13 research outputs found
Spatial Cross-Talk between Oxidative Stress and DNA Replication in Human Fibroblasts
MS-based proteomics has been applied
to a differential network
analysis of the nuclear–cytoplasmic subcellular distribution
of proteins between cell-cycle arrest: (a) at the origin activation
checkpoint for DNA replication, or (b) in response to oxidative stress.
Significant changes were identified for 401 proteins. Cellular response
combines changes in trafficking and in total abundance to vary the
local compartmental abundances that are the basis of cellular response.
Appreciable changes for both perturbations were observed for 245 proteins,
but cross-talk between oxidative stress and DNA replication is dominated
by 49 proteins that show strong changes for both. Many nuclear processes
are influenced by a spatial switch involving the proteins {KPNA2,
KPNB1, PCNA, PTMA, SET} and heme/iron proteins HMOX1 and FTH1. Dynamic
spatial distribution data are presented for proteins involved in caveolae,
extracellular matrix remodelling, TGFβ signaling, IGF pathways,
emerin complexes, mitochondrial protein import complexes, spliceosomes,
proteasomes, and so on. The data indicate that for spatially heterogeneous
cells cross-compartmental communication is integral to their system
biology, that coordinated spatial redistribution for crucial protein
networks underlies many functional changes, and that information on
dynamic spatial redistribution of proteins is essential to obtain
comprehensive pictures of cellular function. We describe how spatial
data of the type presented here can provide priorities for further
investigation of crucial features of high-level spatial coordination
across cells. We suggest that the present data are related to increasing
indications that much of subcellular protein transport is constitutive
and that perturbation of these constitutive transport processes may
be related to cancer and other diseases. A quantitative, spatially
resolved nucleus–cytoplasm interaction network is provided
for further investigations
Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress
We have used a subcellular spatial
razor approach based on LC–MS/MS-based
proteomics with SILAC isotope labeling to determine changes in protein
abundances in the nuclear and cytoplasmic compartments of human IMR90
fibroblasts subjected to mild oxidative stress. We show that response
to mild <i>tert</i>-butyl hydrogen peroxide treatment includes
redistribution between the nucleus and cytoplasm of numerous proteins
not previously associated with oxidative stress. The 121 proteins
with the most significant changes encompass proteins with known functions
in a wide variety of subcellular locations and of cellular functional
processes (transcription, signal transduction, autophagy, iron metabolism,
TCA cycle, ATP synthesis) and are consistent with functional networks
that are spatially dispersed across the cell. Both nuclear respiratory
factor 2 and the proline regulatory axis appear to contribute to the
cellular metabolic response. Proteins involved in iron metabolism
or with iron/heme as a cofactor as well as mitochondrial proteins
are prominent in the response. Evidence suggesting that nuclear import/export
and vesicle-mediated protein transport contribute to the cellular
response was obtained. We suggest that measurements of global changes
in total cellular protein abundances need to be complemented with
measurements of the dynamic subcellular spatial redistribution of
proteins to obtain comprehensive pictures of cellular function
p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress
<p>DNA replication initiation is a key event in the cell cycle, which is dependent on 2 kinases - CDK2 and CDC7. Here we report a novel mechanism in which p53 induces G1 checkpoint and cell cycle arrest by downregulating CDC7 kinase in response to genotoxic stress. We demonstrate that p53 controls CDC7 stability post-transcriptionally via miR-192/215 and post-translationally via Fbxw7β E3 ubiquitin ligase. The p53-dependent pathway of CDC7 downregulation is interlinked with the p53-p21-CDK2 pathway, as p21-mediated inhibition of CDK2-dependent phosphorylation of CDC7 on Thr376 is required for GSK3ß-phosphorylation and Fbxw7ß-dependent degradation of CDC7. Notably, sustained oncogenic high levels of active CDC7 exert a negative feedback onto p53, leading to unrestrained S-phase progression and accumulation of DNA damage. Thus, p53-dependent control of CDC7 levels is essential for blocking G1/S cell-cycle transition upon genotoxic stress, thereby safeguarding the genome from instability and thus representing a novel general stress response.</p
Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint
Depletion of DNA replication initiation factors such
as CDC7 kinase triggers the origin activation checkpoint in healthy
cells and leads to a protective cell cycle arrest at the G1 phase
of the mitotic cell division cycle. This protective mechanism is thought
to be defective in cancer cells. To investigate how this checkpoint
is activated and maintained in healthy cells, we conducted a quantitative
SILAC analysis of the nuclear- and cytoplasmic-enriched compartments
of CDC7-depleted fibroblasts and compared them to a total cell lysate
preparation. Substantial changes in total abundance and/or subcellular
location were detected for 124 proteins, including many essential
proteins associated with DNA replication/cell cycle. Similar changes
in protein abundance and subcellular distribution were observed for
various metabolic processes, including oxidative stress, iron metabolism,
protein translation and the tricarboxylic acid cycle. This is accompanied
by reduced abundance of two karyopherin proteins, suggestive of reduced
nuclear import. We propose that altered nucleo-cytoplasmic trafficking
plays a key role in the regulation of cell cycle arrest. The results
increase understanding of the mechanisms underlying maintenance of
the DNA replication origin activation checkpoint and are consistent
with our proposal that cell cycle arrest is an actively maintained
process that appears to be distributed over various subcellular locations
Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint
Depletion of DNA replication initiation factors such
as CDC7 kinase triggers the origin activation checkpoint in healthy
cells and leads to a protective cell cycle arrest at the G1 phase
of the mitotic cell division cycle. This protective mechanism is thought
to be defective in cancer cells. To investigate how this checkpoint
is activated and maintained in healthy cells, we conducted a quantitative
SILAC analysis of the nuclear- and cytoplasmic-enriched compartments
of CDC7-depleted fibroblasts and compared them to a total cell lysate
preparation. Substantial changes in total abundance and/or subcellular
location were detected for 124 proteins, including many essential
proteins associated with DNA replication/cell cycle. Similar changes
in protein abundance and subcellular distribution were observed for
various metabolic processes, including oxidative stress, iron metabolism,
protein translation and the tricarboxylic acid cycle. This is accompanied
by reduced abundance of two karyopherin proteins, suggestive of reduced
nuclear import. We propose that altered nucleo-cytoplasmic trafficking
plays a key role in the regulation of cell cycle arrest. The results
increase understanding of the mechanisms underlying maintenance of
the DNA replication origin activation checkpoint and are consistent
with our proposal that cell cycle arrest is an actively maintained
process that appears to be distributed over various subcellular locations
Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint
Depletion of DNA replication initiation factors such
as CDC7 kinase triggers the origin activation checkpoint in healthy
cells and leads to a protective cell cycle arrest at the G1 phase
of the mitotic cell division cycle. This protective mechanism is thought
to be defective in cancer cells. To investigate how this checkpoint
is activated and maintained in healthy cells, we conducted a quantitative
SILAC analysis of the nuclear- and cytoplasmic-enriched compartments
of CDC7-depleted fibroblasts and compared them to a total cell lysate
preparation. Substantial changes in total abundance and/or subcellular
location were detected for 124 proteins, including many essential
proteins associated with DNA replication/cell cycle. Similar changes
in protein abundance and subcellular distribution were observed for
various metabolic processes, including oxidative stress, iron metabolism,
protein translation and the tricarboxylic acid cycle. This is accompanied
by reduced abundance of two karyopherin proteins, suggestive of reduced
nuclear import. We propose that altered nucleo-cytoplasmic trafficking
plays a key role in the regulation of cell cycle arrest. The results
increase understanding of the mechanisms underlying maintenance of
the DNA replication origin activation checkpoint and are consistent
with our proposal that cell cycle arrest is an actively maintained
process that appears to be distributed over various subcellular locations
Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint
Depletion of DNA replication initiation factors such
as CDC7 kinase triggers the origin activation checkpoint in healthy
cells and leads to a protective cell cycle arrest at the G1 phase
of the mitotic cell division cycle. This protective mechanism is thought
to be defective in cancer cells. To investigate how this checkpoint
is activated and maintained in healthy cells, we conducted a quantitative
SILAC analysis of the nuclear- and cytoplasmic-enriched compartments
of CDC7-depleted fibroblasts and compared them to a total cell lysate
preparation. Substantial changes in total abundance and/or subcellular
location were detected for 124 proteins, including many essential
proteins associated with DNA replication/cell cycle. Similar changes
in protein abundance and subcellular distribution were observed for
various metabolic processes, including oxidative stress, iron metabolism,
protein translation and the tricarboxylic acid cycle. This is accompanied
by reduced abundance of two karyopherin proteins, suggestive of reduced
nuclear import. We propose that altered nucleo-cytoplasmic trafficking
plays a key role in the regulation of cell cycle arrest. The results
increase understanding of the mechanisms underlying maintenance of
the DNA replication origin activation checkpoint and are consistent
with our proposal that cell cycle arrest is an actively maintained
process that appears to be distributed over various subcellular locations
Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint
Depletion of DNA replication initiation factors such
as CDC7 kinase triggers the origin activation checkpoint in healthy
cells and leads to a protective cell cycle arrest at the G1 phase
of the mitotic cell division cycle. This protective mechanism is thought
to be defective in cancer cells. To investigate how this checkpoint
is activated and maintained in healthy cells, we conducted a quantitative
SILAC analysis of the nuclear- and cytoplasmic-enriched compartments
of CDC7-depleted fibroblasts and compared them to a total cell lysate
preparation. Substantial changes in total abundance and/or subcellular
location were detected for 124 proteins, including many essential
proteins associated with DNA replication/cell cycle. Similar changes
in protein abundance and subcellular distribution were observed for
various metabolic processes, including oxidative stress, iron metabolism,
protein translation and the tricarboxylic acid cycle. This is accompanied
by reduced abundance of two karyopherin proteins, suggestive of reduced
nuclear import. We propose that altered nucleo-cytoplasmic trafficking
plays a key role in the regulation of cell cycle arrest. The results
increase understanding of the mechanisms underlying maintenance of
the DNA replication origin activation checkpoint and are consistent
with our proposal that cell cycle arrest is an actively maintained
process that appears to be distributed over various subcellular locations
Receiver operating characteristics curves for Mcm5 and NMP22 tests for the detection of bladder cancer in all studied patients with valid test results.
<p>Receiver operating characteristics curves for Mcm5 and NMP22 tests for the detection of bladder cancer in all studied patients with valid test results.</p
Performance of Mcm5 and NMP22 tests for bladder carcinoma detection for all patients with test results available.
<p>Abbreviations: CI, 95% confidence interval; NPV, negative predictive value; PPV, positive predictive value; Sens, sensitivity; Spec, specificity.</p