Protein Aggregation Diseases Research Articles

Phage Display Affords Peptides that Modulate β-Amyloid Aggregation

As the population ages, the need to develop methods to understand and intercept the processes responsible for protein aggregation diseases is becoming more urgent. The aggregation of the protein beta-amyloid (Abeta) has been implicated in Alzheimer's Disease (AD); however, whether the toxic species is a large, insoluble aggregate or some lower order form is not yet known. Agents that can modulate the aggregation state of Abeta could resolve this controversy by facilitating our understanding of the consequences of aggregation and its underlying mechanism. To date, however, ligands that bind to specific forms of Abeta have not been identified. To address this deficiency, we tested whether phage display could yield such ligands by screening libraries against Abeta in two different states: monomeric or highly aggregated. Intriguingly, the peptides selected had different effects on Abeta aggregation. Peptides selected for binding to monomeric Abeta did not perturb aggregation, but those selected using highly aggregated Abeta increase the rate of aggregation drastically. The latter also alter the morphology of the resulting aggregate. The ability of a peptide to promote aggregation correlated with its affinity for the N-terminal 10 residues of Abeta. This result indicates that the mechanism by which the peptides influence aggregation is related to their affinity for the Abeta N-terminus. Thus, the identification of compounds that bind to this Abeta section can afford agents that affect aggregation. Moreover, the data suggest that endogenous ligands that interact with the N-terminal region can influence the propensity of Abeta to form aggregates and the morphology of those that form. Our data highlight the utility of phage display for identifying ligands that bind to target proteins in different states, and they indicate that such agents can be used to perturb protein aggregation.

Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins?

Protein fibrillization is implicated in the pathogenesis of most, if not all, age-associated neurodegenerative diseases, but the mechanism(s) by which it triggers neuronal death is unknown. Reductionist in vitro studies suggest that the amyloid protofibril may be the toxic species and that it may amplify itself by inhibiting proteasome-dependent protein degradation. Although its pathogenic target has not been identified, the properties of the protofibril suggest that neurons could be killed by unregulated membrane permeabilization, possibly by a type of protofibril referred to here as the 'amyloid pore'. The purpose of this review is to summarize the existing supportive circumstantial evidence and to stimulate further studies designed to test the validity of this hypothesis.

Design of a mimic of nonamyloidogenic and bioactive human islet amyloid polypeptide (IAPP) as nanomolar affinity inhibitor of IAPP cytotoxic fibrillogenesis.

Protein aggregation into cytotoxic oligomers and fibrils in vivo is linked to cell degeneration and the pathogenesis of >25 uncurable diseases, whereas the high aggregation propensity and insolubility of several bioactive polypeptides and proteins in vitro prevent their therapeutic use. Aggregation of human islet amyloid polypeptide (IAPP) into pancreatic amyloid is strongly associated with the pathogenesis of type II diabetes. IAPP is a 37-residue polypeptide that acts as a neuroendocrine regulator of glucose homeostasis. However, IAPP misfolds and self-associates into cytotoxic aggregates and fibrils even at nanomolar concentrations. Because IAPP aggregation causes beta-cell death and prohibits therapeutic application of IAPP in diabetes, we pursued a minimalistic chemical design approach to generate a molecular mimic of a nonamyloidogenic and bioactive IAPP conformation that would still be able to associate with IAPP and thus inhibit its fibrillogenesis and cytotoxicity. We show that the double N-methylated full length IAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI) is a highly soluble, nonamyloidogenic, and noncytotoxic IAPP molecular mimic and an IAPP receptor agonist. Moreover, IAPP-GI binds IAPP with low nanomolar affinity and completely blocks IAPP cytotoxic self-assembly and fibrillogenesis with activity in the low nanomolar concentration range. Importantly, IAPP-GI dissociates cytotoxic IAPP oligomers and fibrils and is able to reverse their cytotoxicity. Bifunctional soluble IAPP mimics that combine bioactivity with the ability to block and reverse IAPP cytotoxic self-assembly are promising candidates for the treatment of diabetes. Moreover, our amyloid disease inhibitor design concept may be applicable to other protein aggregation diseases.

Autophagy is a prosurvival mechanism in cells expressing an autosomal dominant familial neurohypophyseal diabetes insipidus mutant vasopressin transgene

Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a progressive, inherited neurodegenerative disorder that presents as polydipsia and polyuria as a consequence of a loss of secretion of the antidiuretic hormone vasopressin (VP) from posterior pituitary nerve terminals. VP gene mutations cause adFNDI. Rats expressing an adFNDI VP transgene (Cys67stop) show a neuronal pathology characterized by autophagic structures in the cell body. adFNDI has thus been added to the list of protein aggregation diseases, along with Alzheimer's, Parkinson's and Huntington's, which are associated with autophagy, a bulk process that delivers regions of cytosol to lysosomes for degradation. However, the role of autophagy in these diseases is unclear. To address the relationships between mutant protein accumulation, autophagy, cell survival, and cell death, we have developed a novel and tractable in vitro system. We have constructed adenoviral vectors (Ads) that express structural genes encoding either the Cys67stop mutant protein (Ad-VCAT-Cys67stop) or an epitope-tagged wild-type VP precursor (Ad-VCAT). After infection of mouse neuroblastoma Neuro2a cells, Ad-VCAT encoded material enters neurite processes and accumulates in terminals, while the Cys67stop protein is confined to enlarged vesicles in the cell body. Similar to the intracellular derangements seen in the Cys67stop rats, these structures are of ER origin, and colocalize with markers of autophagy. Neither Ad-VCAT-Cys67stop nor Ad-VCAT expression affected cell viability. However, inhibition of autophagy or lysosomal protein degradation, while having no effect on Ad-VCAT-expressing cells, significantly increased apoptotic cell death following Ad-VCAT-Cys67stop expression. These data suggest that activation of autophagy by the stress of the expression of an adFNDI mutant protein is a prosurvival mechanism.

Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli.

We used preS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43 degrees C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S'-beta-galactosidase aggregation at 43 degrees C. Overexpression of PIMT reduces the amount of preS2-S'-beta-galactosidase found in inclusion bodies at 43 degrees C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S'-beta-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.

Axonal transport defects: a common theme in neurodegenerative diseases

A core pathology central to most neurodegenerative diseases is the misfolding, fibrillization and aggregation of disease proteins to form the hallmark lesions of specific disorders. The mechanisms underlying these brain-specific neurodegenerative amyloidoses are the focus of intense investigation and defective axonal transport has been hypothesized to play a mechanistic role in several neurodegenerative disorders; however, this hypothesis has not been extensively examined. Discoveries of mutations in human genes encoding motor proteins responsible for axonal transport do provide direct evidence for the involvement of axonal transport in neurodegenerative diseases, and this evidence is supported by studies of animal models of neurodegeneration. In this review, we summarize recent findings related to axonal transport and neurodegeneration. Focusing on specific neurodegenerative diseases from a neuropathologic perspective, we highlight discoveries of human motor protein mutations in some of these diseases, as well as illustrate new insights from animal models of neurodegenerative disorders. We also review the current understanding of the biology of axonal transport including major recent findings related to slow axonal transport.

Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone

Aggregation of proteins in tissues is associated with several diseases, including Alzheimer's disease. It is characterized by the accumulation of amyloid beta peptide (Abeta) in the extracellular spaces of the brain cells, resulting in neuronal death and other pathological changes. alpha-Crystallin, a small heat-shock protein in lens, and a peptide chaperone having the functional site sequence DFVIFLDVKHFSPEDLTVK of alphaA-crystallin may inhibit Abeta fibrillogenesis and toxicity. The peptide chaperone (mini-alphaA-crystallin), having an Abeta interacting domain and a complex solubilizing domain, was shown in previous studies to prevent aggregation of several proteins under denaturing conditions. In this in vitro study, using transmission electron microscopy and thioflavin T binding assay, we show that mini-alphaA-crystallin arrests the fibril formation of Abeta peptides. Mini-alphaA-crystallin also suppresses the toxic action of Abeta on rat pheochromocytoma (PC 12) cells. The wide chaperoning capability of the peptide and its ability to inhibit amyloid fibril formation and suppress toxicity suggest that mini-alphaA-crystallin may serve as a universal chaperone in controlling diseases of protein aggregation, including Alzheimer's disease.

Protein aggregation and neurodegenerative disease.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a beta-sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.

The peripheral nervous system and the pathogenesis of prion diseases.

Prion diseases are inevitably fatal neurodegenerative conditions which affect humans and a wide variety of animals. Unlike other protein aggregation diseases such as Alzheimer's, Parkinson's, and polyglutamine repeat diseases, prion diseases are unique in that they are transmissible. Therefore, prion diseases are also called transmissible spongiform encephalopathies. A number of prion diseases are caused by peripheral uptake of the infectious agent. In order to reach their target, the central nervous system, prions enter their host, accumulate and replicate in lymphoid organs, and eventually spread to the central nervous system via peripheral nerves. Once the agent has reached the central nervous system, disease progression is rapid, resulting in neurodegeneration and death. In this article, we review the state of knowledge on the routes of neuroinvasion used by the infectious agent in order to gain access to the central nervous system upon entry into extracerebral sites.

Distinct aggregation and cell death patterns among different types of primary neurons induced by mutant huntingtin protein.

Aggregation of disease proteins is believed to be a central event in the pathology of polyglutamine diseases, whereas the relationship between aggregation and neuronal death remains controversial. We investigated this question by expressing mutant huntingtin (htt) with a defective adenovirus in different types of neurons prepared from rat cerebral cortex, striatum or cerebellum. The distribution pattern of inclusions is not identical among different types of primary neurons. On day 2 after infection, cytoplasmic inclusions are dominant in cortical and striatal neurons, whereas at day 4 the ratio of nuclear inclusions overtakes that of cytoplasmic inclusions. Meanwhile, nuclear inclusions are always predominantly present in cerebellar neurons. The percentage of inclusion-positive cells is highest in cerebellar neurons, whereas mutant htt induces cell death most remarkably in cortical neurons. As our system uses htt exon 1 protein and thus aggregation occurs independently from cleavage of the full-length htt, our observations indicate that the aggregation process is distinct among different neurons. Most of the neurons containing intracellular (either nuclear or cytoplasmic) aggregates are viable. Our findings suggest that the process of mutant htt aggregation rather than the resulting inclusion body is critical for neuronal cell death.

Caspase 8 mediated apoptotic cell death induced by β-sheet forming polyalanine peptides

Expansion of a polyalanine stretch from 10 to 12–17 residues in the N-terminus of the protein PABP2 has been implicated in the genetically acquired disease oculopharyngeal muscular dystrophy, characterized by nuclear protein deposits. Here we report a correlation between the structural properties and cell toxicity of two peptides mimicking the N-terminal domain of PABP2: one containing seven and the other 11 uninterrupted alanine residues. Consistent with earlier observations, the longer peptide (11-ala) was found to adopt β-sheet structure while the shorter one (7-ala) formed α-helix over a wide range of concentrations (∼20–500 μM). We observed that treatment with 11-ala resulted in significantly enhanced death of Chinese hamster V79 cells, compared to the effect of treatment with 7-ala, via the cytochrome c mediated apoptotic pathway. Increases in caspase 8 and caspase 3 activity were also observed in human cells (K562) treated with 11-ala. These results indicate that the toxicity of pathogenic peptides is directly linked to their β-sheet structure and also support recent observations that small oligomeric species of peptides and proteins are the key toxic elements in causing protein aggregation diseases.

Abeta protofibrils possess a stable core structure resistant to hydrogen exchange.

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease.

Structure of the Ubiquitin-associated Domain of p62 (SQSTM1) and Implications for Mutations That Cause Paget's Disease of Bone

The p62 protein (also known as SQSTM1) mediates diverse cellular functions including control of NFkappaB signaling and transcriptional activation. p62 binds non-covalently to ubiquitin and co-localizes with ubiquitylated inclusions in a number of human protein aggregation diseases. Mutations in the gene encoding p62 cause Paget's disease of bone (PDB), a common disorder of the elderly characterized by excessive bone resorption and formation. All of the p62 PDB mutations identified to date cluster within the C-terminal region of the protein, which shows low sequence identity to previously characterized ubiquitin-associated (UBA) domains. We report the first NMR structure of a recombinant polypeptide that contains the C-terminal UBA domain of the human p62 protein (residues 387-436). This sequence, which confers multiubiquitin chain binding, forms a compact three-helix bundle with a structure analogous to the UBA domains of HHR23A but with differences in the loop regions connecting helices that may be involved in binding accessory proteins. We show that the Pro392 --> Leu PDB substitution mutation modifies the structure of the UBA domain by extending the N terminus of helix 1. In contrast to the p62 PDB deletion mutations that remove the UBA domain and ablate multiubiquitin chain binding, the Pro392 --> Leu substitution does not affect interaction of the UBA domain with multiubiquitin chains. Thus, phenotypically identical substitution and deletion mutations do not appear to predispose to PDB through a mechanism dependent on a common loss of ubiquitin chain binding by p62.

Time scale of protein aggregation dictated by liquid-liquid demixing.

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point-mutant form of normal human Hemoglobin (HbA), is the first recognized and best-studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy-HbS aggregates by referring them to the (concentration-dependent) temperature T(s) characterizing the occurrence of the phase transition of liquid-liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter epsilon = (T - T(s)) / T(s), familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter-protein interactions leading to nucleation. Referring to epsilon shows how in a narrow temperature span, that is at T - T(s), nucleation kinetics can undergo orders-of-magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering T(s) and consequently epsilon. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases.

Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions.

For many decades, clinicians have been aware of the formation of insoluble protein aggregates in particular diseases. For example, in sickle cell disease, the polymerization of sickle hemoglobin into fibrils in the deoxygenating environment of the microvasculature causes sickling of red cells and vaso-occlusion (1). In the most common form of α1-antitrypsin deficiency (see Perlmutter, this Perspective series, ref. 2), the presence of α1-antitrypsin inclusions in the endoplasmic reticulum (ER) of hepatocytes is associated with deficient release of the enzyme into the circulation, placing both the inclusion-laden liver and the antitrypsin-deficient lung at risk for damage (3). In Alzheimer disease (see Selkoe, this series, ref. 4), the presence in the CNS of β-amyloid–containing plaques is associated with neurodegeneration and dementia (5). Similarly, other neurodegenerative diseases have recently been discovered to involve protein aggregation. For example, prion diseases such as Creutzfeldt-Jacob disease and bovine spongiform encephalopathy are associated with amyloid deposits of the PrP protein (6). Polyglutamine repeat diseases such as Huntington disease are likewise associated with neuronal cytosolic and intranuclear inclusions (7). These inclusions are composed of fibrils that stain similarly to amyloid (8). Finally, in Parkinson disease, inclusions known as Lewy bodies, found in the cytoplasm of cells of the basal ganglia, include amyloid-like aggregates of the protein α-synuclein (9, 10).

Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions

Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions

Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions

Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions

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.

Abnormal protein aggregation and neurodegenerative diseases

Abnormal protein aggregation or amyloid is the major cause of many neurodegenerative disorders. The present review focuses on the correlation between sequence and structure features of proteins related to the diseases and abnormal protein aggregation. Recent progress has improved our knowledge on understanding the mechanism of amyloid formation. We suggest a nucleation model for ordered protein aggregation, which can also explain pathogenesis mechanisms of these neurodegenerative diseasesin vivo.

8] Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein

Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. When considering presenting an antigen to a mouse with the goal of obtaining specific monoclonal antibodies against a misfolded or aggregated form of a host protein, it is desirable to increase the definition of a subtle conformational difference. This can be achieved by immunizing an antigen knockout mouse that has not developed self-tolerance against the respective antigen. Furthermore, if conformational isoforms and/or oligomeric forms of a protein sequence are understood to exist in an equilibrium, high and pure amounts of recombinant protein may increase the likelihood that a particular population of protein conformation passes an antigenic threshold necessary to start an immunogenic response. Pulling out the monoclonal antibodies by correct screening is essential. Screening against the pure misfolded or aggregated protein is often complicated by its poor solubility and hence the ability to immobilize. In the present case, immobilization of disease-associated PrP on nitrocellulose had been established as a conformation-sensitive screening method, allowing to "freeze" PrP in its distinguishable, disease-associated conformation. We are cautious to generalize conclusions of how to assess the generation of monoclonal antibodies against these particular protein isoforms to other diseases of protein misfolding and/or aggregation, but ultimately the present approach may inspire respective experiments.