Molecular characterization of metal-binding polypeptide domains by electrospray ionization mass spectrometry and metal chelate affinity chromatography

Volz, Jürgen; Bosch, Frank U; Wunderlin, Markus; Schuhmacher, Martina; Melchers, Klaus; Bensch, Klaus W.; Steinhilber, W; Schäfer, Klaus; Tóth, Gábor; Penke, Botond; Przybylski, Michael

doi:10.1016/s0021-9673(97)00877-7

Cited by 36 publications

(27 citation statements)

References 27 publications

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“…The cross-linking reaction was stopped by adding 10 mM Tris and incubating on ice for 15 min before washing and lysis in PBS plus 0.1% Triton X-100. APP has a metal-binding domain that binds copper and cobalt with high affinity (36), and this method has been used previously to purify APP to homogeneity (37). To reduce background inherent in co-immunoprecipitations caused by the heavy chain of IgG (which runs at a similar molecular weight to ubiquilin), we exploited this feature to pull down APP using a cobalt-based metal affinity resin (TALON resin; Clontech).…”

Section: Methodsmentioning

confidence: 99%

Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein

Stieren

Ayadi

Xiao

et al. 2011

Journal of Biological Chemistry

109

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Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein

Stieren

Ayadi

Xiao

et al. 2011

Journal of Biological Chemistry

109

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“…Immobilized nickel ion affinity media have been shown to bind peptides and proteins containing adjacent histidine residues and to bind peptides containing two or more histidines in close proximity, such as H ϫ H and H ϫϫ H (16). In order to see if carboxypeptidase Y can be used to obtain amino acid sequence data from immobilized metal affinity-bound peptides, histatin-5 (Fig.…”

Section: Histatinmentioning

confidence: 99%

Direct Analysis of the Products of Sequential Cleavages of Peptides and Proteins Affinity-Bound to Immobilized Metal Ion Beads by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Qian¹,

Zhou²,

Khaledi³

et al. 1999

Analytical Biochemistry

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“…From solution studies, it is known that Cu(II) forms stable complexes with His-containing peptides. 19,20 Once the speciÐc coordination site, i.e., the imidazole nitrogen of histidine, is occupied by Cu, other high Ðeld ligands in close proximity, such as the amide nitrogens of the peptide backbone will get involved in Cu binding.19, 21 We therefore surmise that protons from backbone NH groups of the peptide are abstracted to compensate for the charge of the Cu ions.…”

Section: Maldi-ms Of Peptide-metal Ion Complexesmentioning

confidence: 99%