Heparin and Other Glycosaminoglycans Stimulate the Formation of Amyloid Fibrils from α-Synuclein in Vitro

200

206

Parkinson's disease is characterized by the aggregation of ␣-synuclein into filamentous forms within affected neurons of the basal ganglia. Fibrillization of purified recombinant ␣-synuclein is inefficient in vitro but can be enhanced by the addition of various agents including glycosaminoglycans and polycations. Here we report that fatty acids and structurally related anionic detergents greatly accelerate fibrillization of recombinant ␣-synuclein at low micromolar concentrations with lag times as short as 11 min and apparent first order growth rate constants as fast as 10.4 h ؊1 . All detergents and fatty acids were micellar at active concentrations because of an ␣-synuclein-dependent depression of their critical micelle concentrations. Other anionic surfaces, such as those supplied by anionic phospholipid vesicles, also induced ␣-synuclein fibrillization, with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate ␣-synuclein fibrillization, that this mechanism underlies the inducer activity of anionic surfactants, and that anionic membranes may serve this function in vivo.

Section: Efficient Induction Of ␣-Synucleinsupporting

confidence: 77%

Section: Efficient Induction Of ␣-Synucleinmentioning

confidence: 99%

Rapid Anionic Micelle-mediated α-Synuclein Fibrillization in Vitro

Necula¹,

Chirita²,

Kuret

2003

200

206

“…mAcP was incubated as described in the legend to Fig. 1A promote amyloid fibril formation in vitro by all studied protein systems (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In particular, it is generally reported that HS causes an increase of the rate by which fibrils are formed (13)(14)(15)(16)(17)22) and of the final ThT fluorescence (14 -17).…”

Section: Discussionmentioning

confidence: 98%

“…The HS-mediated appearance of a new phase in the aggregation process is not a peculiar and exceptional trait of the protein and experimental conditions used here. It is commonly observed in the time course of aggregation of other systems, when HS or its highly sulfated analogue heparin is present (14,18,22,48,49), although in some other cases it is not (16,17).…”

Section: Implications For a Correct Interpretation Of The Hs-inducedmentioning

confidence: 99%

Kinetic Analysis of Amyloid Formation in the Presence of Heparan Sulfate

Motamedi-Shad¹,

Monsellier²,

Torrassa³

et al. 2009

A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and ␤-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis.

“…Thioflavin T forms a complex with fibrils, and the fluorescence intensity is proportional to the amount of fibrils that are present when the concentration of ThT is held constant (16). The effects of ThT buffer solutions on LC structure were evaluated by circular dichroism (CD) and confirmed by TEM and AFM (27)(28)(29)(30).…”

Section: Methodsmentioning

confidence: 99%

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

Ren

Hong

Gong³

et al. 2010

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a 1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo 1 amyloid fibrils. The presence of HS and over-O-sulfatedheparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic 1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length 1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.Amyloid deposition is hypothesized to play a role in many diseases including rheumatoid arthritis and diabetes, as well as the amyloidoses themselves (1). The amyloidoses are a group of systemic and localized diseases that exhibit deposition of insoluble fibrillar proteins in organs and tissues. Primary amyloidosis (AL) 2 is the most common systemic form in the UnitedStates. It results from a plasma cell dyscrasia in which clonal B cells produce an excess of amyloidogenic immunoglobulin light chains (LCs), which circulate and deposit as insoluble fibrils throughout the body (2). A structural rearrangement from nonfibril-prone oligomers to more fibril-prone conformers is required for protofilament formation (3). Nascent protofilaments can nucleate and elongate through the addition of partially folded oligomeric intermediates to the ends of the growing protofilaments. Protofilaments can further intertwine to form protofibrils and fibrils (4, 5). Glycosaminoglycans (GAGs) are one of the major components of the extracellular matrix. They consist of disaccharide repeating units that are unbranched and attached to a serine residue of a core protein through a trisaccharide linker. Each GAG has a unique sequence, and post-assembly modifications, which can be induced by a number of factors, lead to alterations in sulfation, acetylation, or length of chain (6). Recently, Staples et al. (7) showed that the fu...