catalyze covalent isopeptide crosslinks between reactive lysine and glutamine residues in proteins. Higher than normal local concentrations of TGase have been correlated with increased protein aggregation in vivo. These insoluble protein aggregates are the hallmark of several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, although each aggregating protein involved is disease specific. Because TGase is implicated in protein aggregation, there is evidence that its regulation may retard disease progression. Here we report on a laser light transmission technique as an in vitro tool to gauge the efficacy of creatine, a candidate inhibitor, to regulate aggregation. Sedimentation velocities of protein-coated particles in TGase-containing water-glycerol solutions were tracked with different levels of creatine. Sedimentation velocities were converted to apparent aggregate sizes using Stoke's law of sedimentation. The results indicated that creatine promoted up to a 20% reduction in protein aggregation in vitro. This technique may prove to be useful in identifying other functional TGase inhibitors.

Centrifugation, Density Gradient↗

PubMed2004

Investigation of the effects of copper ions on protein aggregation using a model system.

Protein aggregation is a notable feature of various human disorders, including Parkinson's disease, Alzheimer's disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. alpha-synuclein, amyloid beta (Abeta) peptide, beta(2)-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu(2+) produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu(2+)-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu(2+)-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a beta structure introduced by the point mutation. The effect of Cu(2+) on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.

Acid Anhydride Hydrolases↗