Amyloid fibrils from the viewpoint of protein folding

In amyloid related diseases, proteins form fibrillar aggregates with highly ordered -sheet structure regardless of their native conformations. Formation of such amyloid fibrils can be reproducible in vitro using isolated proteins/peptides, suggesting that amyloid fibril formation takes place as a result of protein conformational change. In vitro studies revealed that perturbation of the native structure is important for the fibril formation, and it is suggested that the mechanisms of amyloid fibril formation share the mechanisms of protein folding. In particular, amyloid fibril formation is similar to one of the common features of proteins, i.e. amorphous aggregation upon partial unfolding, which is likely driven by hydrophobic interactions through exposed protein interior. However, these molecular associations are distinct phenomena, and identifying factors that lead to amyloid fibril formation would precede our understanding of the mechanisms of amyloid fibrillization. The necessity of understanding the nature of protein denatured states is also suggested.Received 6 July 2003; accepted 19 August 2003     

Biological activity and pathological implications of misfolded proteins

The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, β2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999     

Proteolytic generation and aggregation of peptides from transmembrane regions: lung surfactant protein C and amyloid β-peptide

The formation of amyloid fibrils is associated with several devastating diseases in humans and animals, including e.g. Alzheimers disease (AD) and the spongiform encephalopathies. Here, we review and discuss the current knowledge on two amyloid peptides: lung surfactant protein C (SP-C) and the amyloid -peptide (A), implicated in human lung disease and in AD, respectively. Both these hydrophobic peptides are derived from the transmembrane region of their precursor protein, and can transit from a monomeric -helical state to a -sheet fibril. The helices of SP-C and A are composed of amino acid residues with inherently higher propensities for strand than helix conformation. Their helical states are stabilized by a membrane environment, and loss of membrane association thus promotes structural conversion and fibril formation. We speculate that the loss of structural context for sequences with a high propensity for formation of sheets may be a common feature of amyloid formation in general.Received 9 July 2003; received after revision 15 August 2003; accepted 3 September 2003     

Cerebral amyloidosis: amyloid subunits, mutants and phenotypes

Cerebral amyloid diseases are part of a complex group of chronic and progressive entities bracketed together under the common denomination of protein folding disorders and characterized by the intra- and extracellular accumulation of fibrillar aggregates. Of the more than 25 unrelated proteins known to produce amyloidosis in humans only about a third of them are associated with cerebral deposits translating in cognitive deficits, dementia, stroke, cerebellar and extrapyramidal signs, or a combination thereof. The familial forms reviewed herein, although infrequent, provide unique paradigms to examine the role of amyloid in the mechanism of disease pathogenesis and to dissect the link between vascular and parenchymal amyloid deposition and their differential contribution to neurodegeneration.     

On the structural definition of amyloid fibrils and other polypeptide aggregates

Amyloid fibrils occur inside the human body, associated with ageing or a group of diseases that includes, amongst others, Alzheimer’s disease, atherosclerosis and type II diabetes. Many natural polypeptide chains are able to form amyloid fibrils in vivo or in vitro, and this ability has been suggested to represent an inherent consequence of the chemical structure of the polypeptide chain. Recent literature has provided a wealth of information about the structure of aggregates, precipitates, amyloid fibrils and other types of fibrillar polypeptide assemblies. However, the biophysical meaning associated with these terms can differ considerably depending on the context of their usage. This overview presents a structural comparison of amyloid fibrils and other types of polypeptide assemblies and defines amyloid fibrils, based on structural considerations, as fibrillar polypeptide aggregates with a cross-β conformation. Received 1 March 2007; received after revision 15 March 2007; accepted 25 April 2007     

Chromosome 13 dementias

The importance of cerebral amyloid deposition in the mechanism of neurodegeneration is still debatable. Classic arguments are usually centered on amyloid β(Aβ) and its role in the neuronal loss characteristic of Alzheimer’s disease, the most common form of human cerebral amyloidosis. Two non-Aβ cerebral amyloidoses, familial British and Danish dementias (FBD and FDD), share many aspects of Alzheimer’s disease, including the presence of neurofibrillary tangles, parenchymal preamyloid and amyloid deposits, cerebral amyloid angiopathy and a variety of amyloid-associated proteins and inflammatory components. Both early-onset conditions are linked to specific mutations at or near the stop codon of the chromosome 13 gene BRI2 that cause generation of longer-than-normal protein products. Furin-like processing of these longer precursors releases two de novo-created peptides, ABri and ADan, which deposit as amyloid fibrils in FBD and FDD, respectively. Due to the similar pathology generated by completely unrelated amyloid subunits, FBD and FDD, collectively referred to as chromosome 13 dementias, constitute alternative models for studying the role of amyloid deposition in the mechanism of neuronal cell death.Received 4 March 2005; received after revision 24 April 2005; accepted 26 April 2005     

Activation of innate immunity by lysozyme fibrils is critically dependent on cross-β sheet structure

Inflammation occurs in many amyloidoses, but its underlying mechanisms remain enigmatic. Here we show that amyloid fibrils of human lysozyme, which are associated with severe systemic amyloidoses, induce the secretion of pro-inflammatory cytokines through activation of the NLRP3 (NLR, pyrin domain containing 3) inflammasome and the Toll-like receptor 2, two innate immune receptors that may be involved in immune responses associated to amyloidoses. More importantly, our data clearly suggest that the induction of inflammatory responses by amyloid fibrils is linked to their intrinsic structure, because the monomeric form and a non-fibrillar type of lysozyme aggregates are both unable to trigger cytokine secretion. These lysozyme species lack the so-called cross-β structure, a characteristic structural motif common to all amyloid fibrils irrespective of their origin. Since fibrils of other bacterial and endogenous proteins have been shown to trigger immunological responses, our observations suggest that the cross-β structural signature might be recognized as a generic danger signal by the immune system.     

Crystallin proteins and amyloid fibrils

Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, α-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer’s and Parkinson’s). In this review, the literature on the interaction of αB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials. Received 13 June 2008; received after revision 29 July 2008; accepted 05 August 2008     

Transthyretin: a review from a structural perspective

Transthyretin (formerly called prealbumin) plays important physiological roles as a transporter of thyroxine and retinol-binding protein. X-ray structural studies have provided information on the active conformation of the protein and the site of binding of both ligands. Transthyretin is also one of the precursor proteins commonly found in amyloid deposits. Both wild-type and single-amino-acid-substituted variants have been identified in amyloid deposits, the variants being more amyloidogenic. Sequencing of the gene and the resulting production of a transgenic mouse model have resulted in progress toward solving the mechanism of amyloid formation and detecting the variant gene in individuals at risk. Received 23 January 2001; received after revision 4 April 2001; accepted 30 April 2001     

Molecular basis of Alzheimer's disease

Despite an exponential production of data, Alzheimer's disease (AD) remains an enigma. Unresolved questions persist in the face of the heterogeneity of this neuropathology. Recent progress in understanding mechanisms for AD results from the study of amyloid precursor protein (APP) metabolism and the involvement of senile plaque-associated proteins. In addition to the amyloid cascade hypothesis, alternative schemes emerge, in which the amyloid peptide is not the primary effector of the disease. Perturbations of vesicular trafficking, the cytoskeletal network, and membrane cholesterol distribution could be central events. Furthermore, since the physiological role of APP, presenilins, and apolipoprotein E in the central nervous system are not completely understood, their involvement in AD etiology remains speculative. New actors have to be found to try to explain sporadic cases and non-elucidated familial cases.     

Transformation of amyloid β(1–40) oligomers into fibrils is characterized by a major change in secondary structure

Alzheimer’s disease (AD) is a neurodegenerative disorder occurring in the elderly. It is widely accepted that the amyloid beta peptide (Aβ) aggregation and especially the oligomeric states rather than fibrils are involved in AD onset. We used infrared spectroscopy to provide structural information on the entire aggregation pathway of Aβ(1–40), starting from monomeric Aβ to the end of the process, fibrils. Our structural study suggests that conversion of oligomers into fibrils results from a transition from antiparallel to parallel β-sheet. These structural changes are described in terms of H-bonding rupture/formation, β-strands reorientation and β-sheet elongation. As antiparallel β-sheet structure is also observed for other amyloidogenic proteins forming oligomers, reorganization of the β-sheet implicating a reorientation of β-strands could be a generic mechanism determining the kinetics of protein misfolding. Elucidation of the process driving aggregation, including structural transitions, could be essential in a search for therapies inhibiting aggregation or disrupting aggregates.     

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.     

Structure of rat tail tendon collagen examined by atomic force microscope

The Atomic Force Microscope (AFM) was used to inspect collagen fibrils deposited on mica sheets at different fibrillogenesis times. Collagen was obtained from rat tail tendon fibers. Various fibril forms were observed, together with the characteristic periodic intra-fibril structure (D-bands). The fibril thickness, width, D-band periodicity and depth were measured and the statistical distribution of these parameters at 1, 2, 5, 10 and 15 days of in vitro fibril formation time was calculated. The fibrils showed an increasing size with time, but the band interval measure remained stable. The band depth, after an initial increase, exhibited a relative steadiness. The results indicate that AFM offers, at low resolution, images qualitatively similar to those obtained with electron microscopy, but with less manipulation of the sample. A quantitative evaluation of collagen structural features in the nanometer scale is made possible by AFM.     

Alzheimer’s disease and CADASIL are heritable,adult-onset dementias that both involve damaged small blood vessels

This essay explores an alternative pathway to Alzheimer’s dementia that focuses on damage to small blood vessels rather than late-stage toxic amyloid deposits as the primary pathogenic mechanism that leads to irreversible dementia. While the end-stage pathology of AD is well known, the pathogenic processes that lead to disease are often assumed to be due to toxic amyloid peptides that act on neurons, leading to neuronal dysfunction and eventually neuronal cell death. Speculations as to what initiates the pathogenic cascade have included toxic abeta peptide aggregates, oxidative damage, and inflammation, but none explain why neurons die. Recent high-resolution NMR studies of living patients show that lesions in white matter regions of the brain precede the appearance of amyloid deposits and are correlated with damaged small blood vessels. To appreciate the pathogenic potential of damaged small blood vessels in the brain, it is useful to consider the clinical course and the pathogenesis of CADASIL, a heritable arteriopathy that leads to damaged small blood vessels and irreversible dementia. CADASIL is strikingly similar to early onset AD in that it is caused by germ line mutations in NOTCH 3 that generate toxic protein aggregates similar to those attributed to mutant forms of the amyloid precursor protein and presenilin genes. Since NOTCH 3 mutants clearly damage small blood vessels of white matter regions of the brain that lead to dementia, we speculate that both forms of dementia may have a similar pathogenesis, which is to cause ischemic damage by blocking blood flow or by impeding the removal of toxic protein aggregates by retrograde vascular clearance mechanisms.     

Structural and biochemical aspects of keratan sulphate in the cornea

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly-thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.     

Independent modulation of collagen fibrillogenesis by decorin and lumican

The leucine-rich proteoglycans (also known as "small, leucine-rich proteoglycans," or SLRPs) lumican and decorin are thought to be involved in the regulation of collagen fibril assembly. Preparation of these proteoglycans in chemical amounts without exposure to denaturants has recently been achieved by infecting HT-1080 cells with vaccinia virus that contains an expression cassette for these molecules. Addition of lumican and decorin to a collagen fibrillogenesis assay based on turbidity demonstrated that lumican accelerated initial fibril formation while decorin retarded initial fibril formation. At the end of fibrillogenesis, both proteoglycans resulted in an overall reduced turbidity, suggesting that fibril diameter was lower. The presence of both proteoglycans had a synergistic effect, retarding fibril formation to a greater degree than either proteoglycan individually. Competitive binding studies showed that lumican did not compete for decorin-binding sites on collagen fibrils. Both proteoglycans increased the stability of fibrils to thermal denaturation to approximately the same degree. These studies show that lumican does not compete for decorin-binding sites on collagen, that decorin and lumican modulate collagen fibrillogenesis, and that, in the process, they also enhance collagen fibril stability.     

Cytosolic factors in mitochondrial protein import

In vitro import studies have confirmed the participation of cytosolic protein factors in the import of various precursor proteins into mitochondria. The requirement for extramitochondrial adenosine triphosphate for the import of a group of precursor proteins seems to be correlated with the chaperone activity of the cytosolic protein factors. One of the cytosolic protein factors is hsp70, which generally recognizes and binds unfolded proteins in the cytoplasm. Hsp70 keeps the newly synthesized mitochondrial precursor proteins in import-competent unfolded conformations. Another cytosolic protein factor that has been characterized is mitochondrial import stimulation factor (MSF), which seems to be specific to mitochondrial precursor proteins. MSF recognizes the mitochondrial precursor proteins, forms a complex with them and targets them to the receptors on the outer surface of mitochondria.     

Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.