Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis

Abstract

Three variants of human ␤ 2 -microglobulin (␤ 2 -m) were compared with wild-type protein. For two variants, namely the mutant R3A␤ 2 -m and the form devoid of the N-terminal tripeptide (⌬N3␤ 2 -m), a reduced unfolding free energy was measured compared with wild-type ␤ 2 -m, whereas an increased stability was observed for the mutant H31Y␤ 2 -m. The solution structure could be determined by 1 H NMR spectroscopy and restrained modeling only for R3A␤ 2 -m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Y␤ 2 -m and ⌬N3␤ 2 -m. Precipitation and unfolding were observed over time periods shorter than 4 -6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for ␤ 2 -m fibrillogenesis. The mechanism is consistent with the previous and present results on ␤ 2 -m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.Over the last several years, an overwhelming number of reports have addressed the phenomenon of amyloidogenesis. The interest in the subject stems not only from the social relevance of amyloid pathologies such as Alzheimer's disease, or spongiform encephalopathy, or the various systemic amyloidoses, but also from the general implications in the issue of protein folding.Amyloidoses have been recognized as conformational diseases that arise from the conversion of globular proteins into insoluble fibrillar aggregates (1). Despite the diversity of the involved proteins, amyloid fibrils exhibit a common structure known as cross-␤ structure, which appears to be a particularly stable, generic protein fold, accessible to many polypeptide chains under specific conditions in vitro and in vivo (2). The amyloid deposition of ␤ 2 -microglobulin (␤ 2 -m), 1 the nonpolymorphic light chain of the class I major histocompatibility complex (MHC-I), is associated to dialysis-related amyloidosis (3). The disease is the result of long term hemodialysis in individuals with chronic renal failure, a widespread pathology with high social costs that are further increased by the inevitable dialysis-related amyloidosis complication. Recently ankylosing spondylitis has also been proposed to originate from ␤ 2 -m deposition (4). We determined the solution structure of isolated ␤ 2 -m by NMR spectroscopy (5) and showed that the most important rearrangements of the protein, with respect to its structure in MHC-I, were observed for strands D and E, interstrand loop D-E, and strand A, including the N-terminal segment. We stated that these modifications can be conside...

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry

“…Materials-Recombinant ␤2m and R3A␤2m were prepared as described previously (14,19). The B subunit of ␣-crystallin was employed for all the experiments except for the DOSY measurements in which bovine eye lens ␣-crystallin (Sigma), composed of ϳ3:1 ␣A-/␣B-crystallin, was used.…”

“…For instance, the fragment of ␤2m devoid of the N-terminal hexapeptide (⌬N6␤2m) that occurs to a significant extent (ϳ30%) in ex vivo deposits of the protein (13) has an enhanced tendency to aggregate and form fibrils, even at neutral pH (14). Furthermore, the Arg-3-to-Ala mutant of ␤2m (R3A␤2m) forms a folded structure very close to that of the wild-type protein, but it exhibits a spontaneous propensity to unfold, aggregate, and precipitate over a period of days to weeks (19).…”

“…In vivo, ␤2m causes amyloidosis via accumulation in the tissues of patients with renal failure as a result of long term hemodialysis (11). The structure of ␤2m and the molecular aspects of its amyloid transition have been extensively investigated in recent years, not only because of the clinical relevance of dialysis-related amyloidosis but also as a model for other amyloidogenic proteins (12)(13)(14)(15)(16)(17)(18)(19).…”

Ultrasonication: An Efficient Agitation for Accelerating the Supersaturation-Limited Amyloid Fibrillation of Proteins

“…[11][12][13][14][15][16][17][18][19] Studies on 2-m illustrate a basic mechanism of amyloid fibrillation. Dialysis-related amyloidosis is a common and serious complication in patients receiving hemodialysis for more than 10 years.…”

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

“…It has been shown that I T contains a non-native trans peptide bond between His-31 and Pro-32 that slowly converts into cis conformation during the final refolding step (3). The isomerization occurs with minor rearrangements of the protein toward the native structure from an already native-like state (3)(4)(5). The native-like intermediate has been proposed as the effective fibril-competent species (4,6).…”