Protein aggregation during overexpression limited by peptide extensions with large net negative charge

Folding of the human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) during overexpression in the Escherichia coli cytoplasm was shown previously to be partially rescued by fusion to a 22-residue C-terminal peptide. Here, peptide sequence features required for solubilization and folding of CAR D1 and similar Ig variable-type domains from two other human membrane proteins were investigated. Peptide extensions with net negative charge > -6 fully solubilized CAR D1, and approximately half of the peptide-solubilized protein was correctly folded. The Ig variable-type domains from human A33 antigen and myelin P-zero proteins were only partially solubilized by peptide extensions with net charge of -12, however, and only the solubilized P-zero domain appeared to fold correctly whereas the A33 domain formed soluble microaggregates of misfolded protein. Our results suggest a model where the large net charge of peptide extensions increases electrostatic repulsion between nascent polypeptides. The resulting decrease in aggregation rate can enable some polypeptides to fold spontaneously into their native protein conformations. Analysis of the solubility and folding status of sets of structurally homologous proteins, such as the Ig variable-type domains described here, during overexpression could provide insights into how amino acid and gene sequences influence the efficiency of spontaneous protein folding.     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins

Understanding the energetic and structural basis of protein folding in a physiological context may represent an important step toward the elucidation of protein misfolding and aggregation events that take place in several pathological states. In particular, investigation of the structure and thermodynamic properties of partially folded intermediate states involved in productive folding or in misfolding/aggregation may provide insight into these processes and suggest novel approaches to prevent misfolding in living organisms. This goal, however, has remained elusive, because such intermediates are often transient and correspond to metastable states that are little populated under physiological conditions. Characterization of these states requires their stabilization by means of manipulation of the experimental conditions, involving changes in temperature, pH, or addition of different types of denaturants. In the past few years, hydrostatic pressure has been increasingly used as a thermodynamic variable in the study of both protein folding and misfolding/aggregation transitions. Compared with other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, allowing the stabilization of partially folded states that are usually not significantly populated under more drastic conditions. Much of the recent work in this field has focused on the characterization of folding intermediates, because they seem to be involved in a variety of disease-causing protein misfolding and aggregation reactions. Here, we review recent examples of the use of hydrostatic pressure as a tool to gain insight into the forces and energetics governing the productive folding or the misfolding and amyloid aggregation of proteions.     

Folding versus aggregation: polypeptide conformations on competing pathways

Protein aggregation has now become recognised as an important and generic aspect of protein energy landscapes. Since the discovery that numerous human diseases are caused by protein aggregation, the biophysical characterisation of misfolded states and their aggregation mechanisms has received increased attention. Utilising experimental techniques and computational approaches established for the analysis of protein folding reactions has ensured rapid advances in the study of pathways leading to amyloid fibrils and amyloid-related aggregates. Here we describe recent experimental and theoretical advances in the elucidation of the conformational properties of dynamic, heterogeneous and/or insoluble protein ensembles populated on complex, multidimensional protein energy landscapes. We discuss current understanding of aggregation mechanisms in this context and describe how the synergy between biochemical, biophysical and cell-biological experiments are beginning to provide detailed insights into the partitioning of non-native species between protein folding and aggregation pathways.     

Native structure protects SUMO proteins from aggregation into amyloid fibrils

SUMO proteins belong to the Ubiquitin-like protein family, all sharing a common fold and a similar mechanism of conjugation to target polypeptides. SUMO is ubiquitous in all eukaryotes and participates in many crucial pathways. Native SUMO proteins are highly soluble, a property that is exploited in biotechnology. Moreover, SUMO regulates the solubility of aggregation-prone proteins in neurodegenerative disorders. Despite these properties, we show here that human SUMO1, SUMO2, and SUMO3 proteins are at risk of aggregation into amyloid structures if their native conformation is perturbed. Aggregation is mediated by specific regions, which overlap with SUMO functional interfaces, illustrating a competition between function and aggregation. Aggregation of SUMOs might have important physiological implications because disruption of the SUMO pathway is lethal in different organisms. It appears that functional constraints make it difficult to avoid the competition between productive folding and deleterious aggregation in globular proteins, even for essential polypeptides.     

Diseases of protein aggregation and the hunt for potential pharmacological agents

Protein aggregation is a ubiquitous phenomenon significant to all aspects of science. Notably, the formation of protein aggregates is frequently encountered in biochemical research and biopharmaceutical industry. Formation of protein aggregates is generally regarded to be associated with partially folded intermediate species that are susceptible to self-association due to the exposure of hydrophobic core. Evidence supports the concept that the formation of aggregates in vitro is a generic property of proteins. In human etiology, more than 20 different devastating human diseases have been reported to be associated with protein aggregation. Although protein aggregation diseases have been the center of intense research, much remains to be learned regarding the underlying molecular mechanisms. In this review, the general background information on protein aggregation is first provided. Next, we summarize the properties, characteristics and causes of protein aggregates. Finally, from the perspectives of epidemiology, pathogenesis, existing mechanisms, relevant hypotheses, and current as well as potential therapeutic approaches, two examples of protein aggregation diseases, Alzheimer's disease and cataract, are briefly discussed. Importantly, while a variety of molecules have been suggested, the effective therapeutic drugs for curing the diseases involving protein aggregation have yet to be identified. We believe that a better understanding of the mechanisms of protein aggregation process and an extensive investigation into the drug penetration, efficacy, and side effects will certainly aid in developing the successful pharmacological agents for these diseases.     

Kinetic partitioning of protein folding and aggregation.

We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the alpha/beta protein acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16-31 and 87-98, each of which has a relatively high hydrophobicity and propensity to form beta-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and beta-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.     

The aggregation properties of Escherichia coli proteins associated with their cellular abundance

Proteins are key players in most cellular processes. Therefore, their abundances are thought to be tightly regulated at the gene-expression level. Recent studies indicate, however, that steady-state cellular-protein concentrations correlate better across species than the levels of the corresponding mRNAs; this supports the existence of selective forces to maintain precise cellular-protein concentrations and homeostasis, even if gene-expression levels diverge. One of these forces might be the avoidance of protein aggregation because, in the cell, the folding of proteins into functional conformations might be in competition with anomalous aggregation into non-functional and usually toxic structures in a concentration-dependent manner. The data in the present work provide support for this hypothesis because, in E. coli, the experimental solubility of proteins correlates better with the cellular abundance than with the gene-expression levels. We found that the divergence between protein and mRNAs levels is low for high-abundance proteins. This suggests that because abundant proteins are at higher risk of aggregation, cellular concentrations need to be stringently regulated by gene expression.     

Influence of denatured and intermediate states of folding on protein aggregation

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.     

Amyloid Formation by Human Carboxypeptidase D Transthyretin-like Domain under Physiological Conditions

Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.     

Transient aggregation of nascent thyroglobulin in the endoplasmic reticulum: relationship to the molecular chaperone, BiP

Because of its unusual length, nascent thyroglobulin (Tg) requires a long time after translocation into the endoplasmic reticulum (ER) to assume its mature tertiary structure. Thus, Tg is an ideal molecule for the study of protein folding and export from the ER, and is an excellent potential substrate for molecular chaperones. During the first 15 min after biosynthesis, Tg is found in transient aggregates with and without interchain disulfide bonds, which precede the formation of free monomers (and ultimately dimers) within the ER. By immunoprecipitation, newly synthesized Tg was associated with the binding protein (BiP); association was maximal at the earliest chase times. Much of the Tg released from BiP by the addition of Mg-ATP was found in aggregates containing interchain disulfide bonds; other BiP-associated Tg represented non-covalent aggregates and unfolded free monomers. Importantly, the immediate precursor to Tg dimer was a compact monomer which did not associate with BiP. The average stoichiometry of BiP/Tg interaction involved nearly 10 BiP molecules per Tg molecule. Cycloheximide was used to reduced the ER concentration of Tg relative to chaperones, with subsequent removal of the drug in order to rapidly restore Tg synthesis. After this treatment, nascent Tg aggregates were no longer detectable. The data suggest a model of folding of exportable proteins in which nascent polypeptides immediately upon translocation into the ER interact with BiP. Early interaction with BiP may help in presenting nascent polypeptides to other helper molecules that catalyze folding, thereby preventing aggregation or driving aggregate dissolution in the ER.     

Folding and aggregation are selectively influenced by the conformational preferences of the alpha-helices of muscle acylphosphatase

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.     

Prion proteins: folding and aggregation properties

The partial unfolding or alternative folding of a class of polypeptides is at the origin of fascinating events in living cells. In their non-native conformation, these constitutive polypeptides called prions are at the origin of a protein-based structural heredity. These polypeptides are closely associated to a class of fatal neurodegenerative illnesses in mammals and to the emergence and propagation of phenotypic traits in baker's yeasts. The structural transition from the correctly folded, native form of a prion protein to a persistent misfolded form that ultimately may cause cell death or the transmission of phenotypic traits is not yet fully understood. The mechanistic models accounting for this structure-based mode of inheritance and the extent of partial unfolding of prions or their alternative folding and the subsequent aggregation process are developed and discussed. Finally, the potential regulation of prion propagation by molecular chaperones is presented.     

Lattice simulations of aggregation funnels for protein folding.

A computer model of protein aggregation competing with productive folding is proposed. Our model adapts techniques from lattice Monte Carlo studies of protein folding to the problem of aggregation. However, rather than starting with a single string of residues, we allow independently folding strings to undergo collisions and consider their interactions in different orientations. We first present some background into the nature and significance of protein aggregation and the use of lattice Monte Carlo simulations in understanding other aspects of protein folding. The results of a series of simulation experiments involving simple versions of the model illustrate the importance of considering aggregation in simulations of protein folding and provide some preliminary understanding of the characteristics of the model. Finally, we discuss the value of the model in general and of our particular design decisions and experiments. We conclude that computer simulation techniques developed to study protein folding can provide insights into protein aggregation, and that a better understanding of aggregation may in turn provide new insights into and constraints on the more general protein folding problem.     

Diseases associated with protein aggregation

The so-called conformational diseases constitute a specific subtype of protein folding diseases that is characterized by abnormal aggregation of improperly folded polypeptides. This review describes a series of examples of such disorders and summarizes the present knowledge on their molecular pathophysiology and new therapeutic strategies.     

Inclusion bodies: specificity in their aggregation process and amyloid-like structure

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.     

Beta-sheet folding of 11-kDa fibrillogenic polypeptide is completely aggregation driven

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) was designed and genetically engineered to form antiparallel beta-strands of GAGAGA repeats. Modulation of pH enables control of solubility, folding, and aggregation of YE8 by control of the overall polypeptide charge, a consequence of the protonation or deprotonation of the glutamic acid and histidine residues. YE8 exhibits all the major properties of a fibrillogenic protein providing an excellent model for detailed study of the fibrillation. At neutral pH, YE8 is soluble in disordered form, yet at pH 3.5 folds into a predominantly beta-sheet conformation that is fibrillogenic. Atomic force microscopy and transmission electron microscopy indicated the formation of fibrillar aggregates on well-defined, hydrophobic surfaces. The beta-sheet folding of YE8 exhibited a lag phase that could be eliminated by seeding or stirring. The strong dependence of lag time on polypeptide concentration established the limiting step in aggregation as initiation of beta-sheet folding.     

Construction and deconstruction of bacterial inclusion bodies

Bacterial inclusion bodies (IBs) are refractile aggregates of protease-resistant misfolded protein that often occur in recombinant bacteria upon gratuitous overexpression of cloned genes. In biotechnology, the formation of IBs represents a main obstacle for protein production since even favouring high protein yields, the in vitro recovery of functional protein from insoluble deposits depends on technically diverse and often complex re-folding procedures. On the other hand, IBs represent an exciting model to approach the in vivo analysis of protein folding and to explore aggregation dynamics. Recent findings on the molecular organisation of embodied polypeptides and on the kinetics of inclusion body formation have revealed an unexpected dynamism of these protein aggregates, from which polypeptides are steadily released in living cells to be further refolded or degraded. The close connection between in vivo protein folding, aggregation, solubilisation and proteolytic digestion offers an integrated view of the bacterial protein quality control system of which IBs might be an important component especially in recombinant bacteria.     

Misfolding and aggregation of vacuolar glycoproteins in plant cells

Phaseolin and lectin-related polypeptides, the abundant oligomeric glycoproteins of bean seeds, are synthesized on the endoplasmic reticulum (ER) and then transported to the storage vacuole via the Golgi apparatus. Glycosylation and folding are among the major modifications these proteins undergo in the ER. Although a recurrent role of N-glycosylation is on protein folding, in previous studies on common bean (Phaseolus vulgaris) seeds we demonstrated that the oligosaccharide side-chains are not required for folding, intracellular transport and activity of storage glycoproteins. We show here that in lima bean (Phaseolus lunatus), incubation of the developing cotyledon with tunicamycin to prevent glycosylation has a dramatic effect on the intracellular transport of the storage glycoproteins. When lacking their glycans, phaseolin and lectin-related polypeptides misfold and are retained in the ER as mixed aggregates to which the chaperone BiP irreversibly associates. The lumen of the ER becomes enlarged to accommodate the aggregated polypeptides. Intracellular transport of legumin, a naturally unglycosylated storage protein, is mostly unaffected by the inhibitor, indicating that the observed phenomenon specifically occurs on glycoproteins. Furthermore, recombinant lima bean phaseolin synthesized in tobacco protoplasts is also correctly folded and matured in the presence of tunicamycin. To our knowledge, this is the first report that describes in detail the block of intracellular transport of vacuolar glycoproteins in plant cells due to aggregation following glycosylation inhibition.