A. Heck
Utrecht University, Pharmaceutical Sciences, Faculty Member
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Proteasome-dependent Degradation of Transcription Factor Activating Enhancer-binding Protein 4 (TFAP4) Controls Mitotic Divisionmore
by Teck Yew Low and A. Heck
TFAP4, a basic helix-loop-helix transcription factor that regulates the expression of a multitude of genes involved in the regulation of cellular proliferation, stemness, and epithelialmesenchymal transition, is up-regulated in... more
TFAP4, a basic helix-loop-helix transcription factor that regulates
the expression of a multitude of genes involved in the
regulation of cellular proliferation, stemness, and epithelialmesenchymal
transition, is up-regulated in colorectal cancer
and a number of other human malignancies. We have found
that, during the G2 phase of the cell division cycle, TFAP4 is targeted
for proteasome-dependent degradation by the SCFTrCP
ubiquitin ligase. This event requires phosphorylation of TFAP4 on
a conserved degron. Expression of a stable TFAP4 mutant unable
to interact with TrCP results in a number of mitotic defects,
including chromosome missegregation and multipolar spindles,
which eventually lead to the activation of the DNA damage
response. Our findings reveal thatTrCP-dependent degradation
of TFAP4 is required for the fidelity of mitotic division.
the expression of a multitude of genes involved in the
regulation of cellular proliferation, stemness, and epithelialmesenchymal
transition, is up-regulated in colorectal cancer
and a number of other human malignancies. We have found
that, during the G2 phase of the cell division cycle, TFAP4 is targeted
for proteasome-dependent degradation by the SCFTrCP
ubiquitin ligase. This event requires phosphorylation of TFAP4 on
a conserved degron. Expression of a stable TFAP4 mutant unable
to interact with TrCP results in a number of mitotic defects,
including chromosome missegregation and multipolar spindles,
which eventually lead to the activation of the DNA damage
response. Our findings reveal thatTrCP-dependent degradation
of TFAP4 is required for the fidelity of mitotic division.
Research Interests:
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Publication Date: 2011
Publication Name: Molecular Systems Biology
Research Interests: Human Embryonic Stem Cells, Mass Spectrometry, Induced Pluripotent Stem Cells (I Psc), Proteomics, Cell Adhesion, and 12 moreHuman embryonic stem cell, Gene expression, Cell line, Humans, Embryonic Stem Cell, Molecular systems biology, Antigen Processing, Embryonic Stem Cells, Proteome, Therapeutic Use, Gene expression profiling, and Differential expression
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Coupled activation and degradation of eEF2K regulates protein synthesis in response to genotoxic stressmore
by Teck Yew Low and A. Heck
The kinase eEF2K [eukaryotic elongation factor 2 (eEF2) kinase] controls the rate of peptide chain elongation by phosphorylating eEF2, the protein that mediates the movement of the ribosome along the mRNA by promoting translocation of the... more
The kinase eEF2K [eukaryotic elongation factor 2 (eEF2) kinase] controls the rate of peptide chain elongation by phosphorylating eEF2, the protein that mediates the movement of the ribosome along the mRNA by promoting translocation of the transfer RNA from the A to the P site in the ribosome. eEF2K-mediated phosphorylation of eEF2 on threonine 56 (Thr⁵⁶) decreases its affinity for the ribosome, thereby inhibiting elongation. Here, we show that in response to genotoxic stress, eEF2K was activated by AMPK (adenosine monophosphate-activated protein kinase)-mediated phosphorylation on serine 398. Activated eEF2K phosphorylated eEF2 and induced a temporary ribosomal slowdown at the stage of elongation. Subsequently, during DNA damage checkpoint silencing, a process required to allow cell cycle reentry, eEF2K was degraded by the ubiquitin-proteasome system through the ubiquitin ligase SCF(βTrCP) (Skp1-Cul1-F-box protein, β-transducin repeat-containing protein) to enable rapid resumption of translation elongation. This event required autophosphorylation of eEF2K on a canonical βTrCP-binding domain. The inability to degrade eEF2K during checkpoint silencing caused sustained phosphorylation of eEF2 on Thr⁵⁶ and delayed the resumption of translation elongation. Our study therefore establishes a link between DNA damage signaling and translation elongation.
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Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly... more
Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.
Publication Date: 2015
Publication Name: Cell Reports
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Targeted SCX Based Peptide Fractionation for Optimal Sequencing by Collision Induced, and Electron Transfer Dissociationmore
by A. Heck and Bas van Breukelen
Publication Date: 2008
Publication Name: Journal of Proteomics & Bioinformatics
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by Bas van Breukelen and A. Heck
Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and... more
Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.
Publication Date: 2014
Publication Name: Molecular Systems Biology
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Quantitative and qualitative proteome characteristics extracted from in-depth integrated genomics and proteomics analysismore
by A. Heck and Bas van Breukelen
Publication Date: 2013
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Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectramore
by Bas van Breukelen and A. Heck
A complicating factor in analyzing electrospray ionization mass spectra of intact macromolecular heterogeneous protein complexes is the potential overlap of ions from different species present in solution. Therefore, it is often not... more
A complicating factor in analyzing electrospray ionization mass spectra of intact macromolecular heterogeneous protein complexes is the potential overlap of ions from different species present in solution. Therefore, it is often not possible to assign all ion signals. With the aim of allowing the more efficient and comprehensive analysis of very complex mass spectra of intact heterogeneous protein complexes we developed a software program: SOMMS. The program uses simple user input parameters together with Gaussian curve fitting to simulate putative mass spectra of protein (sub)complexes within a specified charge state window. In addition, the program can simulate spectra for heterogeneous protein complexes using bi- and multinomial distributions and it can calculate zero-charge spectra and relatively quantify the abundance of each component in a mixture. As a proof of concept we analyzed the complex mass spectra of alpha-glutamate synthase and alphabeta-glutamate synthase from Azosp...
Publication Date: 2006
Publication Name: Rapid communications in mass spectrometry : RCM
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On-the-fly targeted selection of labeled peptides in liquid chromatography/mass spectrometry based quantitative proteomicsmore
by A. Heck and Bas van Breukelen
Publication Date: 2000
Publication Name: Rapid Communications in Mass Spectrometry
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A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusingmore
by Bas van Breukelen and A. Heck
We experimentally demonstrate the use of an in-house developed pI calculator which takes into account peptide PTM such as phosphorylation and N-terminal acetylation. The pI calculator was utilized for a large set of peptides derived from... more
We experimentally demonstrate the use of an in-house developed pI calculator which takes into account peptide PTM such as phosphorylation and N-terminal acetylation. The pI calculator was utilized for a large set of peptides derived from a complex zebrafish lysate fractionated using peptide IEF, whereby a good correlation between the calculated (theoretical) pI and the experimental pI could be established. This pI calculator permits the implementation of optimal pK values depending on the experimental conditions and a reliable calculation of peptide pI which can be utilized as a filtering technique in validating peptide identifications. Our data reveal that the shift due to a phosphorylation or N-terminal acetylation is highly dependent on the presence of acidic or basic residues in the peptide. Furthermore, using this pI calculator, we revealed previously unknown position-specific pKs of asparagine and carbamidomethylated cysteine depending on their location in the peptide. Collectively, this peptide pI calculator is a welcome addition to the versatility and robustness of IEF for the separation and confident identification of (post-translationally modified) peptides.
Publication Date: 2008
Publication Name: PROTEOMICS
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Publication Date: 2012
Publication Name: Molecular & Cellular Proteomics
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Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental... more
Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics. We therefore developed the qcML format, an XML-based standard that follows the design principles of the related mzML, mzIdentML, mzQuantML, and TraML standards from the HUPO-PSI (Proteomics Standards Initiative). In addition to the XML format, we also provide tools for the calculation of a wide range of quality metrics as well as a database format and interconversion tools, so that existing LIMS systems can easily add relational storage of the quality control data to their existing schema. We here describe the qcML specification, along with possible use cases and an illustrative example of the subsequent analysis possibilities. All information about qcML is available at http://code.google.com/p/qcml.
Publication Date: 2014
Publication Name: Molecular & Cellular Proteomics
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Publication Date: 2014
Publication Name: Molecular & Cellular Proteomics
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Deconvolution of overlapping isotopic clusters improves quantification of stable isotope–labeled peptidesmore
by Bas van Breukelen and A. Heck
Publication Date: 2011
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Multiplexed Proteomics Mapping of Yeast RNA Polymerase II and III Allows Near-Complete Sequence Coverage and Reveals Several Novel Phosphorylation Sitesmore
by Bas van Breukelen and A. Heck
Publication Date: 2008
Publication Name: Analytical Chemistry
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LysNDeNovo: An algorithm enabling de novo sequencing of Lys-N generated peptides fragmented by electron transfer dissociationmore
by Bas van Breukelen and A. Heck
Recently, we introduced a novel proteomics method employing a metalloendopeptidase with Lys-N specificity to produce proteolytic peptides. Fragmentation spectra generated by electron transfer dissociation, for a large proportion of the... more
Recently, we introduced a novel proteomics method employing a metalloendopeptidase with Lys-N specificity to produce proteolytic peptides. Fragmentation spectra generated by electron transfer dissociation, for a large proportion of the Lys-N proteolytic peptides, were found to be dominated by extensive series of c-type ions. Taking advantage of this unique spectral property, we developed an algorithm, LysNDeNovo, to facilitate de novo sequencing of these peptides. LysNDeNovo contains simple and naive heuristics to demonstrate a proof of concept, i.e. that Lys-N peptide electron transfer dissociation spectra are perfectly suited for de novo interpretation. A stringent "golden" dataset of peptides identified by conventional database search algorithms was taken to validate the performance of LysNDeNovo. The results on this dataset indicate that LysNDeNovo was able to confidently identify a considerable proportion (42%), without requiring any prior genome or protein sequences. Results of similar quantity and quality could also be obtained on a much more extensive experimental dataset, illustrating the potential for higher throughput de novo sequencing using these methods.
Publication Date: 2010
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Publication Date: 2012
Publication Name: Nature Methods
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Both genomics and proteomics technologies have matured in the last decade to a level where they are able to deliver systemwide data on the qualitative and quantitative abundance of their respective molecular entities, that is DNA/RNA and... more
Both genomics and proteomics technologies have matured in
the last decade to a level where they are able to deliver systemwide
data on the qualitative and quantitative abundance of their
respective molecular entities, that is DNA/RNA and proteins. A
next logical step is the collective use of these technologies,
ideally gathering data on matching samples. The first large
scale so-called proteogenomics studies are emerging, and
display the benefits each of these layers of analysis has on the
other layers to together generate more meaningful insight into
the connection between the phenotype/physiology and
genotype of the system under study. Here we review a selected
number of these studies, highlighting what they can uniquely
deliver. We also discuss the future potential and remaining
challenges, from a somewhat proteome biased perspective.
the last decade to a level where they are able to deliver systemwide
data on the qualitative and quantitative abundance of their
respective molecular entities, that is DNA/RNA and proteins. A
next logical step is the collective use of these technologies,
ideally gathering data on matching samples. The first large
scale so-called proteogenomics studies are emerging, and
display the benefits each of these layers of analysis has on the
other layers to together generate more meaningful insight into
the connection between the phenotype/physiology and
genotype of the system under study. Here we review a selected
number of these studies, highlighting what they can uniquely
deliver. We also discuss the future potential and remaining
challenges, from a somewhat proteome biased perspective.