Abstract
A hallmark of neurodegenerative diseases caused by polyglutamine expansion is the abnormal accumulation of mutant proteins into ubiquitin-positive inclusions. The local build-up of these ubiquitinated proteins suggests that the proteasome machinery inadequately clears misfolded proteins, resulting in their increase to potentially toxic levels. Inclusions may disrupt normal cell homeostasis by sequestering vital cellular factors, such as chaperones, proteasomes and transcription components. Here, we used fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of polyglutamine-expanded ataxin1 and inclusion-associated proteins. These experiments demonstrated that at least two types of ataxin1 inclusions exist; those that undergo rapid and complete exchange with a nucleoplasmic pool and those that contain varying levels of slow-exchanging ataxin1. Slow-exchanging inclusions contain high ubiquitin levels, but surprisingly low proteasome levels, suggesting an impairment in the ability of proteasomes to recognize ubiquitinated substrates. Proteasomes and CBP remained highly dynamic components of inclusions, indicating that although enriched with ataxin1, they are not irreversibly trapped. These results redefine our perception of polyglutamine inclusions and demonstrate the usefulness of FRAP and live cell imaging to study factors that modulate their behaviour.
Publication types
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Amino Acid Sequence / genetics
-
Ataxin-1
-
Ataxins
-
Bacterial Proteins
-
CREB-Binding Protein
-
Cell Nucleus / genetics
-
Cell Nucleus / metabolism*
-
Cell Nucleus / pathology
-
Cysteine Endopeptidases / drug effects
-
Cysteine Endopeptidases / genetics
-
Cysteine Endopeptidases / metabolism
-
Enzyme Inhibitors / pharmacology
-
Fluorescence Recovery After Photobleaching
-
Green Fluorescent Proteins
-
HeLa Cells
-
Humans
-
Inclusion Bodies / genetics
-
Inclusion Bodies / metabolism*
-
Inclusion Bodies / pathology
-
Luminescent Proteins
-
Multienzyme Complexes / drug effects
-
Multienzyme Complexes / genetics
-
Multienzyme Complexes / metabolism
-
Nerve Tissue Proteins / genetics
-
Nerve Tissue Proteins / metabolism*
-
Neurodegenerative Diseases / genetics
-
Neurodegenerative Diseases / metabolism*
-
Neurodegenerative Diseases / physiopathology
-
Nuclear Matrix / genetics
-
Nuclear Matrix / metabolism
-
Nuclear Proteins / genetics
-
Nuclear Proteins / metabolism*
-
Peptides / genetics
-
Peptides / metabolism*
-
Proteasome Endopeptidase Complex
-
Recombinant Fusion Proteins
-
Trans-Activators / genetics
-
Trans-Activators / metabolism
-
Trinucleotide Repeat Expansion / genetics*
-
Ubiquitin / genetics
-
Ubiquitin / metabolism
Substances
-
ATXN1 protein, human
-
Ataxin-1
-
Ataxins
-
Bacterial Proteins
-
Enzyme Inhibitors
-
Luminescent Proteins
-
Multienzyme Complexes
-
Nerve Tissue Proteins
-
Nuclear Proteins
-
Peptides
-
Recombinant Fusion Proteins
-
Trans-Activators
-
Ubiquitin
-
yellow fluorescent protein, Bacteria
-
Green Fluorescent Proteins
-
polyglutamine
-
CREB-Binding Protein
-
CREBBP protein, human
-
Cysteine Endopeptidases
-
Proteasome Endopeptidase Complex