Robin Roychaudhuri

Amyloid β-protein assembly and Alzheimer disease

The biochemistry of amyloid proteins has been a fascinating and important area of research because of its contribution to our understanding of protein folding dynamics and assembly and of the pathogenetic mechanisms of human disease. One such disease is AD,2 the most common neurodegenerative disorder of aging. In AD, Aβ (Fig. 1A), which is expressed normally and ubiquitously throughout life as a 40–42-residue peptide, forms fibrils that deposit in the brain as “amyloid plaques.” This pathologic deposition process led researchers to investigate fibril formation as a target for therapeutic intervention. In doing so, an increasing number of fibril precursors and non-fibrillar Aβ assemblies have been identified, the majority of which are neurotoxic. These findings have altered prevailing fibril-centered views of the pathobiology of amyloid diseases (1) and intensified efforts to understand the early folding and assembly dynamics of Aβ. In the discussion that follows, we seek to introduce the reader to the complex world of Aβ assembly and biological activity, a goal we hope will provide a conceptual framework upon which further knowledge or experimentation may be built. FIGURE 1. Aβ assembly. A, the sequence of Aβ42 is shown in one-letter amino acid code. The side chain charge at neutral pH is color-coded (red, negative; blue, positive). B, nucleation-dependent polymerization, reflecting the unfavorable self-association ...

Mechanism of amyloid β−protein dimerization determined using single−molecule AFM force spectroscopy

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β−protein (Aβ). The two additional C−terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single−molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C−termini, the N−terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N−terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N−terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

Amyloid β-Protein Assembly: Differential Effects of the Protective A2T Mutation and Recessive A2V Familial Alzheimer's Disease Mutation

Oligomeric states of the amyloid β-protein (Aβ) appear to be causally related to Alzheimers disease (AD). Recently, two familial mutations in the amyloid precursor protein gene have been described, both resulting in amino acid substitutions at Ala2 (A2) within Aβ. An A2V mutation causes autosomal recessive early onset AD. Interestingly, heterozygotes enjoy some protection against development of the disease. An A2T substitution protects against AD and age-related cognitive decline in non-AD patients. Here, we use ion mobility-mass spectrometry (IM-MS) to examine the effects of these mutations on Aβ assembly. These studies reveal different assembly pathways for early oligomer formation for each peptide. A2T Aβ42 formed dimers, tetramers, and hexamers, but dodecamer formation was inhibited. In contrast, no significant effects on Aβ40 assembly were observed. A2V Aβ42 also formed dimers, tetramers, and hexamers, but it did not form dodecamers. However, A2V Aβ42 formed trimers, unlike A2T or wild-type (wt) Aβ42. In addition, the A2V substitution caused Aβ40 to oligomerize similar to that of wt Aβ42, as evidenced by the formation of dimers, tetramers, hexamers, and dodecamers. In contrast, wt Aβ40 formed only dimers and tetramers. These results provide a basis for understanding how these two mutations lead to, or protect against, AD. They also suggest that the Aβ N-terminus, in addition to the oft discussed central hydrophobic cluster and C-terminus, can play a key role in controlling disease susceptibility.

Structural dynamics of the amyloid β-protein monomer folding nucleus

Alzheimers disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.

A Critical Role of Ser26 Hydrogen Bonding in Aβ42 Assembly and Toxicity

Amyloid β-protein (Aβ) assembly is a seminal process in Alzheimers disease. Elucidating the mechanistic features of this process is thought to be vital for the design and targeting of therapeutic agents. Computational studies of the most pathologic form of Aβ, the 42-residue Aβ42 peptide, have suggested that hydrogen bonding involving Ser26 may be particularly important in organizing a monomer folding nucleus and in subsequent peptide assembly. To study this question, we experimentally determined structure-activity relationships among Aβ42 peptides in which Ser26 was replaced with Gly, Ala, α-aminobutryic acid (Abu), or Cys. We observed that aliphatic substitutions (Ala and Abu) produced substantially increased rates of formation of β-sheet, hydrophobic surface, and fibrils, and higher levels of cellular toxicity. Replacement of the Ser hydroxyl group with a sulfhydryl moiety (Cys) did not have these effects. Instead, this peptide behaved like native Aβ42, even though the hydropathy of Cys was similar to that of Abu and very different from that of Ser. We conclude that H bonding of Ser26 is the factor most important in its contribution to Aβ42 conformation, assembly, and subsequent toxicity.

Misfolding and interactions of Aß proteins: Insight from single molecule experiments and computational analyses

Background The current model for the development of Alzheimer’s (AD), Parkinson’s, Huntington’s, prion, and other neurodegenerative diseases involves protein misfolding as the early step followed by spontaneous aggregation, with specific proteins identified as the primary initiators for disease development. Therefore, elucidating the properties of the disease-prone misfolded states, understanding the mechanism of their formation, and identification of their most toxic forms will open prospects for the development of early diagnostics and specific therapeutics for these diseases.

Structural Determinants of Amyloid B-Protein Oligomerization

The oligomerization of amyloid β-protein (Aβ) is a seminal event in the neurodegenerative process in Alzheimers disease (AD). Aβ40 and Aβ42, the two predominant forms of Aβ, display different aggregation behavior which underlies the special pathogenetic significance of Aβ42. Previous computational studies have revealed that a turn-like structure exists at the C-terminal of Aβ42 that is not observed in Aβ40. We report here results of studies to define the structure of this turn and to establish its role in Aβ assembly. We used molecular dynamics to simulate the structure of the Aβ C-terminus (Gly31-Val40/Ala42) and discovered that the dihedral angles of residues 36 and 37 tend to be locked into a region of Ramachanran space consistent with type-II β-turns. Aβ(31-42) predominantly formed a β-hairpin-like structure that was stabilized by hydrogen bonds and hydrophobic interactions between residues 31-35 and residues 38-42. In contrast, Aβ(31-40) appeared relatively unstructured. To investigate the possible role of this peptide-specific, β-hairpin-like structure in Aβ assembly, we synthesized a number of Aβ “mutants” containing amino acid substitutions that we postulated would stabilize or destabilize the hairpin. The stabilizing substitutions facilitated hexamer and dodecamer formation by Aβ42, abolishing formation of fibrils. Interestingly, compared to wild type Aβ42, these substituted peptides were equally toxic. When these substitutions were incorporated into Aβ40, the modified Aβ40 oligomerized like Aβ42, instead of an “Aβ40-like” distribution. In addition, the modified Aβ40 was significantly more toxic than wild type Aβ40. Substitutions in Aβ42 that were predicted to destabilize the turn abolished hexamer and dodecamer formation and resulted in an Aβ42 oligomer size distribution similar to that of Aβ40. Our experiments appear to define the structural determinant that “makes Aβ42 Aβ42.” If true, this structure would be an exceptionally important therapeutic target.