Functional Amyloid Formation within Mammalian Tissue

Amyloid is a generally insoluble, fibrous cross-β sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin—a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology

Identification and nanomechanical characterization of the fundamental single-strand protofilaments of amyloid α-synuclein fibrils.

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments' internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly

Rescuing defective vesicular trafficking protects against alpha-synuclein toxicity in cellular and animal models of Parkinson's disease

Studies in yeast are providing critical insights into the mechanisms of neurodegeneration in Parkinson's disease (PD). A recent study shows that disruption of vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi, caused by the overexpression and/or aggregation of alpha-synuclein, is linked to degeneration of dopamine neurons. Overexpression of proteins that are known to enhance ER-to-Golgi transport rescue defective trafficking in yeast, worm, fly, and cellular models of PD

Pathological relevance of post-translationally modified alpha-synuclein (pSer87, pSer129, nTyr39) in idiopathic Parkinson’s disease and Multiple System Atrophy

Aggregated alpha-synuclein (a-synuclein) is the main component of Lewy bodies (LBs), Lewy neurites (LNs), and glial cytoplasmic inclusions (GCIs), which are pathological hallmarks of idiopathic Parkinson’s disease (IPD) and multiple system atrophy (MSA), respectively. Initiating factors that culminate in forming LBs/LNs/GCIs remain elusive. Several species of a-synuclein exist, including phosphorylated and nitrated forms. It is unclear which a-synuclein post-translational modifications (PTMs) appear within aggregates throughout disease pathology. Herein we aimed to establish the predominant a-synuclein PTMs in post-mortem IPD and MSA pathology using immunohistochemistry. We examined the patterns of three a-synuclein PTMs (pS87, pS129, nY39) simultaneously in pathology- affected regions of 15 PD, 5 MSA, 6 neurologically normal controls. All antibodies recognized LBs, LNs, and GCIs, albeit to a variable extent. pS129 a-synuclein antibody was particularly immunopositive for LNs and synaptic dot-like structures followed by nY39 a- synuclein antibody. GCIs, neuronal inclusions, and small threads were positive for nY39 a- synuclein in MSA. Quantification of the LB scores revealed that pS129 a-synuclein was the dominant and earliest a-synuclein PTM followed by nY39 a-synuclein, while lower amounts of pSer87 a-synuclein appeared later in disease progression in PD. These results may have implications for novel biomarker and therapeutic developments

A century-old debate on protein aggregation and neurodegeneration enters the clinic

The correlation between neurodegenerative disease and protein aggregation in the brain has long been recognized, but a causal relationship has not been unequivocally established, in part because a discrete pathogenic aggregate has not been identified. The complexity of these diseases and the dynamic nature of protein aggregation mean that, despite progress towards understanding aggregation, its relationship to disease is difficult to determine in the laboratory. Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered presymptomatically, drastically reduce the incidence of these diseases. The clinical trials could also settle the century-old debate about causality

Molecular electron microscopy approaches to elucidating the mechanisms of protein fibrillogenesis

Electron microscopy (EM) has played a central role in our current understanding of the mechanisms underlying the pathogenesis of several amyloid diseases, including Alzheimer's disease, Parkinson's disease, and prion diseases. In this chapter, we discuss the application of various EM techniques to monitor and characterize quaternary structural changes during amyloid fibril formation in vitro and the potential of extending some of these techniques to characterizing ex vivo material. In particular, we would like to bring to the attention of the reader two very powerful molecular EM techniques that remain under utilized by researchers in the amyloid community, namely scanning transmission electron microscopy and single particle molecular averaging EM. An overview of the strength and limitations of these techniques as tools for elucidating the structural basis of amyloid fibril formation will be presented

Discovery of a novel aggregation domain in the huntingtin protein: implications for the mechanisms of Htt aggregation and toxicity

Aggravating aggregation: an N-terminal domain that is in close proximity to the polyQ domain in the huntingtin protein, htt105-138, is shown to be highly aggregation prone. Potential cross-talk between this domain and the polyQ region may play a central role in regulating the aggregation and toxicity of Htt-N-terminal fragments

Amyloids go genomic: insights regarding the sequence determinants of prion formation from genome-wide studies

[Image: see text] The availability of fully sequenced genomes provides a useful starting point for identifying putative amyloid and prion forming sequences through genome-wide scans. With an inventory in hand, one can assess the amyloid forming potential and the functional consequences of amyloid formation for each sequence. Thus, advancing our understanding of how cells process and utilize deleterious and functional aggregates, respectively

Molecular Structure and Dimeric Organization of the Notch Extracellular Domain as Revealed by Electron Microscopy

Background: The Notch receptor links cell fate decisions of one cell to that of the immediate cellular neighbor. In humans, malfunction of Notch signaling results in diseases and congenital disorders. Structural information is essential for gaining insight into the mechanism of the receptor as well as for potentially interfering with its function for therapeutic purposes. Methodology/Principal Findings: We used the Affinity Grid approach to prepare specimens of the Notch extracellular domain (NECD) of the Drosophila Notch and human Notch1 receptors suitable for analysis by electron microscopy and three-dimensional (3D) image reconstruction. The resulting 3D density maps reveal that the NECD structure is conserved across species. We show that the NECD forms a dimer and adopts different yet defined conformations, and we identify the membrane-proximal region of the receptor and its ligand-binding site. Conclusions/Significance: Our results provide direct and unambiguous evidence that the NECD forms a dimer. Our studies further show that the NECD adopts at least three distinct conformations that are likely related to different functional states of the receptor. These findings open the way to now correlate mutations in the NECD with its oligomeric state and conformation