5 research outputs found
Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome
Formin
mDia2 is a cytoskeleton-regulatory protein that switches
reversibly between a closed, autoinhibited and an open, active conformation.
Although the open conformation of mDia2 induces actin assembly thereby
controlling many cellular processes, mDia2 possesses also actin-independent
and conformation-insensitive scaffolding roles related to microtubules
and p53, respectively. Thus, we hypothesize that mDia2 may have other
unappreciated functions and regulatory modes. Here we identify and
validate proteasome and Ubiquitin as mDia2-interacting partners using
stable isotope labeling with amino acids in cell culture-based quantitative
proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated,
binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome
substrate. Surprisingly, knockdown of mDia2 increases the activity
of the proteasome in vitro, whereas mDia2 overexpression has opposite
effects only when it adopts the open conformation and cannot induce
actin assembly. Consistently, a combination of candidate and unbiased
proteome-wide analyses indicates that mDia2 regulates the cellular
levels of proteasome substrate β-catenin and a number of ubiquitinated
actin-regulatory proteins. Hence, these findings add more complexity
to the mDia2 activity cycle by showing that the open conformation
may control actin dynamics also through actin-independent regulation
of the proteasome
Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome
Formin
mDia2 is a cytoskeleton-regulatory protein that switches
reversibly between a closed, autoinhibited and an open, active conformation.
Although the open conformation of mDia2 induces actin assembly thereby
controlling many cellular processes, mDia2 possesses also actin-independent
and conformation-insensitive scaffolding roles related to microtubules
and p53, respectively. Thus, we hypothesize that mDia2 may have other
unappreciated functions and regulatory modes. Here we identify and
validate proteasome and Ubiquitin as mDia2-interacting partners using
stable isotope labeling with amino acids in cell culture-based quantitative
proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated,
binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome
substrate. Surprisingly, knockdown of mDia2 increases the activity
of the proteasome in vitro, whereas mDia2 overexpression has opposite
effects only when it adopts the open conformation and cannot induce
actin assembly. Consistently, a combination of candidate and unbiased
proteome-wide analyses indicates that mDia2 regulates the cellular
levels of proteasome substrate β-catenin and a number of ubiquitinated
actin-regulatory proteins. Hence, these findings add more complexity
to the mDia2 activity cycle by showing that the open conformation
may control actin dynamics also through actin-independent regulation
of the proteasome
Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome
Formin
mDia2 is a cytoskeleton-regulatory protein that switches
reversibly between a closed, autoinhibited and an open, active conformation.
Although the open conformation of mDia2 induces actin assembly thereby
controlling many cellular processes, mDia2 possesses also actin-independent
and conformation-insensitive scaffolding roles related to microtubules
and p53, respectively. Thus, we hypothesize that mDia2 may have other
unappreciated functions and regulatory modes. Here we identify and
validate proteasome and Ubiquitin as mDia2-interacting partners using
stable isotope labeling with amino acids in cell culture-based quantitative
proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated,
binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome
substrate. Surprisingly, knockdown of mDia2 increases the activity
of the proteasome in vitro, whereas mDia2 overexpression has opposite
effects only when it adopts the open conformation and cannot induce
actin assembly. Consistently, a combination of candidate and unbiased
proteome-wide analyses indicates that mDia2 regulates the cellular
levels of proteasome substrate β-catenin and a number of ubiquitinated
actin-regulatory proteins. Hence, these findings add more complexity
to the mDia2 activity cycle by showing that the open conformation
may control actin dynamics also through actin-independent regulation
of the proteasome
Deep Proteome Profiling of Circulating Granulocytes Reveals Bactericidal/Permeability-Increasing Protein as a Biomarker for Severe Atherosclerotic Coronary Stenosis
Coronary atherosclerosis represents the major cause of
death in
Western societies. As atherosclerosis typically progresses over years
without giving rise to clinical symptoms, biomarkers are urgently
needed to identify patients at risk. Over the past decade, evidence
has accumulated suggesting cross-talk between the diseased vasculature
and cells of the innate immune system. We therefore employed proteomics
to search for biomarkers associated with severe atherosclerotic coronary
lumen stenosis in circulating leukocytes. In a two-phase approach,
we first performed in-depth quantitative profiling of the granulocyte
proteome on a small pooled cohort of patients suffering from chronic
(sub)Âtotal coronary occlusion and matched control patients using stable
isotope peptide labeling, two-dimensional LC–MS/MS and data-dependent
decision tree fragmentation. Over 3000 proteins were quantified, among
which 57 candidate biomarker proteins remained after stringent filtering.
The most promising biomarker candidates were subsequently verified
in the individual samples of the discovery cohort using label-free,
single-run LC–MS/MS analysis, as well as in an independent
verification cohort of 25 patients with total coronary occlusion (CTO)
and 19 matched controls. Our data reveal bactericidal/permeability-increasing
protein (BPI) as a promising biomarker for severe atherosclerotic
coronary stenosis, being down-regulated in circulating granulocytes
of CTO patients
Deep Proteome Profiling of Circulating Granulocytes Reveals Bactericidal/Permeability-Increasing Protein as a Biomarker for Severe Atherosclerotic Coronary Stenosis
Coronary atherosclerosis represents the major cause of
death in
Western societies. As atherosclerosis typically progresses over years
without giving rise to clinical symptoms, biomarkers are urgently
needed to identify patients at risk. Over the past decade, evidence
has accumulated suggesting cross-talk between the diseased vasculature
and cells of the innate immune system. We therefore employed proteomics
to search for biomarkers associated with severe atherosclerotic coronary
lumen stenosis in circulating leukocytes. In a two-phase approach,
we first performed in-depth quantitative profiling of the granulocyte
proteome on a small pooled cohort of patients suffering from chronic
(sub)Âtotal coronary occlusion and matched control patients using stable
isotope peptide labeling, two-dimensional LC–MS/MS and data-dependent
decision tree fragmentation. Over 3000 proteins were quantified, among
which 57 candidate biomarker proteins remained after stringent filtering.
The most promising biomarker candidates were subsequently verified
in the individual samples of the discovery cohort using label-free,
single-run LC–MS/MS analysis, as well as in an independent
verification cohort of 25 patients with total coronary occlusion (CTO)
and 19 matched controls. Our data reveal bactericidal/permeability-increasing
protein (BPI) as a promising biomarker for severe atherosclerotic
coronary stenosis, being down-regulated in circulating granulocytes
of CTO patients