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Research Article
FE65 regulates and interacts with the Bloom syndrome
protein in dynamic nuclear spheres – potential
relevance to Alzheimer’s disease
Andreas Schrötter1, Thomas Mastalski1, Fabian M. Nensa1, Martin Neumann1, Christina Loosse1,
Kathy Pfeiffer1, Fouzi El Magraoui2, Harald W. Platta2, Ralf Erdmann2, Carsten Theiss3, Julian Uszkoreit4,
Martin Eisenacher4, Helmut E. Meyer4, Katrin Marcus1,* and Thorsten Müller1,*,`
1
Functional Proteomics, Medizinisches Proteom-Center, Ruhr-University Bochum, 44801 Bochum, Germany
Institute of Physiological Chemistry, System Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany
Institute of Anatomy and Molecular Embryology, Ruhr-University Bochum, 44780 Bochum, Germany
4
Medizinisches Proteom-Center, Ruhr-University Bochum, 44801 Bochum, Germany
2
3
*These authors contributed equally to this work
`
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 13 March 2013
Journal of Cell Science 126, 2480–2492
2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.121004
Summary
The intracellular domain of the amyloid precursor protein (AICD) is generated following cleavage of the precursor by the c-secretase
complex and is involved in membrane to nucleus signaling, for which the binding of AICD to the adapter protein FE65 is essential.
Here we show that FE65 knockdown causes a downregulation of the protein Bloom syndrome protein (BLM) and the
minichromosome maintenance (MCM) protein family and that elevated nuclear levels of FE65 result in stabilization of the BLM
protein in nuclear mobile spheres. These spheres are able to grow and fuse, and potentially correspond to the nuclear domain 10.
BLM plays a role in DNA replication and repair mechanisms and FE65 was also shown to play a role in DNA damage response in
the cell. A set of proliferation assays in our work revealed that FE65 knockdown in HEK293T cells reduced cell replication. On the
basis of these results, we hypothesize that nuclear FE65 levels (nuclear FE65/BLM containing spheres) may regulate cell cycle reentry in neurons as a result of increased interaction of FE65 with BLM and/or an increase in MCM protein levels. Thus, FE65
interactions with BLM and MCM proteins may contribute to the neuronal cell cycle re-entry observed in brains affected by
Alzheimer’s disease.
Key words: Morbus Alzheimer, Mass spectrometry, Neurodegeneration, Nuclear translocation, Signal transduction, Stable FE65 knockdown model
Introduction
Almost 15 years ago, the interaction of the amyloid precursor
protein (APP) and the FE65 adapter protein family was described
(Fiore et al., 1995; Guénette et al., 1996). It was suggested that
FE65 might have an important impact on APP related
mechanisms and in the pathogenesis of Alzheimer’s disease
(AD). FE65 consists of three functional domains, two
phosphotyrosine binding domains (PTB) and a WW domain
(Ermekova et al., 1997), pointing to a highly interesting molecule
potentially involved in various intracellular pathways. The
YENPTY sequence in APP constitutes the interaction motif,
which is responsible for binding to the PTB2 domain of FE65
(Bressler et al., 1996; Fiore et al., 1995; McLoughlin and Miller,
1996). In a similar fashion, the two other members of the FE65
family, FE65L1 and FE65L2, bind to APP (Duilio et al., 1998;
Guénette et al., 1996). A role for FE65 in AD may result from the
effect of FE65 proteins on APP processing, with FE65 proteins
contributing to Ab generation in neurons and in the brain (Sabo
et al., 1999; Suh et al., 2011; Wang et al., 2004). Alternatively,
the observations that phosphorylation of the APP intracellular
domain (AICD) at T668 is necessary for its interaction with FE65
in the nucleus (Chang et al., 2006), that phosphorylation of T668
leads to FE65 nuclear translocation (Nakaya et al., 2008), and
that pT668 APP is elevated in AD brain (Shin et al., 2007)
suggest a role for nuclear FE65 and AICD in AD. Furthermore,
FE65 knockout mice are impaired for the hidden platform in the
Morris water maze test and showed defective early-phase
LTP (Wang et al., 2009). Although the underlying molecular
mechanisms are unknown, these data suggest a possible role for
FE65 proteins in the learning and memory deficits found in AD.
In support of a role for the FE65/APP interactions in AD, there is
evidence for a protective effect of an FE65 polymorphism in the
AD population over 75 years (Hu et al., 1998). This bi-allelic
polymorphism in intron 13 generates an altered FE65 protein
lacking part of the APP binding site and binds APP less
efficiently than FE65 (Guénette et al., 2000; Hu et al., 2002;
Lambert et al., 2000). At present whether FE65 proteins
contribute to AD is unclear, but a better understanding of the
role of nuclear FE65 is necessary to determine whether it plays a
role in AD pathophysiology.
FE65 forms a multimeric complex with AICD and the histone
acetyltransferase TIP60, which activates transcription of a
reporter gene construct raising the possibility that this complex
regulates transcription (Cao and Südhof, 2001). Furthermore,
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FE65 mediates cell cycle re-entry in AD
FE65 and FE65L1 stimulate APP intracellular domain (AICD)
generation (Chang et al., 2003; Wiley et al., 2007; Xie et al.,
2007). However, the identification of several putative target
genes have been controversially discussed (Baek et al., 2002;
Bimonte et al., 2004; Müller et al., 2007; Telese et al., 2005; von
Rotz et al., 2004). Elucidation of the role of FE65 in a gene
expression complex is further complicated by the observation
that FE65 itself triggers robust gene expression in a reporter
assay (Yang et al., 2006). Microscopy studies revealed that
the above described complex consisting of AICD, FE65 and
TIP60, is visible as speckles in the nucleus (Muresan and
Muresan, 2004; von Rotz et al., 2004). Further evidence for FE65
function in the nucleus was obtained by X-ray treatment of
mouse embryonic fibroblasts isolated from FE65 knockout mice,
which revealed significantly higher levels of the histone gammaH2AX than in controls, a typical cellular response to DNA
damage (Minopoli et al., 2007). Notably, increased nuclear DNA
oxidation is known to occur in AD caused by elevated reactive
oxygen species (Santos et al., 2012). Although our present
knowledge is limited, the presented facts point to a nuclear
function for this adapter protein, as well as to a putative role in
neurodegeneration.
In order to gain further mechanistic insights into FE65
function, we established a FE65 knockdown cell culture model,
which was screened for effects by state-of-the-art proteomics
and functional studies. We were able to show that FE65 plays
an important role in the localization of a certain set of proteins,
including the Bloom syndrome protein (BLM), which is
involved in DNA replication and repair. In FE65 knockdown
cells, BLM and other proteins are massively aggregated in
the ER. Functional studies demonstrate that FE65 knockdown
cells are characterized by a significantly lower level of
proliferation and DNA replication. In contrast, FE65
overexpression results in a re-localization of BLM to nuclear
mobile spheres. Size, growth and fusion of these spheres may be
associated with the cell cycle and DNA replication, for which
FE65 is a key regulator. We hypothesize that FE65 plays a
crucial role in neuronal cell cycle re-entry, which is reported to
occur in AD.
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Results
Knockdown of FE65 significantly disturbs the cellular
proteome
FE65 is an APP binding protein, whose physiological role as well
as pathophysiological role in AD is poorly understood. In order to
gain further functional insights, we generated five stable FE65
HEK293T knockdown clones using selectable shRNAmir
constructs. As a control, we established five cell lines
transfected with random shRNAmir constructs. A significant
knockdown was achieved as demonstrated by immunoblotting of
cell lysate samples (Fig. 1A). Densitometric analysis of five
stable knockdown versus five control cell lines revealed an
effective downregulation of the FE65 protein with high statistical
significance (Fig. 1B, downregulation to 39.6%, P,1022).
Moreover, significant downregulation of the FE65 transcript
(using GAPDH as reference protein) was also proven by
quantitative real-time PCR (qPCR) analysis (Fig. 1C, P,1024,
higher dCt values correspond to lower RNA levels in comparison
to the control) with a calculated remaining gene expression of
40.3% (+3.9%, 23.6%). As a redundancy of the FE65 protein
family is discussed in the literature (Guénette et al., 2006) and in
order to preclude silencing of the FE65-like proteins, we also
determined the expression of FE65L1 (Fig. 1D) and FE65L2
(Fig. 1E) by qPCR. However no significant differences of the
like protein expression in FE65 knockdown cells compared to
controls were evident. Subsequently, total protein extracted from
the five established stable knockdown clones and the five stable
control clones were used for a differential proteome approach
(Fig. 2A), which we successfully applied before (Müller et al.,
2011b; Müller et al., 2011a; Spitzer et al., 2010). Using the LTQ
Orbitrap Velos mass spectrometry instrument, we identified
10,819 proteins in total. Quantification of these proteins was
performed using the label-free spectral count based approach
(Müller et al., 2011b; Müller et al., 2011a; Spitzer et al., 2010).
Among the total identified proteins, 136 revealed significant
abundance changes (supplementary material Table S3, P,0.05),
whereas 30 proteins correspond to the so called black-and-white
list, which are proteins identified only in one group with at least
three measured values (supplementary material Table S3, last
Fig. 1. Generation of a stable FE65 cell culture knockdown
model. (A) Following transfection with selectable shRNAmir
constructs for FE65, stable HEK293T cells showed prominent
knockdown of FE65 in five clones (KD) versus five controls (C) by
immunoblot analysis (stably transfected with a control shRNAmir
construct). (B) Densitometry, using b-actin as a reference, proved the
significance of the knockdown (P,1022, n55). (C) Significant
knockdown was also evident at the FE65 expression level (P,1024,
n55). Significance was calculated using the dCt values, which are
illustrated in the figure (higher dCt values correspond to lower RNA
levels and vice versa). Knockdown efficiency was calculated
according to Livak and Schmittgen (Livak and Schmittgen, 2001).
FE65 levels were silenced to 40.3% (+3.9%, 23.6%). The expression
level of FE65L1 (D) as well as that of FE65L2 (E) was unchanged in
FE65 knockdown cells versus controls.
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Fig. 2. See next page for legend.
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FE65 mediates cell cycle re-entry in AD
section). Black-and-white proteins might be highly interesting
candidate proteins but might also correspond to false-positive
results. Thus, we focused on the proteins with significant
abundance changes for further experiments. The choice of
candidates for subsequent validation experiments was derived
from a ternary strategy: initially, we evaluated the significance of
the results from the label-free study (statistical significance, ratio
of abundance changes as given in supplementary material Table
S3). Next, protein candidate selection was performed with the
help of the pathway analysis software IPA (Ingenuity Pathway
Analysis) and STRING (Search Tool for the Retrieval of
Interacting Genes/Proteins). Detailed data analysis strategy is
described in Materials and Methods. Finally, recent literature
findings with respect to FE65 and AD were manually assessed and
aligned with the pathway results. Data upload in the mentioned
software tools resulted in the pathway cartoons that are given in
Fig. 2B (IPA) and Fig. 2C (STRING, excerpt, please find the full
pathway cartoon in supplementary material Fig. S6), respectively.
Data analysis in IPA pointed to an impairment of a network
involved in DNA replication, recombination and repair (called Top
Network in IPA) including the MCM (minichromosome
maintenance) protein family (MCM2–MCM7), which are all
quantified as significantly less abundant proteins in FE65
knockdown cells. Another top network identified by IPA (not
shown) was similarly entitled with DNA replication, recombination
and repair (cell death, post-translational modifications) and pointed
to the proteins BLM (Bloom syndrome), TERF2 (telomeric repeatbinding factor 2) as potentially interesting candidates. The interplay
of MCM proteins was also evident using STRING for data
interpretation (Fig. 2C). BLM and TERF2 were illustrated as
associated proteins. The candidate KAT7 (TIP60_2) was another
interesting candidate as it belongs to the family of histone
acetyltransferases of which KAT5 (TIP60) is known to interact
with FE65 (Cao and Südhof, 2001).
Abundance of proteins involved in DNA replication and
repair are significantly reduced upon FE65 knockdown
For subsequent experiments we selected the MCM3 protein as
representative for the MCM family as this protein was identified
with high abundance (spectral index .45 on average). Moreover,
MCM3 was found 1.5-fold less abundant in the knockdown cells
with high significance (P,0.025). As a second candidate and
representative of proteins involved in DNA repair, we selected
the BLM protein, which was 7.6-fold less abundant in the FE65
knockdown cells (P,0.01). Finally, KAT7 was selected, as this
Fig. 2. Label-free proteomics pipeline with subsequent pathway analysis.
(A) For the identification of differentially abundant proteins in stable FE65
knockdown clones versus controls, total cell lysates were separated using
SDS-PAGE. Each lane (10 lanes in total) was cut three times (a, b, c),
resulting in 30 gel pieces, which were subsequently digested using trypsin.
Extracted peptides were identified using the LTQ Orbitrap Velos mass
spectrometer. Differentially abundant proteins were identified using an MS
label-free analysis followed by data confirmation and functional studies.
(B) Candidate protein lists were uploaded into IPA (for details see Materials
and Methods) revealing changes in cellular mechanisms for DNA replication,
recombination and repair. (C) In a similar fashion, the software STRING was
used to identify cellular networks altered by FE65 loss. Symbols in B and C
correspond to protein abbreviations, which were assembled in a network by
the corresponding pathway tool.
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protein might be able to bind FE65 similar to its family member
protein KAT5.
MCM3 downregulation was also evident by immunoblotting
samples from the five knockdown clones versus five controls
(Fig. 3A). Using b-actin as reference, densitometric analysis
revealed significance of our findings for MCM3 (Fig. 3B,
P,0.05). Gene expression changes caused by FE65 and its
interacting proteins have been discussed in several publications
(for review see Müller et al., 2008). In order to identify whether
MCM3 is regulated at the mRNA level, we performed
quantitative real-time PCR with GAPDH as expression control.
Indeed, qPCR revealed a significant reduction in MCM3 gene
expression to 73.6% (+2.2%, 22.2%; Fig. 3C, P,1023). In order
to study the subcellular localization of the MCM3 protein, we
next stained fixed FE65 knockdown and control cells using
immunofluorescence (Fig. 3D). MCM3 was predominantly
localized to the nucleus, and a differing localization in
knockdown and control cells was not evident. In a similar
fashion, we were able to validate our findings of different
BLM protein abundance using immunoblotting (Fig. 3E).
Densitometry revealed significant lower BLM abundance in the
knockdown cells (Fig. 3F, P,0.05). BLM regulation was also
evident at the mRNA level supporting the significant
downregulation of BLM in KD cells to 71.5% (+4.3%, 24%;
Fig. 3G, P,1023; higher dCt values indicate downregulation).
However in contrast to the MCM3 protein, a significantly
different subcellular localization of the BLM protein was
observed in the FE65 knockdown cells by immunofluorescence
staining (Fig. 3H, lower row, right). Stable control cells (upper
row) revealed a nuclear background staining of BLM with
distinct nuclear spots (nuclear foci) in all cells (Hoechst stain was
used to visualize the nucleus of cells) similar to the phenotype of
AICD-, FE65-, TIP60-transfected cells. In contrast, nuclear spots
were completely absent in the FE65 knockdown cells (lower
row). Instead, a single distinct perinuclear signal was evident
potentially localized to the ER.
In order to test whether the ER conformation might be affected
by FE65 in general, we used an ER live cell tracker in control and
knockdown cells (supplementary material Fig. S1A). The
knockdown cells revealed a prominent focused perinuclear ER
staining in contrast to the control cells, in which the ER-tracker
showed a more uniform pattern around the nucleus.
To further investigate the hypothesis of ER protein localization
changes caused by the FE65 knockdown, we investigated the
protein PRDX4 (peroxiredoxin 4), a family member of PRDX
proteins known to be located in the ER (Tavender et al., 2008).
PRDX4 signal was preferentially detected in the ER in control
cells (supplementary material Fig. S1B, upper row). Similar to
the BLM protein, a prominent change in PRDX4 localization was
evident in the fixed knockdown cells with structures compatible
to the ER live cell tracker stain. To confirm that these changes in
protein distribution were dependent on FE65, we overexpressed a
FE65-dsRED construct in the knockdown cells in order to
evaluate PRDX4 localization following FE65 reconstitution
(supplementary material Fig. S2). As expected, PRDX4
localization in rescued KD cells was similar to control cells
(green arrow).
In contrast to MCM3 and BLM, validation experiments for
the candidate KAT7 failed (detailed results are given in
supplementary material Fig. S3). Taken together, our mass
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Fig. 3. MCM3 and BLM regulation in stable FE65 knockdown
cells. (A) Immunoblotting, using b-actin as control, revealed a lower
abundance of MCM3 in the knockdown cells (KD1–KD5) versus
controls (C1–C5). (B) Densitometry proved the significance of our
findings (P,0.05; five knockdown versus five control samples;
n55). (C) qPCR analysis of MCM3 indicated downregulation at the
mRNA level as well (P,1023, n55). Significance was calculated
using the dCt values (higher dCt values correspond to lower RNA
levels and vice versa). (D) Immunofluorescence staining of MCM3
(red) revealed prominent nuclear staining without any signal
localization difference between knockdown and control cells
[Hoechst (blue) was used as counterstain]. (E) Lower abundance in
knockdown cells versus controls was also validated for BLM.
(F) Densitometry proved the significance (P,0.05, n55).
(G) qPCR analysis indicated a significant downregulation at the
mRNA level (P,1023, n55; comparable to C; higher dCt values
indicate downregulation in qPCR). (H) Immunofluorescence
staining of BLM revealed prominent differences between control
and knockdown cells. Multiple nuclear spots were evident in the
controls. No nuclear spot-like signal was detectable in the FE65
knockdown cells. In contrast, an extranuclear signal was evident.
spectrometry based approach is not errorless but was able to
identify FE65 knockdown dependently regulated proteins.
FE65 knockdown resulted in a significant decrease in
cell proliferation
The redistribution of BLM, which is involved in DNA
replication, from the nucleus to the ER may have functional
consequences for cell proliferation. In addition, a significantly
reduced abundance of MCM proteins in FE65 knockdown cells
might impact DNA replication and repair. In order to examine
cell proliferation in FE65 knockdown cells, we plated equal
numbers of cells in culture dishes and visualized the same field
over 96 hours of growth (Fig. 4A). Knockdown cells revealed
less proliferation within this timeframe, whereas control cells
were confluent after 96 hours. As control and knockdown cells
were established with a similar vector background containing a
turbo GFP cassette, we next evaluated and quantified cell
proliferation using the GFP signal. As outlined in Fig. 4B the
GFP fluorescence ratio (control/knockdown) significantly
increased over time (96 hours) providing evidence for elevated
proliferation of the control cells compared to knockdown cells.
The difference in GFP signal between control and FE65 KD after
96 hours of growth was highly significant (Fig. 4B, P,1028).
Finally, we used the xCELLigence System (Roche, Switzerland)
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FE65 mediates cell cycle re-entry in AD
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Fig. 4. FE65 deficiency reduces cell proliferation. (A) An equal number of control and FE65 knockdown cells were plated and monitored for cell growth and
proliferation over 96 hours. Control cells were grown to complete confluence in 96 hours whereas proliferation was slower in FE65 knockdown cells (n54).
(B) GFP fluorescence of control and FE65 knockdown cells (both stable clones contain a turbo GFP cassette) was monitored from 12 to 96 hours after plating.
GFP intensity ratio (control/knockdown) revealed prominent higher proliferation in the controls over time. Quantification of the GFP fluorescence after 96 hours
demonstrated highly significant differences between the two clones (P,1028, n54). (C) Cell index doubling time was determined using the xCELLigence
System (Roche, Switzerland). Doubling time from 12 to 96 hours after plating the cells was significantly higher in the knockdown cells (P,1024, n54).
(D) Extent of DNA synthesis/replication was studied using EdU, which incorporates into newly synthesized DNA of proliferating cells (BrdU analogue).
Following Alexa Fluor 594 staining, the signal intensity in FE65 knockdown cells was clearly lower than in control cells (n54).
to quantify the cell doubling time in control versus knockdown
cells (Fig. 4C). Calculated for a timeframe from 12 to 96 hours,
the cell index doubling time was significantly higher in FE65 KD
than control cells (P,1024). Since proliferation is accompanied
by synthesis of new DNA, we next studied DNA replication using
the EdU (BrdU analogue) cell assay. Indeed, knockdown cells
revealed less EdU incorporation than control cells (Fig. 4D,
right) and Hoechst stain was unaffected in both conditions
(Fig. 4D, middle). Collectively, these data indicate that DNA
replication/proliferation is significantly reduced in FE65
knockdown cells.
BLM and MCM3 might play a crucial role in FE65-dependent
DNA replication/repair mechanisms. Interestingly, the nuclear
localization of BLM resembles the nuclear spot-like structures
known to originate in AICD-, FE65-, TIP60-transfected cells
(von Rotz et al., 2004).
BLM colocalizes with FE65 in nuclear structures and binds
to FE65 in co-immunoprecipitation assays
We hypothesized that the similar nuclear staining pattern of BLM
and FE65 may be due to a physical interaction between these two
proteins. To test this hypothesis, we overexpressed different
combinations of AICD, FE65 and TIP60 in HEK293 cells until
we obtained the reported nuclear spot phenotype (Müller et al.,
2013). As transfection of FE65-EGFP/TIP60 resulted in the
largest percentage of cells with nuclear spots, we selected these
cells to determine whether FE65 and BLM colocalized. As
demonstrated in Fig. 5A, FE65–EGFP/TIP60-positive cells
(GFP, green) reveal a clear colocalization with BLM (red),
which is also evident in the overlay as well as in the zoom image in
Fig. 5C. A similar phenotype is also observed by single
overexpression of a BLM-EGFP construct (Fig. 5D) and the
colocalization of BLM and TIP60 is also evident in FE65-EGFP/
TIP60-transfected cells (Fig. 5B). We next sought evidence for a
BLM–FE65 interaction using co-immunoprecipitation (Fig. 5E).
Using combined lysates from FE65 and BLM–EGFP expressing
cells, we precipitated BLM with anti-BLM or anti-GFP antibody.
In both samples FE65 was co-immunoprecipitated. As the control
revealed some unspecific BLM interaction to the beads we
performed another co-immunoprecipitation for FE65. BLM could
be identified in the FE65 IP as well supporting the interaction of
both proteins.
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Fig. 5. BLM colocalizes and interacts with FE65.
(A) FE65-EGFP/TIP60-transfected cells are characterized
by a spot-like phenotype in the nucleus (GFP) (see also
supplementary material Fig. S5). BLM colocalizes to the
nuclear structures (BLM in red, Hoechst nuclear stain in
blue). (C) An enlarged view of part of the overlay image
shows nuclear ring-like structures in contrast to the spots
described in the literature. (B) BLM also colocalizes to TIP60
in FE65-GFP/TIP60-transfected cells. (D) The phenotype of
BLM-EGFP-transfected cells resembles that of FE65-EGFP/
TIP60-transfected cells. (E) Co-immunoprecipitation
supports the interaction of FE65 to BLM. (F) In contrast,
MCM3 did not show clear colocalization to the FE65 spots
but this does not preclude an interaction of both proteins in
nuclear spheres.
In contrast to BLM, MCM3 nuclear localization is not
restricted to FE65-positive nuclear spots (Fig. 5F), but this does
not preclude an interaction between the two proteins. The KAT7
protein did not show colocalization with the FE65 signal
(supplementary material Fig. S4).
BLM and FE65 localize to dynamic nuclear spheres
In order to better understand the nature of the nuclear foci in which
FE65 localizes, we re-examined FE65-GFP/TIP60-transfected
cells (Fig. 6A). A higher magnification of FE65-positive nuclear
foci revealed a ring-like structure, which was also evident in the
brightfield microscopy image. The shape of the FE65-positive
nuclear structure was further examined with serial digital confocal
microscopy sections. The sections from top to bottom (Fig. 6BI)
and a profile view image (Fig. 6BII) of the EGFP structure show a
hollow sphere. The diameter of the largest spheres were measured
and the average size is of 2.5 mm. Microscopy observations of
these structures in living cells showed that these sphere-like
structures are highly mobile. In order to evaluate the dynamics of
these circular objects, we used live cell fluorescence imaging and
tracked the GFP signal in FE65/TIP60-transfected cells (Fig. 6C
and supplementary material Movie 1). Video microscopy
demonstrated that smaller dots are able to grow and fuse to
larger compartments appearing as spheres. In addition to the
mobility of the nuclear spheres, the different size of these objects
in cells was striking. We have classified the FE65–EGFP spheres
into two different types, either small (supplementary material Fig.
S4, red arrow) or large, with the later showing a clear hollow
sphere structure (supplementary material Fig. S4, yellow arrow).
Each cell shows similar types of FE65–EGFP-positive spheres,
but the average sphere size varies from cell to cell (supplementary
material Fig. S4). Finally, we monitored the growth and fusion of
different spheres as shown in frames 1 to 4 in Fig. 6C (see also
supplementary material Movie 1). The fusion of three different
spheres (indicated by the yellow arrow) occurs within 6 minutes
(movie was recorded for 3 hours in total). Following fusion, the
size of these objects in frame 4 is clearly enlarged in comparison
to frame 1. The lower images correspond to upper images
processed by the EMBOSS filter in order to further improve image
quality by relief transformation. The size of the hollow is also
enlarged after sphere fusion. Using electron microscopy we
subsequently monitored the nuclear spheres in more detail
(Fig. 6D). The first image in the upper row demonstrates large
circular structures with a reduced electron density in the center of
some structures. Other cells bear a greater number of smaller
spheres (middle), whereas control-vector-transfected cells (right)
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Fig. 6. FE65/TIP60 as well as FE65 alone is present in nuclear
mobile spheres, which are able to grow and fuse with each
other. (A) FE65–EGFP and TIP60 were overexpressed (OE) in
HEK293T cells. GFP fluorescence signal (middle) revealed nuclear
ring-like structures, which are also evident in the brightfield image
(left and overlay right). Higher magnification (lower row) shows the
nuclear ring-like structures in more detail, raising suspicion of a
sphere-like structure. (B) Serial confocal microscope images from
the top (I) to the bottom (VI) of these nuclear objects were recorded
in order to identify the underlying geometric structure in more
detail. Serial sections as well as side-view image (B9) point to a
sphere-like structure. The diameter of the largest spheres was
2.5 mm on average with no EGFP signal in the center of these
objects. (C) Live-cell fluorescence microscopy monitoring EGFP
was undertaken in order to monitor the sphere dynamics (lower row
correspond to images in upper row but were processed using the
EMBOSS filter generating a relief structure). Some cells had
smaller spheres, some enlarged circular objects with no EGFP
signal in the center, but most cells had a mixture of different sized
spheres within the nucleus, putatively pointing to a cell-cycledependent effect. Cells were monitored over a total time period of
3 hours (see supplementary material Movie 1). Spheres were high
mobility within seconds and able to grow and fuse. Fusion occurred
within few minutes as shown in frames 1–4 (corresponding to
6 minutes). Three circular objects marked by the yellow arrow
sequentially fused ending up in an enlarged sphere with a larger
inner hole compared to that in frame 1. (D) Electron microscopy
imaging of FE65-EGFP/TIP60-transfected cells validated the
finding of large spheres (left) with low electron density in the center
(brightfield image of FE65-EGFP/TIP60-transfected cells is given
in lower row for comparison). Other cells (middle) had more
smaller spheres per nucleus. In contrast, control-vector-transfected
cells did not show any nuclear sphere aggregation. (E) The
described phenotype is evident in FE65-EGFP-transfected cells.
However, the number of sphere-positive cells is remarkably
enriched by the transfection of the FE65 interacting protein TIP60.
Thus, we used FE65-EGFP/TIP60 transfections for most
microscopy experiments in order to obtain a higher percentage of
cells with the spheres.
did not show any nuclear sphere-like structures. Our brightfield
microscopy data are in agreement with the EM data (lower row).
Both types of spheres (small, large with inner hole) are evident in
FE65-EGFP-transfected cells (Fig. 6E, left). However, the extent
of sphere-positive cells is remarkably increased by co-transfection
of FE65 with TIP60 (Fig. 6E, right). The nuclear sphere-like
structures demonstrate highly dynamic changes within seconds,
growth and fusion within a few minutes as well as different sizes
in different nuclei. The identification of BLM as an FE65-binding
protein whose nuclear localization and dynamics within the
nucleus is FE65 dependent, provides interesting insights into the
possible consequences of nuclear FE65.
Discussion
In the present study, the proteomic analysis of stable FE65
knockdown cells revealed the downregulation of MCM3 and
BLM proteins. Furthermore, FE65 and BLM are able to interact
in the co-immunoprecipitation assay. In FE65 knockdown cells,
BLM localization was shifted from the nucleus presumably to the
ER and overexpression of FE65 restored BLM localization to
nuclear spheres. These nuclear spheres are able to grow and fuse
and are highly dynamic. FE65 knockdown also produced a
reduction in cellular proliferation due to an increase in the
doubling time possibly due to slower DNA replication resulting
from BLM mislocalization.
BLM is a DNA helicase present in nuclear foci that are sites of
DNA synthesis during the late S and G2/M phases of the cell
cycle (Bhattacharyya et al., 2009). The MCM (minichromosome
maintenance) protein family plays an important role in S-phase
genome stability in the context of DNA replication, damage and
repair (Bailis and Forsburg, 2004; Mincheva et al., 1994). MCM
is a hexamer of six related proteins (MCM2–7) forming a nuclear
circular structure (Cortez et al., 2004) and loss of MCM function
causes DNA damage and genome instability (Bailis and
Forsburg, 2004). Both BLM and members of the MCM family
have been identified with significantly lower abundance in FE65
knockdown cells suggesting a functional relationship between
these proteins and FE65.
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Journal of Cell Science 126 (11)
Journal of Cell Science
The present knowledge for FE65 function includes a role for
FE65 in DNA repair, especially following DNA double strand
breaks (Minopoli et al., 2007; Stante et al., 2009). However, the
molecular mechanism is unknown. Our work suggests that the
contribution of FE65 to DNA repair may be mediated by its
effects on BLM and MCM proteins, which is further strengthened
by the finding that the localization of BLM is changed from a
nuclear spot-like phenotype to localization presumably in the ER
in the FE65 knockdown cells. In cells lacking FE65, BLM
mislocalization likely prevents its participation in important
nuclear DNA replication or repair processes (Fig. 7). Our work
also demonstrates that BLM colocalizes with FE65 in nuclear
sphere-like structures and that the two proteins interact. As BLM
Fig. 7. Suggested mechanism for the role of FE65 in neurodegeneration.
FE65 plays a pivotal role in DNA replication putatively causing neuronal
cell-cycle re-entry in Alzheimer’s disease. FE65 is an important binding
protein of APP. Interaction of the FE65 PTB2 domain with the YENPTY
motif in APP depends on the phosphorylation status of T668 in the VTPE
motif of APP as well as on APP cleavage. As a result, binding of FE65 to APP
is weakened, and liberated FE65 translocates into the nucleus stabilizing
the Bloom syndrome protein BLM. The interaction is associated with DNA
replication and cell proliferation changes. Putatively involved proteins are
TERF2 (telomeric repeat-binding factor 2) and the MCM protein family
(minichromosome maintenance). In contrast, the knockdown of FE65 results
in ER protein aggregation including BLM and PRDX4 (peroxiredoxin 4).
In neurons, re-initiating DNA replication (neuronal cell cycle re-entry) is
known to result in apoptosis. Thus, higher neuronal FE65 levels (known to be
present in AD brains) or elevation of nuclear FE65 results in
neurodegeneration.
contains a PPKP amino acid motif near its C-terminal end, we
hypothesize that the interaction occurs at the WW domain in
FE65, which is already known to interact with the PPLP motif in
Mena (Ermekova et al., 1997). Thus, the reduced BLM protein
levels in nuclear spheres appears to result from the
downregulation of its interacting protein FE65 and may also be
due to reduced BLM expression. FE65 also seems to influence the
expression of MCM3 at the mRNA level by an as yet unknown
mechanism.
Different microscopic techniques revealed the existence of
FE65/TIP60 and BLM in highly mobile nuclear spheres that
are able to grow and fuse with each other. Nuclear AICD,
FE65, TIP60-dependent spot-like structures have already been
described (Konietzko et al., 2010). Here, we show colocalization
of the BLM protein with FE65/TIP60 in nuclear foci suggesting
that these foci correspond to the nuclear domain 10 (ND10),
which is a structure of 0.2 to 1 mm size composed of proteins
such as PML (promyelocytic leukemia protein) and BLM
(Yankiwski et al., 2001). However, the largest spheres we
observed correspond to a diameter of 2.5 mm on average. The
discrepancy in the size of these structures might point to an
unknown nuclear structure, or a consequence of overexpression
of FE65/Tip60 on ND10 foci. The possibility of these structures
being different from ND10 is further supported by the finding
that BLM is not part of ND10 during DNA synthesis when it
colocalizes instead with the Werner syndrome gene product
(Yankiwski et al., 2000). Thus, the size as well as the fusion of
FE65-positive nuclear spheres may depend on the cell cycle with
tiny spheres being part of ND10 and large spheres being separate
from ND10. Collectively, our data suggest that nuclear FE65
plays a pivotal role in cellular DNA replication/repair
mechanisms mediated by BLM and MCM proteins.
DNA replication and repair appears to be of central relevance
in brain development since BLM expression is closely related to
neuronal development (Hachiya et al., 2001; Morimoto et al.,
2002). FE65 proteins also play an important role during brain
development, in cortical plate formation and axonal path finding
(Guénette et al., 2006). DNA replication and repair may also be
of central relevance to AD pathogenesis. Given that higher
nuclear levels of FE65 protein result from T668 phosphorylation
of APP and that pT668 APP is increased in AD, higher nuclear
FE65 levels may occur in AD and may impair BLM and the
MCM protein family function in DNA replication and repair.
Functionality of DNA repair is highly important in AD, where
DNA damage by oxidation possibly involving Ab or reactive
oxygen species, belongs to the earliest detectable events during
the progression from healthy aging to dementia (Coppedè and
Migliore, 2010; Kruman et al., 2002). Notably, the number of
BLM foci increases in response to ionizing radiation (Wu et al.,
2001), which is able to induce reactive oxygen species
(Yamamori et al., 2012), and BLM overexpression exacerbates
the sensitivity to DNA damaging agents (Mirzaei et al., 2011).
DNA damage is normally repaired by mechanisms involving
BLM and the MCM family, responsible for genome replication
but also repair. Presently, the relationship between oxidative
damage in AD and nuclear FE65 protein levels in neurons is
unknown.
Slower proliferation and a reduction in DNA replication were
observed in the FE65 knockdown cells. The molecular
mechanism may involve lower BLM and MCM protein levels
in the nucleus. Putatively elevated nuclear FE65 levels in AD
Journal of Cell Science
FE65 mediates cell cycle re-entry in AD
2489
might promote proliferation (and thereby cell cycle re-entry), for
which the interaction of BLM with FE65 might be essential. Cell
cycle re-entry in neurons occurs in AD (Herrup, 2012; Lopes
et al., 2009b) and results in neuronal cell death (Folch et al.,
2011). Cell cycle re-entry in AD is characterized by the
association of MCM2 with neurofibrillary tangles (Bonda et al.,
2009), kinase upregulation, cytoskeletal alterations (Lopes et al.,
2009b) and re-expression of cell cycle related proteins like
CDK11 (Bajić et al., 2011). The re-entry was also reported not to
be induced by Ab or Tau pathology (Lopes et al., 2009a). Finally
tetraploidy, a putative initial result of the re-entry is also a known
AD feature (Frade and López-Sánchez, 2010). Notably, BLM
colocalizes with TERF2 (telomeric repeat-binding factor 2) in
foci actively synthesizing DNA during late S and G2/M phases of
the cell cycle, which has been described for immortalized
cells using the alternative lengthening of telomere pathway
(Bhattacharyya et al., 2009; Yankiwski et al., 2000). TERF2 was
also identified as a differentially abundant protein in FE65
knockdown cells supporting a role for FE65 in cell cycle
regulation. We hypothesize that elevated or diminished nuclear
FE65 levels may contribute to neuronal cell cycle re-entry in AD
causing neurodegeneration as neurons are hardly able to
proliferate. Thus, the intervention of FE65 nuclear translocation
may be a promising therapeutic approach for AD. Targeting
FE65 localization or BLM–FE65 interaction might also
correspond to a strategy for Bloom syndrome treatment – a
disease that is characterized by a high risk of cancer in affected
individuals caused by mutations in the BLM gene (Amor-Gueret,
2006).
Science, Australia). RNA lysates from cell culture were gained using the
NucleoSpin RNA II kit (Machery Nagel, Germany) according to the
manufacturer’s protocol. Template cDNA was synthesized from 2 mg total RNA
using the RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific, USA)
and random hexamer primers following manufacturer’s instructions. Cycling
conditions were 95 ˚C for 15 minutes, followed by 45 cycles of 95 ˚C for
15 seconds, 56 ˚C for 30 seconds, and 72 ˚C for 30 seconds. The dCt values were
calculated using GAPDH as control. Experiments were performed in triplicates for
each clone analyzed. Melting curve analysis confirmed that only one product was
amplified. For statistical analysis of all quantitative PCR experiments, normal
distribution of data was assured and remaining gene expression was calculated by
the method of Livak and Schmittgen (Livak and Schmittgen, 2001). For
quantification of mRNA levels of MCM3, BLM and KAT7, the same qPCR
procedure was used. All sense and antisense primer sequences used in qPCR
experiments are shown in supplementary material Table S2.
Materials and Methods
Microscopy
Cell culture
For immunofluorescence microscopy, cells were washed with PBS+/+, fixed with
4% paraformaldehyde (PFA), permeabilized with standard 0.5% Triton X-100 and
incubated with the corresponding antibody as follows: MCM3 antibody (dilution
factor 1:50; cat. no: ab4460; Abcam, UK); BLM antibody (dilution factor 1:50;
cat. no: ab2179; Abcam, UK); KAT7 antibody (dilution factor 1:50; cat. no:
ab37289; Abcam, UK), KAT5/TIP60 antibody (1:50; cat. no: sc-5727, Santa Cruz,
USA), secondary TRITC antibody (dilution factor 1:200; cat. no: T5268 or
T6028; Sigma-Aldrich, USA), secondary DyLight405 (1:200; cat. no: 3063-1;
Epitomics, USA); nuclear visualization was carried out with Hoechst Staining.
Images were obtained using the fluorescence microscope IX51 (Olympus
Microscopy, UK). To confirm presumed endoplasmic reticulum (ER) signals, a
commercially available ER-TrackerTM Blue-White DPX (cat. no: E-12353;
Invitrogen, USA) was used. Cell preparation and ER live-cell staining was
performed according to the manufacturer’s instructions. For confocal laser
scanning microscopy, HEK293T cells, 48 hours after seeding, were transfected
with FE65-GFP/TIP60 (equal-molar mixture of pFE65-EGFP and p2N3T-TIP60
vectors), pBLM-EGFP vector, or pFE65 (without EGFP fusion) vector using
Lipofectamine reagent METAFECTENETM (Biontex, Germany) according to the
manufacturer’s instructions. FE65-GFP/TIP60-transfected cells were analyzed by
confocal laser scanning microscopy (LSM 510, Zeiss) in combination with Zeiss
636 (Plan-Neofluar, NA 1.4) oil immersion lenses. Time-lapse imaging was done
for 3 hours, frames were taken every 2 minutes. Progress of moving nuclear
spheres was monitored by phase-contrast optics using a phase-contrast microscope
(Zeiss) equipped with a CCD camera (Princeton Instruments, USA) and
Metamorph image software (Vistron, Germany). For transmission electron
microscopy, specimen were fixed in 2.5% GA in PB, postfixed in OsO4,
dehydrated, embedded in Epon and examined (Philips EM 410).
The FE65 knockdown was established by transfection with a GIPZ lentiviral
shRNAmir vector (Open Biosystems, USA) in HEK293T cells. For the control
state, a non-silencing GIPZ lentiviral shRNAmir vector (Open Biosystems, USA)
was used. First 1806104 cells were seeded on 10 cm cell culture dishes (TPP,
Switzerland) using five dishes for control and five dishes for the knockdown
condition (n55). Cells were cultivated at 37 ˚C and 5% CO2 in 10 ml minimum
essential medium (MEM; Sigma-Aldrich, USA) supplemented with 1% penicillin/
streptomycin and 10% fetal calf serum (FCS; Invitrogen, USA). 48 hours after
seeding, HEK293T cells were transfected using Lipofectamine reagent
METAFECTENETM (Biontex, Germany). For each cell culture dish, 36 ml
METAFECTENETM and 36 mg total DNA was used whereby 5 ml MEM was
removed before transfection. The transfection procedure was performed according
to the manufacturer’s instructions. The hairpin sequences of the applied GIPZ
lentiviral shRNAmir vectors (Open Biosystems, USA) are shown in supplementary
material Table S1. Puromycin was used to improve transfection efficiency and to
get rid of untransfected cells. 24 hours after transfection, the final MEM dish
volume was adjusted to 7 ml total MEM and 12 mg/ml puromycin was added in
each cell culture dish. Cells were selected for 96 hours followed by MEM filtration
through a Filtropur S 0.2 sterile filter (Sarstedt, Germany). The filtered MEM was
added again to the selected cells. 12 hours after filtration procedure, cells were
washed with 7 ml cold (4 ˚C) GIBCOTM phosphate-buffered saline +/+ (PBS+/+;
Invitrogen, USA) to initiate the generation of stable cell lines. Single colonies were
picked and transferred to larger cell culture dishes (TPP, Switzerland). Cells were
cultivated at 37 ˚C and 5% CO2 in 10 ml MEM supplemented with 1% penicillin/
streptomycin and 10% FCS for 1–1.5 months. After this incubation period, cells
were washed with 7 ml cold (4 ˚C) PBS+/+ and harvested with 8 ml cold PBS+/+ .
2 ml were used for RNA and 6 ml for protein isolation. Subsequently, PBS+/+ was
removed by centrifugation at 2006g at 4 ˚C. For protein isolation, cell pellets were
sonicated in DIGE buffer [7 M urea, 2 M thiourea, 2% CHAPS, 130 mM
dithiothreitol (DTT), 30 mM Tris-HCl, pH 8.5] and the cellular extracts were
centrifuged at 15.500 g for 15 minutes at 4 ˚C. The supernatants were used for
subsequent experiments and the protein concentration was determined by amino
acid analysis (ASA) on an HPLC Alliance 2695 instrument (Waters, USA).
Quantitative PCR (qPCR)
To quantify mRNA levels of FE65, FE65L1 and FE65L2, SYBR Green real-time
PCR assays were performed on a RotorGene RG-3000 device (Corbett Life
Immunoblotting
Total protein was separated by SDS-PAGE using 4–12% NuPAGETM Bis-Tris gels
(Invitrogen, USA) and proteins were transferred to nitrocellulose. The Odyssey
Infrared Imaging System (LI-COR Biosciences, USA) was used for protein
detection. For the FE65 blot, 25 mg total protein were used for each lane, the blot
was probed with a FE65 antibody (cat. no: sc-33155; Santa Cruz Biotechnology,
USA) and an antibody dilution of 1:200 in StartingBlockTM TBS blocking buffer
(Thermo Scientific, USA) was applied. The MCM3 blot was probed with a MCM3
antibody (cat. no: ab4460; Abcam, UK) using a dilution of 1:3000 in
StartingBlockTM TBS blocking buffer and 20 mg total protein. The BLM blot
was incubated with a BLM antibody (dilution factor 1:1000, 20 mg total protein;
cat. no: ab2179; Abcam, UK). The KAT7 blot was probed with a KAT7 antibody
(dilution factor 1:200 in StartingBlockTM TBS blocking buffer, 20 mg total protein;
cat no: ab37289; Abcam, UK). Additionally, all immunoblots were incubated with
a b-actin antibody (dilution factor 1:10000; cat. no: A1978; Sigma Aldrich,
Germany). The b-actin signal intensity was used for normalization. Secondary
antibodies were used as follows: IRDyeTM 800CW antibody (dilution factor
1:15000; cat. no: 926-32211; LI-COR Biosciences, USA). IRDyeTM 680CW
antibody (dilution factor 1:15000; cat. no: 926-32220; LI-COR Biosciences, USA).
Quantification was carried out by densitometry with Odyssey Application
Software version 3.0.21 (LI-COR Biosciences, USA).
Mass spectrometry, spectral count based label-free proteomics
Total cell lysates (20 mg each lane) were separated by SDS-PAGE with 4–12%
NuPAGETM Bis-Tris Gel (Invitrogen, USA) and stained with ImperialTM Protein
Stain (Thermo Scientific, USA). Following destaining, the complete gel was
reduced using DTT and alkylated with iodacetamide (IAA). The gel was cut into
three horizontal slices and afterwards every lane was excised (compare Fig. 2A).
In-gel digestion was performed overnight at 37 ˚C with trypsin (Promega, USA) in
10 mM HCl and 50 mM ammonium hydrogen carbonate (NH4HCO3) at pH 7.8.
Resulting peptides were extracted once with 100 ml of 1% FA, and twice with
100 ml of 5% FA, 50% ACN. Extracts were combined and ACN was removed in
2490
Journal of Cell Science 126 (11)
vacuo. For LC-MS analysis, a final volume of 40 ml was prepared by addition of
0.1% TFA.
Nano-HPLC-MS/MS was performed on an UltiMate 3000 RSLCnano LC
system (Dionex, Idstein, Germany). Samples were loaded on a trap column
(Dionex, 75 mm62 cm, particle size 3 mm, pore size 100 Å) with 0.1% TFA (flow
rate 10 ml/minute). After washing, the trap column was connected with an
analytical C18 column (Dionex, 75 mm625 cm, particle size 2 mm, pore size
100 Å). Peptides were separated with a flow rate of 400 nl/minute using the
following solvent system: (A) 0.1% FA; (B) 84% ACN, 0.1% FA. In a first step, a
gradient from 5% B to 40% B (95 minutes) was used, followed by a second
gradient from 40% B to 95% B within 5 minutes and finally a gradient from 95%
to 5% B within 25 minutes. ESI-MS/MS was performed on a LTQ Orbitrap Velos
(Thermo Fisher Scientific), which was directly coupled to the HPLC system.
MS spectra were scanned between 300 and 2000 m/z with a resolution of 30,000
and a maximal acquisition time of 500 ms. The m/z values initiating
MS/MS were set on a dynamic exclusion list for 35 seconds. Lock mass
polydimethylcyclosiloxane (m/z 445.120) was used for internal recalibration. The
20 most intensive ions (charge .1) were selected for MS/MS-fragmentation in the
ion trap. Fragments were generated by low-energy collision-induced dissociation
(CID) on isolated ions with collision energy of 35% and maximal acquisition time
of 50 ms.
visible in the mid of wells in brightfield microscopy (Fig. 4A). Simultaneously
GFP fluorescence of control and knockdown cells (both stable clones contain a
turbo GFP cassette) was monitored from 12 to 96 hours with the InfiniteTM 200
PRO device (excitation at 395 nm, emission at 509 nm, Tecan Group,
Switzerland) and plotted with a Boltzmann sigmoidal fit. To determine cell
index doubling times, we used the xCELLigence System (Roche, Switzerland).
5000 cells were seeded on an xCELLigence System microplate and cell index
doubling times was monitored from 12 to 96 hours. DNA replication assay was
done by EdU labeling (BrdU analogue) with the Click-iT EdU Alexa Fluor 594
Imaging Kit (Invitrogen, USA) according to manufacturer’s protocol with Hoechst
counterstaining.
Acknowledgements
We thank Prof. T. Russo (Dipartimento di Biochimica e
Biotecnologie Mediche, Università di Napoli Federico II, Italy) for
FE65 expression vectors and Prof. J. Groden (Department of
Molecular Virology, Immunology and Medical Genetics, Ohio
State University, USA) for providing the BLM expression vector.
We thank Prof. Suzanne Guénette from MGH, Harvard for her
critical revision of the manuscript.
Journal of Cell Science
Data processing and database searches
Raw files were transformed to *.mgf-files (ProteomDiscoverer 1.3, Thermo
Scientific Fisher), imported in ProteinScapeTM (version 2.1, Bruker Daltonics,
Bremen, Germany), and analyzed using Mascot (Matrixscience, London, UK) with
a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.5 Da.
Searches were performed allowing one missed cleavage site after tryptic digestion.
Carbamidomethylation (C), oxidation (M), and phosphorylation (S,T,Y) were
considered as variable modifications. All data were searched against a database
created by DecoyDatabaseBuilder (Reidegeld et al., 2008) containing the whole
human ipi (release 2011/06, v3.84 human, 90166 entries) with one additional
shuffled decoy for each protein.
Protein quantification
After peptide identification, an algorithm using a given minimal peptide score
(minPepScore) and a minimal number of peptides per protein (minNrPeps) was
applied. The algorithm performs the following steps: score calculation for all
proteins by adding up the Mascot ion scores of the protein’s peptides, which have a
score of at least minPepScore. Here, a peptide is defined by an amino acid
sequence and its modifications. If two peptides are equal except for the score, only
the higher score is taken. Reporting the highest scoring protein group (a group
consists of all proteins in the database containing the same set of identified
peptides) which has at least minNrPeps peptides not yet flagged as used up and
flag all the peptides of the reported proteins as used up. Repetition of step 2 was
done until no more protein groups get reported. A local false discovery rate (FDR)
was calculated for each protein group, regarding a group as decoy, if it consists of
decoy proteins only. With this strategy the minPepScore was calculated, which
yielded the list with the most target (opposed to decoy) groups beneath an FDRthreshold of 5%. For the given data a minNrPeps of 2 was used to exclude ‘one hit
wonders’, which yielded a minPepScore of 22 for the longest list. Among the
proteins in this list, every peptide spectrum match (PSM) was extracted.
These PSMs were further processed using the Pivot table function of Microsoft
Excel resulting in a table representing spectral counts for every peptide belonging
to a certain protein. Processed spectral counts (PSC) based on spectral and peptide
counts were calculated as described previously (22,23) and subsequently used as
basis for label-free quantification. In brief, PSC calculation was performed by
summing up all spectral counts belonging to the respective protein. To identify
differentially expressed proteins, the ratio between the averaged spectral indices of
the knockdown samples and controls was calculated and Student’s t-test was
conducted for each protein. In order to control the FDR, the resulting p-values
were adjusted for multiple testing according to Benjamini and Hochberg
(Benjamni and Hochberg, 1995). However, adjusted P-value calculation was
inappropriate for our approach as the number of identified proteins (which is a
determining factor for the correction calculation) is extremely large in our study
caused by the use of a sensitive MS instrument. Thus, all proteins reported to be
relevant in this work have been validated by independent methods. Initially, a
significant t-test (,0.05) and a spectral index ratio .1.8 was used to assign a
protein as potential candidate for subsequent experiments.
The pathway analysis was done with two independent softwares, IPA 9.0
(Ingenuity Pathway Analysis, Ingenuity Systems, USA, http://www.ingenuity.
com) and STRING 9.0 (Search Tool for the Retrieval of Interacting Genes/
Proteins, EMBL Institute, Europe, http://string-db.org) according to our experience
of pathway tools in proteomics (Müller et al., 2011c).
Cell proliferation and DNA replication assay
At first, cell proliferation was examined visually over 96 hours using a 96-well
microplate format. Equal well position was ensured by centering the black shading
Author contributions
A.S. performed the experiments assisted by T.M., F.M.N., M.N.,
C.L., K.P., and F. El M.; T.M. designed the experiments, analyzed
the data and wrote the manuscript. All authors discussed the data.
Funding
This work was funded by Forschungsförderung Ruhr-Universität
Bochum Medizinische Fakultät [grant numbers AZ-F616-2008 and
AD F680-2009 to T. Müller]; and the federal state of North RhineWestphalia within the Protein Research Unit Ruhr within Europe
project.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.121004/-/DC1
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