Extended polyglutamine tracts cause aggregation and structural perturbation of an adjacent beta barrel protein

Formation of fibrillar intranuclear inclusions and related neuropathologies of the CAG-repeat disorders are linked to the expansion of a polyglutamine tract. Despite considerable effort, the etiology of these devastating diseases remains unclear. Although polypeptides with glutamine tracts recapitulate many of the observed characteristics of the gene products with CAG repeats, such as in vitro and in vivo aggregation and toxicity in model organisms, extended polyglutamine segments have also been reported to structurally perturb proteins into which they are inserted. Additionally, the sequence context of a polyglutamine tract has recently been shown to modulate its propensity to aggregate. These findings raise the possibility that indirect influences of the repeat tract on adjacent protein domains are contributory to pathologies. Destabilization of an adjacent domain may lead to loss of function, as well as favoring non-native structures in the neighboring domain causing them to be prone to intermolecular association and consequent aggregation. To explore these phenomena, we have used chimeras of a well studied globular protein and exon 1 of huntingtin. We find that expansion of the polyglutamine segment beyond the pathological threshold (>35 glutamines) results in structural perturbation of the neighboring protein whether the huntingtin exon is N- or C-terminal. Elongation of the polyglutamine region also substantially increases the propensity of the chimera to aggregate, both in vitro and in vivo, and in vitro aggregation kinetics of a chimera with a 53-glutamine repeat follow a nucleation polymerization mechanism with a monomeric nucleus.     

Protein aggregation/crystallization and minor structural changes: universal versus specific aspects

Protein association covers wide interests in biophysics, protein science, and biotechnologies, and it is often viewed as governed by conformation details. More recently, the existence of a universal physical principle governing aggregation/crystallization processes has been suggested by a series of experiments and shown to be linked to the universal scaling properties of concentration fluctuations occurring in the proximity of a phase transition (spinodal demixing in the specific case). Such properties have provided a quantitative basis for capturing kinetic association data on a universal master curve, ruled by the normalized distance of the state of the system from its instability region. Here we report new data on lysozyme crystal nucleation. They strengthen the evidence in favor of universality and show that the system enters the region of universal behavior in a stepwise manner as a result of minor conformation changes. Results also show that the link between conformation details and universal behavior is actuated by interactions mediated by the solvent. Outside the region of universal behavior, nucleation rates become unpredictable and undetectably long.     

Protein misfolding and aggregation

Interest in the problem of protein misfolding and aggregation has exploded in recent years for two reasons: (1) the sharp rise in the number and volume of therapeutic proteins produced commercially and (2) the recognition of the central role of protein aggregates in degenerative diseases. The systematic study of protein aggregation presents major challenges to both the experimentalist and the theoretician. Much of the work retains an empirical flavor due to the experimental complexities; the sensitivity of protein aggregation to the slightest change in protein amino acid composition, solvent properties, or protein concentration; and the lack of robust theoretical models of misfolding and aggregation. Novel experimental and computational approaches are being developed, and we anticipate substantial progress will be made in the near future. Several presentations describing the latest advances in protein misfolding and aggregation were given at the American Chemical Society meeting (BIOT division) held in September, 2006 in San Francisco.     

Effects of a homogeneous magnetic field on erythrocyte sedimentation and aggregation

Effects of a homogeneous static magnetic field on erythrocyte sedimentation rate (ESR) have been assessed by using the standard Westergren method. A magnetic field of 6.3 T in the vertical direction only slightly enhanced ESR in saline solution, which was consistent with an effect on cell orientation. On the other hand, the magnetic field greatly enhanced ESR in plasma. It took a long time (about 20 min) for an ESR change to occur in plasma in response to the magnetic field. The effects in plasma were too large to originate only from cell orientation and were clearly distinct from a magnetic field-induced Boycott effect under an inhomogeneous magnetic field. A morphological examination and the nonlinear time course of the sedimentation in plasma indicated that the magnetic field increased cell aggregation and thereby enhanced ESR in plasma. Bioelectromagnetics 18:215–222, 1997. © 1997 Wiley-Liss, Inc.     

High-density,homogeneous endospore monolayer deposition on test surfaces

Bacillus subtilis spores were deposited in high-density single layers on metal, glass, and polymer substrates using vacuum filtration followed by a wetted filter transfer step. Quantitative analysis of spore transfer was performed using culture-based and germinability assays, and spore distributions were observed with electron microscopy.     

Protein aggregation and its impact on product quality

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Protein aggregation in silico

Protein aggregation is a challenge to the successful manufacture of protein therapeutics; it can impose severe limitations on purification yields and compromise formulation stability. Advances in computer power, and the wealth of computational studies pertaining to protein folding, have facilitated the development of molecular simulation as a tool to investigate protein misfolding and aggregation. Here, we highlight the successes of protein aggregation studies carried out in silico, with a particular emphasis on studies related to biotechnology. To conclude, we discuss future prospects for the field, and identify several biotechnology-related problems that would benefit from molecular simulation.     

Protein aggregation in Huntington's disease

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.     

Protein aggregation in crowded environments

The generic tendency of proteins to aggregate into non-functional, and sometimes cytotoxic, structures poses a universal problem for all types of cell. This tendency is greatly exacerbated by the high total concentration of macromolecules found within most intracellular compartments, a phenomenon referred to as macromolecular crowding. This review discusses the quantitative effects of crowding on protein aggregation and the role of molecular chaperones in combating this problem.     

Protein intake by gypsy moth larvae on homogeneous and heterogeneous diets

Abstract. Food selection behaviour, food utilization efficiency and growth performance of a generalist insect, the gypsy moth ( Lymantria dispar (L.), Lepidoptera: Lymantriidae), were examined with respect to variation in food nitrogen concentration. The results suggest that gypsy moth do not suffer physiologically and in fact may benefit from intraplant variation by selective feeding. When provided with diet cubes containing identical nitrogen concentrations, control larvae tended to consume food from a single cube. This behaviour contrasted with that of larvae provided cubes differing in nitrogen concentration. These larvae tended to consume more from the high nitrogen cube, but allocated feeding more evenly among diet cubes than did control larvae. Overall, larvae mixed foods so as to obtain a mean concentration of 2.9-3.2% nitrogen, a concentration assumed to approximate the 'intake target'. Larvae confined to single nitrogen concentrations mitigated the impact of imbalanced diets on body composition via both pre-ingestive and post-ingestive compensation. When confined to a specific nitrogen concentration, larvae adjusted their intake to the point of best compromise. In this case, this was the geometrically closest point to the estimated intake target. Larvae with a choice of foods that deviated more than ±1% from each other in nitrogen concentration grew as well as or better than larvae without a choice but given identical mean nitrogen concentrations. These results demonstrate that selectivity and nitrogen consumption by gypsy moth larvae are altered according to the particular choices available. Insects may benefit from intraplant variation in food quality because such variation provides the opportunity to choose foods and mix them in ways that permit close matching with the intake target. Variation may be particularly important to insects which must offset changing nutritional demands.     

Protein aggregation: mechanisms and functional consequences

Understanding the mechanisms underlying protein misfolding and aggregation has become a central issue in biology and medicine. Compelling evidence show that the formation of amyloid aggregates has a negative impact in cell function and is behind the most prevalent human degenerative disorders, including Alzheimer's Parkinson's and Huntington's diseases or type 2 diabetes. Surprisingly, the same type of macromolecular assembly is used for specialized functions by different organisms, from bacteria to human. Here we address the conformational properties of these aggregates, their formation pathways, their role in human diseases, their functional properties and how bioinformatics tools might be of help to study these protein assemblies.     

Protein stability and aggregation in Parkinson's disease

Parkinson's disease (PD), the second most common age-related neurodegenerative disease, results in abnormalities in motor functioning. Many fundamental questions regarding its aetiology remain unanswered. Pathologically, it is not until 70-80% of the dopaminergic neurons from the substantia nigra pars compacta are lost before clinical symptoms are observed. Thus research into PD is complicated by this apparent paradox in that what appears to be the beginning of the disease at the clinical level is really the end point neurochemically. Consequently, we can only second guess when the disease started and what initiated it. The causation is probably complex, with contributions from both genetic and environmental factors. Intracellular proteinaceous inclusions, Lewy bodies and Lewy neurites, found in surviving dopaminergic neurons, are the key pathological characteristic of PD. Their presence points to an inability within these terminally differentiated cells to deal with aggregating proteins. Recent advances in our knowledge of the underlying disease process have come about from studies on models based on genes associated with rare hereditary forms of PD, and mitochondrial toxins that mimic the behavioural effects of PD. The reason that dopaminergic neurons are particularly sensitive may be due to the additional cellular stress caused by the breakdown of the inherently chemically unstable neurotransmitter, dopamine. In the present review, I discuss the proposal that in sporadic disease, interlinked problems of protein processing and inappropriate mitochondrial activity seed the foundation for age-related increased levels of protein damage, and a reduced ability to deal with the damage, leading to inclusion formation and, ultimately, cell toxicity.     

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin’s antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.     

Protein aggregation: in silico algorithms and applications

Protein aggregation is a topic of immense interest to the scientific community due to its role in several neurodegenerative diseases/disorders and industrial importance. Several in silico techniques, tools, and algorithms have been developed to predict aggregation in proteins and understand the aggregation mechanisms. This review attempts to provide an essence of the vast developments in in silico approaches, resources available, and future perspectives. It reviews aggregation-related databases, mechanistic models (aggregation-prone region and aggregation propensity prediction), kinetic models (aggregation rate prediction), and molecular dynamics studies related to aggregation. With a multitude of prediction models related to aggregation already available to the scientific community, the field of protein aggregation is rapidly maturing to tackle new applications.     

Protein aggregation and its consequences for human disease

Protein molecules have emerged through evolution so that they are able to remain in their functional and soluble states under normal physiological conditions, although in other situations they often have a high propensity to aggregate. Aggregation in vivo is associated with a wide range of human disorders, including Alzheimer's disease and type II diabetes, medical conditions that are becoming increasingly common in the modern world. In such diseases, aggregated proteins can often be observed as highly intractable thread-like species known as amyloid fibrils. This article provides an overview of our present knowledge of the nature of these fibrillar aggregates and the manner in which they form, and discusses the origins and potential means of suppression of the pathogenic properties with which they and their precursors are associated.