Biomedical subjects

Jeroen Krijgsveld

Publications and source records attributed to Jeroen Krijgsveld.

At least 19 recordsLinked to original sources

PubMed2026

Hypertranscription caused by p53 deficiency triggers nucleotide insufficiency that induces replication stress and genomic instability.

p53 plays a central role in the DNA damage response, inducing repair, cell-cycle arrest or apoptosis. Its loss is associated with replication stress and genomic instability. While several underlying mechanisms were suggested, the primary triggers of catastrophic genomic events like chromothripsis, a known driver of tumorigenesis linked with p53 loss, are still unclear. Using p53-depleted epithelial cells and fibroblasts, as well as patient-derived fibroblasts with germline p53 variants that spontaneously undergo chromothripsis, we found that p53 loss causes hypertranscription and increased nucleotide consumption. The resulting nucleotide shortage induces replication stress, causing telomere dysfunction, micronuclei formation, and chromothripsis. These effects were rescued by nucleoside supplementation or normalization of transcription levels, demonstrating a causal link between transcriptional activity, nucleotide availability, and genome stability. Emerging chromothriptic clones displayed restored DNA replication, telomere stabilization, and extrachromosomal DNA, suggesting key features that support clonal selection. We identify nucleotide pool homeostasis as a critical p53 function that suppresses replication stress, prevents chromothripsis, and protects against early tumorigenesis.

Genomic Instability↗

PubMed2006

Novel binding partners of Ldb1 are required for haematopoietic development.

Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.

Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.

Amino Acid Sequence↗

PubMed2006

A quest for human and mouse embryonic stem cell-specific proteins.

Embryonic stem cells (ESCs) are of immense interest as they can proliferate indefinitely in vitro and give rise to any adult cell type, serving as a potentially unlimited source for tissue replacement in regenerative medicine. Extensive analyses of numerous human and mouse ESC lines have shown generic similarities and differences at both the transcriptional and functional level. However, comprehensive proteome analyses are missing or are restricted to mouse ESCs. Here we have used an extensive proteomic approach to search for ESC-specific proteins by analyzing the differential protein expression profiles of human and mouse ESCs and their differentiated derivatives. The data sets comprise 1,775 non-redundant proteins identified in human ESCs, 1,532 in differentiated human ESCs, 1,871 in mouse ESCs, and 1,552 in differentiated mouse ESCs with a false positive rate of <0.2%. Comparison of the data sets distinguished 191 proteins exclusively identified in both human and mouse ESCs but not in their differentiated derivatives. Besides well known ESC benchmarks, this subset included many uncharacterized proteins, some of which may be novel ESC-specific markers. To complement the mass spectrometric approach, differential expression of a selection of these proteins was confirmed by Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting. Additionally two other independently isolated and cultured human ESC lines as well as their differentiated derivatives were monitored for differential expression of selected proteins. Some of these proteins were identified exclusively in ESCs of all three human lines and may thus serve as generic ESC markers. Our wide scale proteomic approach enabled us to screen thousands of proteins rapidly and select putative ESC-associated proteins for further analysis. Validation by three independent conventional protein analysis techniques shows that our methodology is robust, provides an excellent tool to characterize ESCs at the protein level, and may disclose novel ESC-specific benchmarks.

Animals↗

PubMed2006

In-gel isoelectric focusing of peptides as a tool for improved protein identification.

In the analysis of proteins in complex samples, pre-fractionation is imperative to obtain the necessary depth in the number of reliable protein identifications by mass spectrometry. Here we explore isoelectric focusing of peptides (peptide IEF) as an effective fractionation step that at the same time provides the added possibility to eliminate spurious peptide identifications by filtering for pI. Peptide IEF in IPG strips is fast and sharply confines peptides to their pI. We have evaluated systematically the contribution of pI filtering and accurate mass measurements on the total number of protein identifications in a complex protein mixture (Drosophila nuclear extract). At the same time, by varying Mascot identification cutoff scores, we have monitored the false positive rate among these identifications by searching reverse protein databases. From mass spectrometric analyses at low mass accuracy using an LTQ ion trap, false positive rates can be minimized by filtering of peptides not focusing at their expected pI. Analyses using an LTQ-FT mass spectrometer delivers low false positive rates by itself due to the high mass accuracy. In a direct comparison of peptide IEF with SDS-PAGE as a pre-fractionation step, IEF delivered 25% and 43% more proteins when identified using FT-MS and LTQ-MS, respectively. Cumulatively, 2190 non redundant proteins were identified in the Drosophila nuclear extract at a false positive rate of 0.5%. Of these, 1751 proteins (80%) were identified after peptide IEF and FT-MS alone. Overall, we show that peptide IEF allows to increase the confidence level of protein identifications, and is more sensitive than SDS-PAGE.

Animals↗

PubMed2006

Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors.

Seven cathepsin B-like cysteine proteases (CBLs) were identified from the immunoprotective excretory-secretory products of Haemonchus contortus. Two-dimensional (2-D) zymography and biotinylated inhibitors were employed to localize active CBLs in 2-D protein gels. Mass spectrometry provided the identification of AC-4, HMCP1, HMCP2, and GCP7 as well as three novel CBLs encoded by clustered expressed sequence tags.

Amino Acid Sequence↗

PubMed2006

Embryonic stem cell proteomics.

Human embryonic stem cells potentially represent an unlimited source of cells and tissues for regenerative medicine. Understanding signaling events that drive proliferation and specialization of these cells into various differentiated derivatives is of utmost importance for controlling their behavior in vitro. Major progress has been made in unraveling these signaling events with large-scale studies at the transcriptional level, but analysis of protein expression, interaction and modification has been more limited, since it requires different strategies. Recent advances in mass spectrometry-based proteomics indicate that proteome characterization can contribute significantly to our understanding of embryonic stem cell biology. In this article, we review mass spectrometry-based studies of human and mouse embryonic stem cells and their differentiated progeny, as well as studies of conditioned media that have been reported to support self-renewal of the undifferentiated cells in the absence of the more commonly used feeder cells. In addition, we make concise comparisons with related transcriptome profiling reports.

Animals↗

PubMed2005

GATA-1 forms distinct activating and repressive complexes in erythroid cells.

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.

Animals↗

PubMed2005

GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7.

The packaging of eukaryotic genomic DNA into chromatin is modulated through a range of posttranslational histone modifications. Among these, the role of histone ubiquitylation remains poorly understood. Here, we show that the essential Drosophila ubiquitin-specific protease 7 (USP7) contributes to epigenetic silencing of homeotic genes by Polycomb (Pc). We purified USP7 from embryo nuclear extracts as a stable heteromeric complex with guanosine 5'-monophosphate synthetase (GMPS). The USP7-GMPS complex catalyzed the selective deubiquitylation of histone H2B, but not H2A. Biochemical assays confirmed the tight association between USP7 and GMPS in Drosophila embryo extracts. Similar to USP7, mutations in GMPS acted as enhancers of Pc in vivo. USP7 binding to GMPS was required for histone H2B deubiquitylation and strongly augmented deubiquitylation of the human tumor suppressor p53. Thus, GMPS can regulate the activity of a ubiquitin protease. Collectively, these results implicate a biosynthetic enzyme in chromatin control via ubiquitin regulation.

Animals↗

PubMed2004

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

Animals↗

PubMed2004

Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research.

Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression. However, more recently a need has been recognized for investigating cancer development directly at the protein level. In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer. This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC makes use of (12)C- and (13)C-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively. Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry. The incorporation of stable isotopes also allows for a quantitative comparison between the two samples. Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential. Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts. Western blotting provided further confirmation of the mass spectrometry-based quantification. Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines.

Actinin↗

PubMed2004

Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes.

The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. Here, we report the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (i) the BAP complex, defined by the presence of OSA and the absence of Polybromo and BAP170, and (ii) the PBAP complex, containing Polybromo and BAP170 but lacking OSA. Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. We conclude that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. Our results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes.

Amino Acid Sequence↗

PubMed2004

Mass spectrometry-based quantitative proteomics.

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.

Animals↗

PubMed2003

Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.

A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.

Animals↗

PubMed2003

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

Animals↗

PubMed2003

Recent liquid chromatographic-(tandem) mass spectrometric applications in proteomics.

Conventional proteomics makes use of two-dimensional gel electrophoresis followed by mass spectrometric analysis of typtic fragments derived from in-gel digestion of proteins. Although being a very strong technique capable of separating and visualizing hundreds of proteins, 2D-gel electrophoresis has some well-documented disadvantages as well. More recently, liquid chromatographic-(tandem) mass spectrometric techniques have been developed to overcome some of the shortcomings of 2D-gel electrophoresis. In this review we have described several recent applications of liquid chromatography-(tandem) mass spectrometry in the field of proteomics and especially in the field of membrane proteomics, quantitative proteomics and in the analysis of post-translational modifications.

Chromatography, Liquid↗

PubMed2003

Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition.

Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 90% protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs from Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues of Caenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies.

Amino Acid Sequence↗

PubMed2003

Lung proteome alterations in a mouse model for nonallergic asthma.

A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.

Actins↗