10 research outputs found
Huntington's disease protein contributes to RNA-mediated gene silencing through association with Argonaute and P bodies
Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes
A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12
The bacterial ribosomal protein S12 contains a universally conserved
D88 residue on a loop region thought to be critically involved in
translation due to its proximal location to the A site of the 30S
subunit. While D88 mutants are lethal this residue has been found
to be post-translationally modified to β-methylthioaspartic
acid, a post-translational modification (PTM) identified in S12 orthologs
from several phylogenetically distinct bacteria. In a previous report
focused on characterizing this PTM, our results provided evidence
that this conserved loop region might be involved in forming multiple
proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic
and transcriptomic approach reveals new insight into betamethylthiolation
of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199).
To follow-up on this study, the D88 containing loop was probed to
identify candidate binders employing a two-step complementary affinity
purification strategy. The first involved an endogenously expressed
S12 protein containing a C-terminal tag for capturing S12 binding
partners. The second strategy utilized a synthetic biotinylated peptide
representing the D88 conserved loop region for capturing S12 loop
interaction partners. Captured proteins from both approaches were
detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem
mass spectrometry. The results presented in this report revealed proteins
that form direct interactions with the 30S subunit and elucidated
which are likely to interact with S12. In addition, we provide evidence
that two proteins involved in regulating ribosome and/or mRNA transcript
levels under stress conditions, RNase R and Hfq, form direct interactions
with the S12 conserved loop, suggesting that it is likely part of
a protein binding interface
A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12
The bacterial ribosomal protein S12 contains a universally conserved
D88 residue on a loop region thought to be critically involved in
translation due to its proximal location to the A site of the 30S
subunit. While D88 mutants are lethal this residue has been found
to be post-translationally modified to β-methylthioaspartic
acid, a post-translational modification (PTM) identified in S12 orthologs
from several phylogenetically distinct bacteria. In a previous report
focused on characterizing this PTM, our results provided evidence
that this conserved loop region might be involved in forming multiple
proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic
and transcriptomic approach reveals new insight into betamethylthiolation
of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199).
To follow-up on this study, the D88 containing loop was probed to
identify candidate binders employing a two-step complementary affinity
purification strategy. The first involved an endogenously expressed
S12 protein containing a C-terminal tag for capturing S12 binding
partners. The second strategy utilized a synthetic biotinylated peptide
representing the D88 conserved loop region for capturing S12 loop
interaction partners. Captured proteins from both approaches were
detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem
mass spectrometry. The results presented in this report revealed proteins
that form direct interactions with the 30S subunit and elucidated
which are likely to interact with S12. In addition, we provide evidence
that two proteins involved in regulating ribosome and/or mRNA transcript
levels under stress conditions, RNase R and Hfq, form direct interactions
with the S12 conserved loop, suggesting that it is likely part of
a protein binding interface
A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12
The bacterial ribosomal protein S12 contains a universally conserved
D88 residue on a loop region thought to be critically involved in
translation due to its proximal location to the A site of the 30S
subunit. While D88 mutants are lethal this residue has been found
to be post-translationally modified to β-methylthioaspartic
acid, a post-translational modification (PTM) identified in S12 orthologs
from several phylogenetically distinct bacteria. In a previous report
focused on characterizing this PTM, our results provided evidence
that this conserved loop region might be involved in forming multiple
proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic
and transcriptomic approach reveals new insight into betamethylthiolation
of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199).
To follow-up on this study, the D88 containing loop was probed to
identify candidate binders employing a two-step complementary affinity
purification strategy. The first involved an endogenously expressed
S12 protein containing a C-terminal tag for capturing S12 binding
partners. The second strategy utilized a synthetic biotinylated peptide
representing the D88 conserved loop region for capturing S12 loop
interaction partners. Captured proteins from both approaches were
detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem
mass spectrometry. The results presented in this report revealed proteins
that form direct interactions with the 30S subunit and elucidated
which are likely to interact with S12. In addition, we provide evidence
that two proteins involved in regulating ribosome and/or mRNA transcript
levels under stress conditions, RNase R and Hfq, form direct interactions
with the S12 conserved loop, suggesting that it is likely part of
a protein binding interface
A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12
The bacterial ribosomal protein S12 contains a universally conserved
D88 residue on a loop region thought to be critically involved in
translation due to its proximal location to the A site of the 30S
subunit. While D88 mutants are lethal this residue has been found
to be post-translationally modified to β-methylthioaspartic
acid, a post-translational modification (PTM) identified in S12 orthologs
from several phylogenetically distinct bacteria. In a previous report
focused on characterizing this PTM, our results provided evidence
that this conserved loop region might be involved in forming multiple
proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic
and transcriptomic approach reveals new insight into betamethylthiolation
of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199).
To follow-up on this study, the D88 containing loop was probed to
identify candidate binders employing a two-step complementary affinity
purification strategy. The first involved an endogenously expressed
S12 protein containing a C-terminal tag for capturing S12 binding
partners. The second strategy utilized a synthetic biotinylated peptide
representing the D88 conserved loop region for capturing S12 loop
interaction partners. Captured proteins from both approaches were
detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem
mass spectrometry. The results presented in this report revealed proteins
that form direct interactions with the 30S subunit and elucidated
which are likely to interact with S12. In addition, we provide evidence
that two proteins involved in regulating ribosome and/or mRNA transcript
levels under stress conditions, RNase R and Hfq, form direct interactions
with the S12 conserved loop, suggesting that it is likely part of
a protein binding interface
A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12
The bacterial ribosomal protein S12 contains a universally conserved
D88 residue on a loop region thought to be critically involved in
translation due to its proximal location to the A site of the 30S
subunit. While D88 mutants are lethal this residue has been found
to be post-translationally modified to β-methylthioaspartic
acid, a post-translational modification (PTM) identified in S12 orthologs
from several phylogenetically distinct bacteria. In a previous report
focused on characterizing this PTM, our results provided evidence
that this conserved loop region might be involved in forming multiple
proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic
and transcriptomic approach reveals new insight into betamethylthiolation
of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199).
To follow-up on this study, the D88 containing loop was probed to
identify candidate binders employing a two-step complementary affinity
purification strategy. The first involved an endogenously expressed
S12 protein containing a C-terminal tag for capturing S12 binding
partners. The second strategy utilized a synthetic biotinylated peptide
representing the D88 conserved loop region for capturing S12 loop
interaction partners. Captured proteins from both approaches were
detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem
mass spectrometry. The results presented in this report revealed proteins
that form direct interactions with the 30S subunit and elucidated
which are likely to interact with S12. In addition, we provide evidence
that two proteins involved in regulating ribosome and/or mRNA transcript
levels under stress conditions, RNase R and Hfq, form direct interactions
with the S12 conserved loop, suggesting that it is likely part of
a protein binding interface
A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12
The bacterial ribosomal protein S12 contains a universally conserved
D88 residue on a loop region thought to be critically involved in
translation due to its proximal location to the A site of the 30S
subunit. While D88 mutants are lethal this residue has been found
to be post-translationally modified to β-methylthioaspartic
acid, a post-translational modification (PTM) identified in S12 orthologs
from several phylogenetically distinct bacteria. In a previous report
focused on characterizing this PTM, our results provided evidence
that this conserved loop region might be involved in forming multiple
proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic
and transcriptomic approach reveals new insight into betamethylthiolation
of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199).
To follow-up on this study, the D88 containing loop was probed to
identify candidate binders employing a two-step complementary affinity
purification strategy. The first involved an endogenously expressed
S12 protein containing a C-terminal tag for capturing S12 binding
partners. The second strategy utilized a synthetic biotinylated peptide
representing the D88 conserved loop region for capturing S12 loop
interaction partners. Captured proteins from both approaches were
detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem
mass spectrometry. The results presented in this report revealed proteins
that form direct interactions with the 30S subunit and elucidated
which are likely to interact with S12. In addition, we provide evidence
that two proteins involved in regulating ribosome and/or mRNA transcript
levels under stress conditions, RNase R and Hfq, form direct interactions
with the S12 conserved loop, suggesting that it is likely part of
a protein binding interface