Abstract
The ubiquitin mutant UBB+1 has been identified as a hallmark of neurodegenerative diseases. In this study, we characterize polyubiquitinated forms of UBB+1 in vitro and from patient samples. The ability of UBB+1 to be readily ubiquitinated by several E2 enzymes provided a mechanism for the controlled synthesis and purification of defined conjugates. This allowed us to utilize polyUb-UBB+1 conjugates for biochemical assays, as well as solution NMR. Coupled with our immunoassay for detection of ubiquitinated forms of UBB+1 in patient blood samples, we gain a clearer picture of the molecular mechanisms underlying neurodegenerative diseases.
Keywords:
UBB +1; biomolecular NMR; deubiquitinase; neurodegeneration; polyubiquitin.
© 2016 Federation of European Biochemical Societies.
MeSH terms
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Aged
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Alzheimer Disease / genetics*
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Alzheimer Disease / metabolism
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Alzheimer Disease / pathology
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Case-Control Studies
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Cysteine Endopeptidases / chemistry
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / metabolism
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Deubiquitinating Enzymes
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Endopeptidases / chemistry
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Endopeptidases / genetics
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Endopeptidases / metabolism
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Endosomal Sorting Complexes Required for Transport / chemistry
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Endosomal Sorting Complexes Required for Transport / genetics
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Endosomal Sorting Complexes Required for Transport / metabolism
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Female
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Fluoresceins / chemistry
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Fluorescent Dyes / chemistry
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Gene Expression Regulation
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Humans
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Male
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Middle Aged
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Models, Molecular
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Neurons / metabolism*
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Neurons / pathology
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Nuclear Magnetic Resonance, Biomolecular
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Polyubiquitin / chemistry*
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Polyubiquitin / genetics
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Polyubiquitin / metabolism
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Proteasome Endopeptidase Complex / metabolism
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Protein Isoforms / chemistry
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Structure, Secondary
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Staining and Labeling
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Ubiquitin / chemistry*
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Ubiquitin / genetics
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Ubiquitin / metabolism
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Ubiquitin Thiolesterase / chemistry
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Ubiquitin Thiolesterase / genetics
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Ubiquitin Thiolesterase / metabolism
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Ubiquitin-Specific Proteases / chemistry
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Ubiquitin-Specific Proteases / genetics
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Ubiquitin-Specific Proteases / metabolism
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Ubiquitination
Substances
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Endosomal Sorting Complexes Required for Transport
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Fluoresceins
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Fluorescent Dyes
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Protein Isoforms
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STAMBP protein, human
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UBB protein, human
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Ubiquitin
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Polyubiquitin
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fluorescein 5-maleimide
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Endopeptidases
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Deubiquitinating Enzymes
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OTUB1 protein, human
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OTUD3 protein, human
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USP2 protein, human
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Ubiquitin Thiolesterase
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Ubiquitin-Specific Proteases
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex