Ubiquitin Stability and the Lys 63‐Linked Polyubiquitination Site Are Compromised on Copper Binding

Milardi, Danilo; Arnesano, Fabio; Grasso, Giulia; Magrı̀, Antonio; Tabbı̀, Giovanni; Scintilla, Simone; Natile, Giovanni; Rizzarelli, Enrico

doi:10.1002/anie.200701987

Cited by 38 publications

(47 citation statements)

References 21 publications

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“…We have recently shown that Cu II binds to Ub with 0.1 µM affinity and compromises protein stability [20]. The Cu II affinity of Ub is comparable to that of amyloidogenic proteins involved in Parkinson's, Alzheimer's, and prion diseases [21], and of α-syn itself [22].…”

Section: Introductionmentioning

confidence: 99%

Copper-Triggered Aggregation of Ubiquitin

et al. 2009

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Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80∶20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing β-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates.

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Section: Introductionmentioning

confidence: 99%

Copper-Triggered Aggregation of Ubiquitin

et al. 2009

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“…However, in the native structure both the N-terminus and sulfur in the side-group of Met1 are hydrogen-bonded to the carbonyl group of Val17 and the amide of Lys63, respectively [22]. It has been suggested that disruption of these key hydrogen-bonds could destabilize the native form of Ub [21]. probably resulting in the new folded 5+ or 6+ conformers being molten globules: compact forms of Ub that do not retain its biological function [4].…”

Section: Im-ms Analysis Of Ub and Pd-ub Conformersmentioning

confidence: 99%

“…A study of Cu(II) binding to Ub in solution established that the preferred anchoring site is the N-terminus of Met1 followed by the His68 site [21]. On anchoring to Met1 the Cu(II) also binds to three oxygen donor ligands to complete a tetragonal binding geometry.…”

Section: Introductionmentioning

confidence: 99%

“…On anchoring to Met1 the Cu(II) also binds to three oxygen donor ligands to complete a tetragonal binding geometry. Potential oxygen sites were identified as Met1 and Val17 (carbonyl groups), and Glu16 and Glu18 (carboxylate groups) [21]. The Cu(II) study concluded that the lack of additional donor atoms near His68 made it a less preferred anchoring site than the Met1 site.…”

Section: Introductionmentioning

confidence: 99%

“…The Cu(II) study concluded that the lack of additional donor atoms near His68 made it a less preferred anchoring site than the Met1 site. The binding of Cu(II) to the N-terminus may also destabilize Ub by interfering with two key hydrogen bonds between the N-terminus and the CO of Val17, and the sulfur atom of Met1 and the amide NH of Lys63 [21,22]. A further X-ray crystallography study of human Ub with Zn(II), Cd(II), or Hg(II) determined the preferred anchoring sites in order were His68, Met1/Glu16, and Glu18 [23].…”

Section: Introductionmentioning

confidence: 99%

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