title: Homogentisic acid induces aggregation and fibrillation of amyloidogenic proteins.
abstract: Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis.
  
  
  
  Gel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aβ(1-42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA.
  
  
  
  We found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA.
  
  
  
  HGA might be an important amyloid co- component playing significant roles in AKU amyloidosis.
  
  
  
  Our results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking.
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Braconi
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Lia
authors.initials: L
authors.lastname: Millucci
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bernini
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Ottavia
authors.initials: O
authors.lastname: Spiga
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Lupetti
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Marzocchi
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Neri
authors.initials: N
authors.lastname: Niccolai
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bernardini
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100, Siena, Italy. Electronic address: annalisa.santucci@unisi.it.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Santucci
mesh_terms: D000474:Alkaptonuria / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000378:metabolism; D058227:Amyloidogenic Proteins / Q000378:metabolism*; D000686:Amyloidosis / Q000378:metabolism; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D003238:Connective Tissue / Q000187:drug effects / Q000378:metabolism; D050560:Homogentisate 1,2-Dioxygenase / Q000378:metabolism; D006713:Homogentisic Acid / Q000494:pharmacology*; D006801:Humans; D009794:Ochronosis / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011228:Prealbumin / Q000378:metabolism; D066263:Protein Aggregation, Pathological / Q000139:chemically induced*; D000685:Serum Amyloid A Protein / Q000378:metabolism; D051844:alpha-Synuclein / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D058227:Amyloidogenic Proteins; D011228:Prealbumin; D000685:Serum Amyloid A Protein; D051844:alpha-Synuclein; D009320:Atrial Natriuretic Factor; D050560:Homogentisate 1,2-Dioxygenase; D006713:Homogentisic Acid
keywords: Amyloid; Congo Red; Gel electrophoresis; Inborn error of metabolism; Metabolic disease; Serum Amyloid A