Jeroen Demmers
Erasmus MC, Proteomics Center, Faculty Member
During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the... more
During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG) box, which we termed male-specific transcript (MST)-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneup...
ABSTRACT
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Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene... more
Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic...
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The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the Facioscapulohumeral muscular dystrophy locus. Although homology with canonical... more
The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the Facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves LRIF1-mediated interaction with HP1γ at H3K9me3-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together our results provide key insights into SMCH...
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As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major... more
As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.
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Research Interests: Engineering, Physics, Chemistry, Biology, Medicine, and 15 moreMultidisciplinary, Humans, Mice, Animals, PLoS one, Cell nucleus, Chip, Endothelial cell, Chromatin Immunoprecipitation, DNA binding, Base Sequence, Gene Expression Regulation, Nuclear Localization, Molecular Sequence Data, and Signaling pathway
The (pro)renin receptor [(P)RR] interacts with (pro)renin at concentrations that are >1000 times higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin-system (RAS)-independent... more
The (pro)renin receptor [(P)RR] interacts with (pro)renin at concentrations that are >1000 times higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin-system (RAS)-independent functions for (P)RR related to its association with the vacuolar H+-ATPase (V-ATPase). To uncover RAS-independent functions of the (P)RR. We used a proteomics-based approach to purify and identify (P)RR-interacting proteins. This resulted in identification of sortilin-1 (SORT1) as a high-confidence (P)RR-interacting protein, a finding which was confirmed by co-immunoprecipitation of endogenous (P)RR and SORT1. Functionally, silencing (P)RR expression in hepatocytes decreased SORT1 and low-density-lipoprotein (LDL) receptor (LDLR) protein abundance and, as a consequence, resulted in severely attenuated cellular LDL uptake. In contrast to LDL, endocytosis of EGF or transferrin remained unaffected by silencing of the (P)RR. Importantly, reduction of LDLR a...
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Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In... more
Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome in all female somatic cells. Upregulation of Xist transcription on the future inactive X chromosome acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to X-chromosome inactivation. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is upregulated in differentiating mouse embryonic stem cells and activates Xist transcription and X-chromosome inactivation. Here we identify the pluripotency factor REX1 as a key target of RNF12 in the mechanism of X-chromosome inactivation. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout embryonic stem cells show an increased level of REX1. Using chromatin immunoprecipitation sequencing, REX1 binding sites were detected in Xist and Tsix regulatory regions. Overexpression of REX1 in female embryonic stem cells was found to inhibit Xist transcription and X-chromosome inactivation, whereas male Rex1(+/-) embryonic stem cells showed ectopic X-chromosome inactivation. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate X-chromosome inactivation. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and X-chromosome inactivation.
Research Interests: Genetics, Developmental Biology, Molecular Biology, Genomics, Evolution, and 15 moreLong Noncoding Rna, Transcription Factors, Multidisciplinary, Nature, Mice, Female, Animals, Male, Embryonic Stem Cells, Amino Acid Sequence, Protein Binding, Gene Expression Regulation, Ubiquitination, Molecular Sequence Data, and binding sites
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Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we... more
Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at micr...
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Research Interests: Developmental Biology, Flow Cytometry, Induced Pluripotent Stem Cells (I Psc), Cell Cycle, DNA damage, and 14 moreApoptosis, Western blotting, Biological Sciences, Mice, Female, Animals, Male, Embryonic Stem Cells, Genomic instability, Cell cycle checkpoints, ICM, Cell Survival, Pluripotent Stem Cells, and reverse transcriptase polymerase chain reaction
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Research Interests: MicroRNA, Proteomics, Gene expression, Hematopoiesis, Signal Transduction, and 17 moreHematopoietic Stem Cells, Acute Myeloid Leukemia, Cell Differentiation, Humans, Mice, Animals, Blood, Mitogen Activated Protein Kinase, Clinical Sciences, microRNAs, Protein Degradation, Heat Shock Proteins, Cell Proliferation, Base Sequence, Protein Binding, Cell Expansion, and Gene expression profiling
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In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of... more
In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.
Research Interests: Biochemistry, Biophysics, Photochemistry, Mass Spectrometry, Membrane Proteins, and 15 moreOccupational Medicine, Text, Peptides, Phenylalanine, UV light, Lipid bilayers, Gel electrophoresis, Amino Acid Sequence, Membrane Protein, Polyacrylamide Gel Electrophoresis, Lipid Bilayer, Biochemistry and cell biology, Molecular Sequence Data, Sodium Dodecyl Sulfate, and Ultraviolet Rays
Research Interests: Skeletal muscle biology, Cell line, Humans, Mice, Animals, and 15 moreEndoplasmic Reticulum, Biochemical, Physical chromosome mapping, Molecular cloning, Skeletal Muscle, Isoenzymes, phospholipases A2, Amino Acid Profile, Amino Acid Sequence, Cytoplasm, Full Length Movies, Biochemistry and cell biology, Phospholipase C, Molecular Sequence Data, and Cell Membrane
An oligonucleotide-based assay (OBA) was used to identify novel co-factors that can be recruited by the deoxyribonucleic acid (DNA)-bound androgen receptor (AR). Nuclear extracts obtained from LNCaP cells, after incubation with R1881,... more
An oligonucleotide-based assay (OBA) was used to identify novel co-factors that can be recruited by the deoxyribonucleic acid (DNA)-bound androgen receptor (AR). Nuclear extracts obtained from LNCaP cells, after incubation with R1881, were incubated with biotinylated oligonucleotides bound to streptavidin coated beads. The oligonucleotides contain 3 copies in tandem of the androgen responsive element ARE1 from the prostate specific antigen (PSA) gene promoter. As control incubation, a scrambled version of the tandem ARE1 was used. Immunoblots of the eluents revealed that the AR was bound to the ARE1 oligonucleotide and to a much lesser extent to the scrambled oligonucleotide. Proteins eluted from the oligonucleotides, were separated on a 5-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel, followed by identification using mass spectrometry. Identified proteins were scored for having one or more of the following known properties: nuclear localization, involved in transcription regulation, involvement in steroid hormone receptor (SHR) function, or specifical involvement in AR function. A total number of 85 nuclear proteins were found in two separate OBAs. Based on peptide counting, we found enrichment of 7 proteins eluted from the ARE1 oligonucleotide, compared to the scrambled oligonucleotide. Taken together with the obtained scores, these proteins are considered putative AR co-factors. One of these proteins, DDX17, is known to be a co-factor for estrogen receptor alpha (ERalpha), but has never been associated with AR function. The results indicate that the ARE oligonucleotide-based assay may allow enrichment of new candidate DNA-bound AR interacting proteins.
Research Interests: Mass Spectrometry, Estrogen Receptor, Biological Sciences, DNA, Humans, and 15 moreDeoxyribonucleic Acid, Male, Physical sciences, Biotin, Streptavidin, Androgen Receptor, Polyacrylamide Gel Electrophoresis, Protein Binding, Prostate Specific Antigen, T, Response Elements, Oligonucleotides, Nuclear Localization, Ad, and Sodium Dodecyl Sulfate
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CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS.... more
CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS. LL5beta and ELKS form a complex that colocalizes with CLASPs at the cortex of HeLa cells as well as at the leading edge of motile fibroblasts. LL5beta is required for cortical CLASP accumulation and microtubule stabilization in HeLa cells, while ELKS plays an accessory role in these processes. LL5beta is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein, and its recruitment to the cell cortex is influenced by PI3 kinase activity but does not require intact microtubules. Cortical clusters of LL5beta and ELKS do not overlap with focal adhesions but often form in their vicinity and can affect their size. We propose that LL5beta and ELKS can form a PIP3-regulated cortical platform to which CLASPs attach distal microtubule ends.
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Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and... more
Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent 5-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival a...
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The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we... more
The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provi...
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The investigation of the human follicle fluid proteome has gained much interest in the search of new markers as predictors for in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) treatment outcome. Follicular fluid... more
The investigation of the human follicle fluid proteome has gained much interest in the search of new markers as predictors for in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) treatment outcome. Follicular fluid folate, as substrate of one carbon (1-C) metabolism, affects follicular metabolism and oocyte and embryo quality. From this background, we aim to identify a folate-related follicle fluid proteome that associates with IVF/ICSI treatment outcome. In a nested case-control study embedded in a periconception cohort, we performed qualitative and quantitative proteomic analyses using nanoflow LC-MS/MS and TMT labelling in 30 monofollicular fluid samples from women undergoing IVF/ICSI treatment of which 15 used and 15 did not use a folic acid supplement. The protein data are analysed using scaffold proteome Software and differential abundances are expressed as Log2-fold change. Blood samples were obtained before and after treatment for determination of biomarkers of 1-C metabolism and estradiol. We identified 227 uniquely expressed proteins in follicular fluid. In folic acid supplement users compared to nonusers, we established a lower abundance of C-reactive protein (-2·03; P = < 0·01) and higher abundances of apolipoproteins from high-density lipoprotein (HDL), most notably A-I (+1·28; P = < 0·01) and C-I (+1·11; P = 0·016). Preconception folic acid supplement use is associated with suppression of the inflammatory pathway and upregulation of the HDL pathway in human follicular fluid, being a preferential source of cholesterol for steroid hormone synthesis. Studies are needed on the tissue specificity and on the beneficial effects of embryo quality and IVF/ICSI treatment outcome of the proteome of these pathways.
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In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and... more
In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and proteins. This methodology, in which partly deuterated peptide ions are subjected to collision-induced dissociation in the vacuum of a mass spectrometer, has emerged as a useful tool in structural biology. It may potentially provide quantitatively the extent of deuterium incorporation per individual amino acid in peptides and proteins, thus providing detailed structural information related to protein structure and folding. We report that this general methodology has limitations caused by the fact that the incorporated deuterium atoms migrate prior or during the CID MS analysis. Our data are focused on a variety of transmembrane peptides, incorporated in a lipid bilayer, in which the near-terminal amino acids that anchor at the lipid-water interface are s...
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The ordered assembly of a functional pre-initiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA... more
The ordered assembly of a functional pre-initiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional crosstalk between GATA1 and TAF10. Additionally, we analyzed the composition of TFIID and SAGA complexes by mass spectrometry in mouse and human cells and found that their global integrity is maintained, wi...
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The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of... more
The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.
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Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional... more
Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional differences underlie this tumour-type specific mutation spectrum. We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximity ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the exp...
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Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and... more
Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent 5-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival a...
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One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone... more
One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two funda...
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Embryonic stem (ES) cell self-renewal is regulated by transcription factors, including Oct4, Sox2, and Nanog. A number of additional transcriptional regulators of ES cell self-renewal have recently been identified, including the orphan... more
Embryonic stem (ES) cell self-renewal is regulated by transcription factors, including Oct4, Sox2, and Nanog. A number of additional transcriptional regulators of ES cell self-renewal have recently been identified, including the orphan nuclear receptor estrogen-related receptor beta (Esrrb). However, the mode of action of Esrrb in ES cells is unknown. Here, using an Oct4 affinity screen, we identify Esrrb as an Oct4 partner protein. Esrrb can interact with Oct4 independently of DNA. Esrrb is recruited near the Oct-Sox element in the Nanog proximal promoter, where it positively regulates Nanog expression. Esrrb recruitment to the Nanog promoter requires both the presence of Oct4 and a degenerate estrogen-related receptor DNA element. Consistent with its role in Nanog regulation, expression of the Esrrb protein within the Oct4-positive ES cell population is mosaic and correlates with the mosaic expression of the Nanog protein. Together with previous reports that Nanog may regulate Esr...
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During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β)... more
During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of an...
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The ability of preferredoxin to inactivate a 50-pS anion channel of the chloroplast inner membrane in the presence of an energy source was investigated using single-channel recordings. It was found that preferredoxin cannot inactivate the... more
The ability of preferredoxin to inactivate a 50-pS anion channel of the chloroplast inner membrane in the presence of an energy source was investigated using single-channel recordings. It was found that preferredoxin cannot inactivate the channel when GTP is the only energy source present. From this it is concluded that the precursor has to interact with the, translocon of the inner membrane of chloroplasts (Tic) complex to be able to inactivate the 50-pS anion channel. The ability of two mutants of preferredoxin with deletions in their transit sequence to inactivate the channel was also tested. Both mutants have been shown to have a similar binding affinity for the chloroplast envelope, but only one is able to fully translocate. The mutants were both able to inactivate the channel in a similar manner. From this it is concluded that full translocation is not necessary for the inactivation of the channel. It is also shown that preferredoxin is capable of inactivating the 50-pS anion channel in the chloroplast-attached configuration as was previously found in the inside-out configuration. From this it is concluded that stromal factors do not influence the protein-import-induced inactivation of the 50-pS anion channel of the chloroplast inner membrane. Finally the effect of the anion channel blocker 4, 4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the channel activity and on protein import was investigated. It was found that DIDS blocked the channel. Furthermore the addition of the channel blocker reduces the efficiency of import to 52%. This leads to the conclusion that correct functioning of the channel is important for protein import.
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Sp1 and Sp3 belong to the Specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell... more
Sp1 and Sp3 belong to the Specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell cycle and growth control, metabolic pathways and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, while the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double knockout mice. We employed flow cytometry, cell culture and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form p...
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ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for... more
ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, ...
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Neurite extension is regulated by multiple signaling cascades that ultimately converge on the actin and microtubule networks [1]. Rho GTPases, molecular switches that oscillate between an inactive, GDP-bound state and an active, GTP-bound... more
Neurite extension is regulated by multiple signaling cascades that ultimately converge on the actin and microtubule networks [1]. Rho GTPases, molecular switches that oscillate between an inactive, GDP-bound state and an active, GTP-bound state, play a pivotal role in controlling actin cytoskeleton dynamics in the growth cone, whereas the dynamic behavior and interactions of microtubules are largely regulated by proteins called plus-end-tracking proteins (+TIPs), which associate with the ends of growing microtubules. Here, we show that the +TIP Navigator 1 (NAV1) is important for neurite outgrowth and interacts and colocalizes with TRIO, a Rho guanine nucleotide exchange factor that enables neurite outgrowth by activating the Rho GTPases Rac1 and RhoG. We find that binding of NAV1 enhances the affinity of TRIO for Rac1 and RhoG, and that NAV1 regulates TRIO-mediated Rac1 activation and neurite outgrowth. TRIO is also a +TIP, as it interacts with the core +TIP EB1 and tracks microtubule plus ends via EB1 and NAV1. Strikingly, the EB1-mediated recruitment of TRIO to microtubule ends is required for proper neurite outgrowth, and stabilization of the microtubule network by paclitaxel affects both the TRIO-NAV1 interaction and the accumulation of these proteins in neurite extensions. We propose that EB1-labeled ends of dynamic microtubules facilitate the formation and localization of functional NAV1-TRIO complexes, which in turn regulate neurite outgrowth by selectively activating Rac1. Our data reveal a novel link between dynamic microtubules, actin cytoskeleton remodeling, and neurite extension.