The PrP-like Protein Doppel Binds Copper

Qin, Ken; Coomaraswamy, Janaky; Mastrangelo, Peter; Yang, Ying; Lugowski, Stan; Petromilli, Chris; Prusiner, Stanley B.; Fraser, Paul E.; Goldberg, Jonathan M.; Chakrabartty, Avijit; Westaway, David

doi:10.1074/jbc.m210875200

Cited by 61 publications

(70 citation statements)

References 44 publications

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“…Mutational Analysis of Neuroprotective Activity of PrP CBecause (i) copper binding is a potential mechanism whereby PrP C and Dpl might compete (18), (ii) the major Cu(II) binding domain maps within the octarepeat region of PrP (16,45), and (iii) internal N-terminal deletions of PrP are stably expressed but nontoxic (46) (whereas larger deletions such as PrP⌬32-121 and PrP⌬32-134 are toxic (25)), we hypothesized that one neuroprotective region within PrP might lie within the octarepeat sequences. Accordingly, we tested the activity of a cotransfected PrP⌬51-90 allele lacking all five octarepeats, as well as an allele where each histidine residue within the copper binding octarepeats was converted to a glycine residue (PHGG(G/S)WGQ 4 3 PGGG(G/S)WGQ 4 ).…”

Section: Resultsmentioning

confidence: 99%

“…This region is also implicated in targeting, in the form of a weak nuclear localization signal (perhaps only germane to the pathogenesis of PRNP alleles encoding truncated forms of PrP C (63)), in controlling a transition from raft-like domains to clathrin-coated pits (47), and in dynein-mediated retrograde axonal transport (64). Features of the octarepeat sequences include selective Cu(II) binding in vitro (65,66) and again a relationship to trafficking signals. Thus octarepeat sequences serve to facilitate basal and/or Cu(II)-stimulated endocytosis (67)(68)(69)(70) and kinesin-mediated anterograde axonal transport (64).…”

Section: Activity Determinants In Prp C and Dplmentioning

confidence: 99%

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Genetic Mapping of Activity Determinants within Cellular Prion Proteins

Drisaldi

Coomaraswamy

Mastrangelo

et al. 2004

Journal of Biological Chemistry

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The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP C . Internally deleted forms of PrP C resembling Dpl such as PrP⌬32-121 produce a similar PrP C -sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP C , and PrP⌬132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP C was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrP⌬23-28), by deletion of all five octarepeats (PrP⌬51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B (Dpl⌬101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP C function in vivo and place constraints upon current hypotheses to explain Dpl/PrP C antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Activity Determinants In Prp C and Dplmentioning

confidence: 99%

Genetic Mapping of Activity Determinants within Cellular Prion Proteins

Drisaldi

Coomaraswamy

Mastrangelo

et al. 2004

Journal of Biological Chemistry

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“…This observation is consistent with previous studies of other proteins that show that Cu(II) binding protects residues from DEPC modification. 33,34 Six residues in β2m are found to be modified upon reaction with DEPC. Three of the four histidine residues are modified; only the buried His84 was unreactive.…”

Section: Depc Reactions Of β2mmentioning

confidence: 99%

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

2008

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The reliability and information content of diethylpyrocarbonate (DEPC) as a covalent probe of protein surface structure has been improved when used appropriately with mass spectrometric detection. Using myoglobin, cytochrome c, and β-2-microglobulin as model protein systems, we demonstrate for the first time that DEPC can modify Ser and Thr residues in addition to His and Tyr residues. This result expands the capability of DEPC as a structural probe because about 25% of the sequence of the average protein can now be covered using this covalent labeling reagent. In addition, we establish a new approach based on mass spectrometry to ensure the structural integrity of proteins during amino acid-specific covalent labeling reactions. This approach involves monitoring the extent of modification as a function of reagent concentration and allows any small-scale or local perturbations caused by the covalent label to be readily identified and avoided. Results indicate that these dose-response plots are much more reliable and generally applicable probes of possible protein structural changes than fluorescence or circular dichroism spectroscopies. These dose-response plots also provide a means of quantitatively comparing the reactivity of each modified residue. Based on comparisons to known X-ray crystal structures, we find that the solvent accessibility of the reactive atom in the side chain and the presence of a nearby charged residue most affect modification rates. Finally, this improved surface mapping method has been used to determine the effect of Cu(II) binding on the structure of β-2-microglobulin. Results confirm that Cu(II) binds His31, but not any of the other three His residues, and changes the solvent accessibility of residues near His31 and near the N-terminus.

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“…11 Both proteins bind copper [Cu(II)] ions 12,13 and the solution structure of recombinant Dpl displays a similar topology to PrP C such that it closely resembles an N-terminally truncated PrP C lacking the octapeptide repeat region. 14 It is of note that N-terminally truncated PrP C is incapable of rescuing Dpldependent ataxia and can indeed cause a similar ataxic phenotype in the absence of the wild-type PrP C protein.…”

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confidence: 99%

Male Infertility and DNA Damage in Doppel Knockout and Prion Protein/Doppel Double-Knockout Mice

Paisley

Banks

Selfridge

et al. 2004

The American Journal of Pathology

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The prion protein (PrP) and Doppel (Dpl) have many structural and biochemical properties in common, leading to the suggestion that the lack of an obvious phenotype in PrP-deficient mice maybe because of compensation by Dpl. To test this hypothesis and also investigate the function of Dpl we have generated Prnd ؊/؊ and Prnp ؊/؊ /Prnd ؊/؊ mouse lines. Both develop normally and display an identical male sterility phenotype that differs from that reported for another Prnd ؊/؊ mouse line. Sperm from both our mutant lines were present at normal concentrations, had normal motility, and no morphological abnormalities. Despite only rarely fertilizing oocytes in vivo, because of an inability to perform the acrosome reaction, mutant sperm were capable of fertilization in vitro, albeit at reduced rates compared to wild type. Elevated levels of oxidative DNA damage were found in both types of mutant sperm and resulting embryos failed at an early stage. Therefore we found no evidence that Dpl compensates for the loss of PrP function in mutant mouse lines, but it does have an important anti-oxidant function necessary for sperm integrity and male fertility.

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The PrP-like Protein Doppel Binds Copper

Cited by 61 publications

References 44 publications

Genetic Mapping of Activity Determinants within Cellular Prion Proteins

Genetic Mapping of Activity Determinants within Cellular Prion Proteins

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

Male Infertility and DNA Damage in Doppel Knockout and Prion Protein/Doppel Double-Knockout Mice

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