Avijit Chakrabartty

STABILITY OF ALPHA -HELICES

Publisher Summary This chapter focuses on the mechanism of helix formation in an isolated peptide and the factors that determine the stability of a peptide helix. Helix propensities are considered together with N-cap and C-cap propensities, because measurement of helix propensities requires knowing values of the N-cap and C-cap propensities, and vice versa. The chapter considers side-chain interactions: these include both the interaction of a charged side chain with the helix macrodipole and specific interactions between a particular pair of side chains, such as ion pair and H-bond interactions. Measurement of these interactions is of interest for two reasons: their values are needed to relate the stability of a peptide helix to its amino acid composition and sequence; and peptide helices provide one of the best systems, and probably the most sensitive system, for quantifying the energetics of side-chain interactions. It also considers briefly the present status of the Chou-Fasman hypothesis and the relation between the mechanism of α-helix formation in peptides and proteins. It is necessary to use helix-coil transition theory to understand the populated intermediates and to analyze the energetics of helix formation. The two closely related theories of α-helix formation are the Zimm-Bragg theory and the Lifson-Roig theory.

An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.

Manipulating the amyloid-beta aggregation pathway with chemical chaperones.

Amyloid-β (Aβ) assembly into fibrillar structures is a defining characteristic of Alzheimers disease that is initiated by a conformational transition from random coil to β-sheet and a nucleation-dependent aggregation process. We have investigated the role of organic osmolytes as chemical chaperones in the amyloid pathway using glycerol to mimic the effects of naturally occurring molecules. Osmolytes such as the naturally occurring trimethylamine N-oxide and glycerol correct folding defects by preferentially hydrating partially denatured proteins and entropically stabilize native conformations and polymeric states. Trimethylamine N-oxide and glycerol were found to rapidly accelerate the Aβ random coil-to-β-sheet conformational change necessary for fiber formation. This was accompanied by an immediate conversion of amorphous unstructured aggregates into uniform globular and possibly nucleating structures. Osmolyte-facilitated changes in Aβ hydration also affected the final stages of amyloid formation and mediated transition from the protofibrils to mature fibers that are observed in vivo. These findings suggest that hydration forces can be used to control fibril assembly and may have implications for the accumulation of Aβ within intracellular compartments such as the endoplasmic reticulum and in vitro modeling of the amyloid pathway.

Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form

Amyotrophic lateral sclerosis (ALS) is a conformational disease in which misfolding and aggregation of proteins such as SOD1 (familial ALS) and TDP-43 (sporadic ALS) are central features. The conformations adopted by such proteins within motor neurons in affected patients are not well known. We have developed a novel conformation-specific antibody (USOD) targeted against SOD1 residues 42–48 that specifically recognizes SOD1 in which the beta barrel is unfolded. Use of this antibody, in conjunction with the previously described SEDI antibody that recognizes the SOD1 dimer interface, allows a detailed investigation of the in vivo conformation of SOD1 at the residue-specific level. USOD and SEDI immunohistochemistry of spinal cord sections from ALS cases resulting from SOD1 mutations (A4V and ΔG27/P28) shows that inclusions within remaining motor neurons contain SOD1 with both an unfolded beta barrel and a disrupted dimer interface. Misfolded SOD1 can also be immunoprecipitated from spinal cord extracts of these cases using USOD. However, in ten cases of sporadic ALS, misfolded SOD1 is not detected by either immunohistochemistry or immunoprecipitation. Using the amyloid-specific dyes, Congo Red and Thioflavin S, we find that SOD1-positive inclusions in familial ALS, as well as TDP-43- and ubiquitin-positive inclusions in sporadic ALS, contain non-amyloid protein deposits. We conclude that SOD1 misfolding is not a feature of sporadic ALS, and that both SOD1-ALS and sporadic ALS, rather than being amyloid diseases, are conformational diseases that involve amorphous aggregation of misfolded protein. This knowledge will provide new insights into subcellular events that cause misfolding, aggregation and toxicity.

Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrPSc). Exposure to PrPSc from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer–octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide. An in vitro model of preamyloid.

Deposition of amyloid-β (Aβ) aggregates in the brain is a defining characteristic of Alzheimers disease (AD). Fibrillar amyloid, found in the cores of senile plaques, is surrounded by dystrophic neurites. In contrast, the amorphous Aβ (also called preamyloid) in diffuse plaques is not associated with neurodegeneration. Depending on the conditions, Aβ will also form fibrillar or amorphous aggregates in vitro. In this present study, we sought to characterize the properties of the amorphous aggregate and determine whether we could establish an in vitro model for amorphous Aβ. CD data indicated that Aβ40 assembled to form either a β-structured aggregate or an unfolded aggregate with the structured aggregate forming at high peptide concentrations and the unstructured aggregate forming at low Aβ40 levels. The critical concentration separating these two pathways was 10 μm. Fluorescence emission and polarization showed the structured aggregate was tightly packed containing peptides that were not accessible to water. Peptides in the unstructured aggregate were loosely packed, mobile, and accessible to water. When examined by electron microscopy, the structured aggregate appeared as protofibrillar structures and formed classic amyloid fibrils over a period of several weeks. The unstructured aggregate was not visible by electron microscopy and did not generate fibrils. These findings suggest that the unstructured aggregate shares many properties with the amorphous Aβ of AD and that conditions can be established to form amorphous Aβ in vitro. This would allow for investigations to better understand the relationship between fibrillar and amorphous Aβ and could have significant impact upon efforts to find therapies for AD.

Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants.

BackgroundAmyloid precursor protein (APP) is enzymatically cleaved by γ-secretase to form two peptide products, either Aβ40 or the more neurotoxic Aβ42. The Aβ42/40 ratio is increased in many cases of familial Alzheimers disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.ResultsUsing synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.ConclusionThe dissociation constants are correlated to both the Aβ42/Aβ40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Aβ production and Aβ42/Aβ40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and γ-secretase components, potentially revealing a new class of γ-secretase inhibitors.

Protein misfolding in the late-onset neurodegenerative diseases: common themes and the unique case of amyotrophic lateral sclerosis.

Enormous strides have been made in the last 100 years to extend human life expectancy and to combat the major infectious diseases. Today, the major challenges for medical science are age‐related diseases, including cancer, heart disease, lung disease, renal disease, and late‐onset neurodegenerative disease. Of these, only the neurodegenerative diseases represent a class of disease so poorly understood that no general strategies for prevention or treatment exist. These diseases, which include Alzheimers disease, Parkinsons disease, Huntingtons disease, the transmissible spongiform encephalopathies, and amyotrophic lateral sclerosis (ALS), are generally fatal and incurable. The first section of this review summarizes the diversity and common features of the late‐onset neurodegenerative diseases, with a particular focus on protein misfolding and aggregation—a recurring theme in the molecular pathology. The second section focuses on the particular case of ALS, a late‐onset neurodegenerative disease characterized by the death of central nervous system motor neurons, leading to paralysis and patient death. Of the 10% of ALS cases that show familial inheritance (familial ALS), the largest subset is caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). The unusual kinetic stability of SOD1 has provided a unique opportunity for detailed structural characterization of conformational states potentially involved in SOD1‐associated ALS. This review discusses past studies exploring the stability, folding, and misfolding behavior of SOD1, as well as the therapeutic possibilities of using detailed knowledge of misfolding pathways to target the molecular mechanisms underlying ALS and other neurodegenerative diseases. Proteins 2013; 81:1285–1303.

Amyloid β-protein (Aβ) associated with lipid molecules: immunoreactivity distinct from that of soluble Aβ

We previously identified a novel amyloid β‐protein (Aβ), that binds to GM1 ganglioside, in brains exhibiting the early pathological changes of AD. In this study, we raised monoclonal antibodies, using membrane fractions containing abundant GM1 ganglioside‐bound Aβ as antigens. Monoclonal antibody 4396, produced in this study, immunoprecipitates Aβ42 in the membrane fractions of brains with diffuse plaques, but does not react with soluble Aβ42 or GM1 ganglioside. Furthermore, this antibody recognizes the Aβ bound to lipid vesicles containing GM1 ganglioside, and unexpectedly, phosphatidylinositol. In contrast, a control anti‐Aβ monoclonal antibody does not recognize the Aβ bound to these lipid vesicles. These results indicate that Aβ associated with lipids has an immunoreactivity distinct from that of soluble A.

Targeting of Monomer/Misfolded SOD1 as a Therapeutic Strategy for Amyotrophic Lateral Sclerosis

There is increasing evidence that toxicity of mutant superoxide dismutase-1 (SOD1) in amyotrophic lateral sclerosis (ALS) is linked to its propensity to misfold and to aggregate. Immunotargeting of differently folded states of SOD1 has provided therapeutic benefit in mutant SOD1 transgenic mice. The specific region(s) of the SOD1 protein to which these immunization approaches target are, however, unknown. In contrast, we have previously shown, using a specific antibody [SOD1 exposed dimer interface (SEDI) antibody], that the dimer interface of SOD1 is abnormally exposed both in mutant SOD1 transgenic mice and in familial ALS cases associated with mutations in the SOD1 gene (fALS1). Here, we show the beneficial effects of an active immunization strategy using the SEDI antigenic peptide displayed on a branched peptide dendrimer to target monomer/misfolded in SOD1G37R and SOD1G93A mutant SOD1 transgenic mice. Immunization delayed disease onset and extended disease duration, with survival times increased by an average of 40 d in SOD1G37R mice. Importantly, this immunization strategy favored a Th2 immune response, thereby precluding deleterious neuroinflammatory effects. Furthermore, the beneficial effects of immunization correlated with a reduction in accumulation of both monomer/misfolded and oligomeric SOD1 species in the spinal cord, the intended targets of the immunization strategy. Our results support that SOD1 misfolding/aggregation plays a central role in SOD1-linked ALS pathogenesis and identifies monomeric/misfolded SOD1 as a therapeutic target for SOD1-related ALS.