Frode Selheim | University of Bergen (original) (raw)
Papers by Frode Selheim
Current pharmaceutical biotechnology, 2016
Methods in Molecular Biology, 2015
In biological and medical sciences, high throughput analytical methods are now commonly used to i... more In biological and medical sciences, high throughput analytical methods are now commonly used to investigate samples of different conditions, e.g., patients versus controls. Systemic functional analyses emerged as a reference method to go beyond a list of regulated compounds, and identify activated or inactivated biological functions. This approach holds the promise for a better understanding of biological systems, of the mechanisms involved in disease progression, and thus improved diagnosis, prognosis, and treatment. In this chapter, we present a simple workflow to conduct pathway analyses on biological data using the freely available Reactome platform ( http://www.reactome.org ).
Methods in Molecular Biology, 2015
In quantitative proteomics, large lists of identified and quantified proteins are used to answer ... more In quantitative proteomics, large lists of identified and quantified proteins are used to answer biological questions in a systemic approach. However, working with such extensive datasets can be challenging, especially when complex experimental designs are involved. Here, we demonstrate how to post-process large quantitative datasets, detect proteins of interest, and annotate the data with biological knowledge. The protocol presented can be achieved without advanced computational knowledge thanks to the user-friendly Perseus interface (available from the MaxQuant website, www.maxquant.org ). Various visualization techniques facilitating the interpretation of quantitative results in complex biological systems are also highlighted.
Current pharmaceutical biotechnology, 2016
The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applica... more The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can...
Current pharmaceutical biotechnology, Jan 16, 2015
Modern analytical techniques provide an unprecedented insight to biomedical samples, allowing an ... more Modern analytical techniques provide an unprecedented insight to biomedical samples, allowing an in depth characterization of cells or body fluids, to the level of genes, transcripts, peptides, proteins, metabolites, or metallic ions. The fine grained picture provided by such approaches holds the promise for a better understanding of complex pathologies, and consequently the personalization of diagnosis, prognosis and treatment procedures. In practice however, technical limitations restrict the resolution of the acquired data, and thus of downstream biomedical inference. As a result, the study of complex diseases like leukemia and other types of cancer is impaired by the high heterogeneity of pathologies as well as patient profiles. In this review, we propose an introduction to the general approach of characterizing samples and inferring biomedical results. We highlight the main limitations of the technique with regards to complex and heterogeneous pathologies, and provide ways to o...
Current Pharmaceutical Biotechnology, 2015
The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applica... more The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging.
Oncogene, Jan 23, 2015
Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, m... more Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, mainly provided by the system Xc(-) antiporter, is a requirement for glioma cell synthesis of glutathione (GSH) which has a critical role in scavenging free radicals, for example, after radiotherapy. Thus, we hypothesized that the Xc(-)-inhibitor sulfasalazine (SAS) could potentiate the efficacy of radiotherapy against gliomas. Here, we show that the catalytic subunit of system Xc(-), xCT, was uniformly expressed in a panel of 30 human GBM biopsies. SAS treatment significantly reduced cystine uptake and GSH levels, whereas it significantly increased the levels of reactive oxygen species (ROS) in glioma cells in vitro. Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the antioxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and gamma knife radiosurgery (GKRS) synergistically prolonged surviv...
Journal of Proteomics & Bioinformatics, 2009
The hitherto proteome of the human neuroblastoma cell line SH-SY5Y consists of 1029 unique protei... more The hitherto proteome of the human neuroblastoma cell line SH-SY5Y consists of 1029 unique proteins. By using subcellular fractionation, SDS PAGE and nanoLC-Q-TOF-MS/MS we enlarged the proteome with additionally 72.2%, resulting in 3707 unique proteins.
As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukem... more As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.
Platelets, 2000
Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and... more Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-protein-linked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P2, respectively. The late accumulation of PtdIns(3,4)P2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha IIb beta 3 + fibrinogen-dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks.
Leukemia Research, 2009
Sustained ligand stimulation of the receptor tyrosine kinase Flt3 resulted in its downregulation ... more Sustained ligand stimulation of the receptor tyrosine kinase Flt3 resulted in its downregulation and a refractory signaling phase in primary acute myeloid leukemia (AML) cells and in the AML cell line THP-1. Stable isotope amino acid labeling in cell culture and mass spectrometry were used to compare protein expression patterns in THP-1 before and after Flt3-downregulation. 375 distinct proteins were identified where ATP-dependent RNA helicase DDX3, HNRPU, Matrin-3, Importin-7 and Bax were among the 25 most upregulated proteins and Hausp/UBP7, UBE2N and ERp29 among the 17 most downregulated. THP-1 cells with receptor downregulation were sensitized to idarubicin-induced apoptosis but not cytarabine. We hypothesize that FL-induced receptor modulation may chemosensitize selected AML subsets.
FEBS Letters, 2003
Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protei... more Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protein kinase (cAPK, PKA), but a growing number of cAMP e¡ects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of ¢ne-tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.
Current Topics in Medicinal Chemistry, 2011
The prototype second messenger cAMP and its major mediator, the cAMP-dependent protein kinase (PK... more The prototype second messenger cAMP and its major mediator, the cAMP-dependent protein kinase (PKA), is able to control simultaneously multiple processes within the same cell. This appears to be achieved through its unique dissociative regulation and the spatiotemporal regulation of both cAMP and PKA. The widespread tissue distribution and physiological function of this pathway makes it an attractive, but challenging pharmacological target. We will discuss current progress in manipulating the fine-tuning of PKA, and outline so far underexploited possibilities for therapy, such as novel ways to target specific substrates and catalytic cycle intermediates of PKA. An attractive strategy to achieve a more focused pharmacological treatment is to combine more traditional targeting of extracellular receptors or ligands with that of intracellular signaling pathway components. The cAMP signaling pathway provides a variety of possibilities for such an approach.
Current Pharmaceutical Biotechnology, 2006
Cellular signaling lies at the core of cellular behavior, and is central for the understanding of... more Cellular signaling lies at the core of cellular behavior, and is central for the understanding of many pathologic conditions. To comprehend how signal transduction is orchestrated at the molecular level remains the ultimate challenge for cell biology. In the last years there has been a revolution in the development of high-throughput methodologies in proteomics and genomics, which have provided extensive knowledge about expression profiles and molecular interaction-networks. However, these methods have typically provided qualitative and static information. This is about to turn, and several high-throughput methods are now available that provide quantitative and temporal information. These data are well suited for analysis by computational methods and bioinformatics, which are becoming increasingly valuable tools to grasp the complexity of cellular networks. At present, several cellular pathways have been modeled in silico and the analysis provides new understanding of the underlying properties that contribute to their dynamic features. Here, we review methodologies that are used for in silico modeling as well as methods to obtain large-scale quantitative data, and discuss how they can be integrated to generate powerful and predictive models of cellular processes. We argue that the generation of such models provide powerful tools to understand how systems properties emerges in healthy and pathologic states, and to generate efficient strategies for pharmacological intervention.
Current Pharmaceutical Biotechnology, 2006
Analytica Chimica Acta, 2012
Several issues need to be considered concerning chemical labeling strategies in proteomics. Some ... more Several issues need to be considered concerning chemical labeling strategies in proteomics. Some of these are labeling specificity, possible side reactions, completeness of reaction, recovery rate, conserving integrity of sample, hydrolysis of peptide bonds at high pH, and signal suppression in mass spectrometry (MS). We tested the effects of different reaction conditions for 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag) derivatization of the ε-amine group of lysine (K) residues. By using nanoflow LC-electrospray ionization-MS (LC-ESI-MS) and MS/MS in combination with MSight 2-D image analysis, we found that standard Lys Tag derivatization processes and conditions induce side reactions such as (i) Lys Tag labeling of the N-terminus, (ii) methylation of internal aspartic acid (D), glutamic acid (E) and C- and N-peptide termini and (iii) deamidation of asparagine (N) and glutamine (Q). We found temperature and pH to be the main variables to control side reactions. Lowering the reaction temperature from 55°C to room temperature reduced deamidation from 22.8±1.4% (SEM) to 7.7±5.5% (SEM) and almost totally blocked methylation (7.0±1.2% (SEM) to 0.4±0.4% (SEM) of the internal acidic amino acids (D and E) at high pH. We conclude that lowering the reaction temperature minimizes undesired side reactions during Lys Tag derivatization in solution.
Journal of Proteome Research, 2013
Mini-reviews in Medicinal Chemistry, 2006
In this review we present recent studies on the effects of the protein phosphatase inhibitor micr... more In this review we present recent studies on the effects of the protein phosphatase inhibitor microcystin on mammalian cells. Whereas high concentrations of microcystin promote liver cell death induced by ROS signalling without the involvement of typical apoptotic proteins, intermediate doses activate classic apoptotic pathways. Low concentrations however, increase liver cell survival and proliferation, and can cause primary liver cancer.
Thrombosis Research, 1999
We previously have demonstrated that human platelets have functionally active platelet-derived gr... more We previously have demonstrated that human platelets have functionally active platelet-derived growth factor α-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether
Current pharmaceutical biotechnology, 2016
Methods in Molecular Biology, 2015
In biological and medical sciences, high throughput analytical methods are now commonly used to i... more In biological and medical sciences, high throughput analytical methods are now commonly used to investigate samples of different conditions, e.g., patients versus controls. Systemic functional analyses emerged as a reference method to go beyond a list of regulated compounds, and identify activated or inactivated biological functions. This approach holds the promise for a better understanding of biological systems, of the mechanisms involved in disease progression, and thus improved diagnosis, prognosis, and treatment. In this chapter, we present a simple workflow to conduct pathway analyses on biological data using the freely available Reactome platform ( http://www.reactome.org ).
Methods in Molecular Biology, 2015
In quantitative proteomics, large lists of identified and quantified proteins are used to answer ... more In quantitative proteomics, large lists of identified and quantified proteins are used to answer biological questions in a systemic approach. However, working with such extensive datasets can be challenging, especially when complex experimental designs are involved. Here, we demonstrate how to post-process large quantitative datasets, detect proteins of interest, and annotate the data with biological knowledge. The protocol presented can be achieved without advanced computational knowledge thanks to the user-friendly Perseus interface (available from the MaxQuant website, www.maxquant.org ). Various visualization techniques facilitating the interpretation of quantitative results in complex biological systems are also highlighted.
Current pharmaceutical biotechnology, 2016
The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applica... more The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can...
Current pharmaceutical biotechnology, Jan 16, 2015
Modern analytical techniques provide an unprecedented insight to biomedical samples, allowing an ... more Modern analytical techniques provide an unprecedented insight to biomedical samples, allowing an in depth characterization of cells or body fluids, to the level of genes, transcripts, peptides, proteins, metabolites, or metallic ions. The fine grained picture provided by such approaches holds the promise for a better understanding of complex pathologies, and consequently the personalization of diagnosis, prognosis and treatment procedures. In practice however, technical limitations restrict the resolution of the acquired data, and thus of downstream biomedical inference. As a result, the study of complex diseases like leukemia and other types of cancer is impaired by the high heterogeneity of pathologies as well as patient profiles. In this review, we propose an introduction to the general approach of characterizing samples and inferring biomedical results. We highlight the main limitations of the technique with regards to complex and heterogeneous pathologies, and provide ways to o...
Current Pharmaceutical Biotechnology, 2015
The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applica... more The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging.
Oncogene, Jan 23, 2015
Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, m... more Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, mainly provided by the system Xc(-) antiporter, is a requirement for glioma cell synthesis of glutathione (GSH) which has a critical role in scavenging free radicals, for example, after radiotherapy. Thus, we hypothesized that the Xc(-)-inhibitor sulfasalazine (SAS) could potentiate the efficacy of radiotherapy against gliomas. Here, we show that the catalytic subunit of system Xc(-), xCT, was uniformly expressed in a panel of 30 human GBM biopsies. SAS treatment significantly reduced cystine uptake and GSH levels, whereas it significantly increased the levels of reactive oxygen species (ROS) in glioma cells in vitro. Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the antioxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and gamma knife radiosurgery (GKRS) synergistically prolonged surviv...
Journal of Proteomics & Bioinformatics, 2009
The hitherto proteome of the human neuroblastoma cell line SH-SY5Y consists of 1029 unique protei... more The hitherto proteome of the human neuroblastoma cell line SH-SY5Y consists of 1029 unique proteins. By using subcellular fractionation, SDS PAGE and nanoLC-Q-TOF-MS/MS we enlarged the proteome with additionally 72.2%, resulting in 3707 unique proteins.
As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukem... more As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.
Platelets, 2000
Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and... more Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-protein-linked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P2, respectively. The late accumulation of PtdIns(3,4)P2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha IIb beta 3 + fibrinogen-dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks.
Leukemia Research, 2009
Sustained ligand stimulation of the receptor tyrosine kinase Flt3 resulted in its downregulation ... more Sustained ligand stimulation of the receptor tyrosine kinase Flt3 resulted in its downregulation and a refractory signaling phase in primary acute myeloid leukemia (AML) cells and in the AML cell line THP-1. Stable isotope amino acid labeling in cell culture and mass spectrometry were used to compare protein expression patterns in THP-1 before and after Flt3-downregulation. 375 distinct proteins were identified where ATP-dependent RNA helicase DDX3, HNRPU, Matrin-3, Importin-7 and Bax were among the 25 most upregulated proteins and Hausp/UBP7, UBE2N and ERp29 among the 17 most downregulated. THP-1 cells with receptor downregulation were sensitized to idarubicin-induced apoptosis but not cytarabine. We hypothesize that FL-induced receptor modulation may chemosensitize selected AML subsets.
FEBS Letters, 2003
Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protei... more Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protein kinase (cAPK, PKA), but a growing number of cAMP e¡ects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of ¢ne-tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.
Current Topics in Medicinal Chemistry, 2011
The prototype second messenger cAMP and its major mediator, the cAMP-dependent protein kinase (PK... more The prototype second messenger cAMP and its major mediator, the cAMP-dependent protein kinase (PKA), is able to control simultaneously multiple processes within the same cell. This appears to be achieved through its unique dissociative regulation and the spatiotemporal regulation of both cAMP and PKA. The widespread tissue distribution and physiological function of this pathway makes it an attractive, but challenging pharmacological target. We will discuss current progress in manipulating the fine-tuning of PKA, and outline so far underexploited possibilities for therapy, such as novel ways to target specific substrates and catalytic cycle intermediates of PKA. An attractive strategy to achieve a more focused pharmacological treatment is to combine more traditional targeting of extracellular receptors or ligands with that of intracellular signaling pathway components. The cAMP signaling pathway provides a variety of possibilities for such an approach.
Current Pharmaceutical Biotechnology, 2006
Cellular signaling lies at the core of cellular behavior, and is central for the understanding of... more Cellular signaling lies at the core of cellular behavior, and is central for the understanding of many pathologic conditions. To comprehend how signal transduction is orchestrated at the molecular level remains the ultimate challenge for cell biology. In the last years there has been a revolution in the development of high-throughput methodologies in proteomics and genomics, which have provided extensive knowledge about expression profiles and molecular interaction-networks. However, these methods have typically provided qualitative and static information. This is about to turn, and several high-throughput methods are now available that provide quantitative and temporal information. These data are well suited for analysis by computational methods and bioinformatics, which are becoming increasingly valuable tools to grasp the complexity of cellular networks. At present, several cellular pathways have been modeled in silico and the analysis provides new understanding of the underlying properties that contribute to their dynamic features. Here, we review methodologies that are used for in silico modeling as well as methods to obtain large-scale quantitative data, and discuss how they can be integrated to generate powerful and predictive models of cellular processes. We argue that the generation of such models provide powerful tools to understand how systems properties emerges in healthy and pathologic states, and to generate efficient strategies for pharmacological intervention.
Current Pharmaceutical Biotechnology, 2006
Analytica Chimica Acta, 2012
Several issues need to be considered concerning chemical labeling strategies in proteomics. Some ... more Several issues need to be considered concerning chemical labeling strategies in proteomics. Some of these are labeling specificity, possible side reactions, completeness of reaction, recovery rate, conserving integrity of sample, hydrolysis of peptide bonds at high pH, and signal suppression in mass spectrometry (MS). We tested the effects of different reaction conditions for 2-methoxy-4,5-dihydro-1H-imidazole (Lys Tag) derivatization of the ε-amine group of lysine (K) residues. By using nanoflow LC-electrospray ionization-MS (LC-ESI-MS) and MS/MS in combination with MSight 2-D image analysis, we found that standard Lys Tag derivatization processes and conditions induce side reactions such as (i) Lys Tag labeling of the N-terminus, (ii) methylation of internal aspartic acid (D), glutamic acid (E) and C- and N-peptide termini and (iii) deamidation of asparagine (N) and glutamine (Q). We found temperature and pH to be the main variables to control side reactions. Lowering the reaction temperature from 55°C to room temperature reduced deamidation from 22.8±1.4% (SEM) to 7.7±5.5% (SEM) and almost totally blocked methylation (7.0±1.2% (SEM) to 0.4±0.4% (SEM) of the internal acidic amino acids (D and E) at high pH. We conclude that lowering the reaction temperature minimizes undesired side reactions during Lys Tag derivatization in solution.
Journal of Proteome Research, 2013
Mini-reviews in Medicinal Chemistry, 2006
In this review we present recent studies on the effects of the protein phosphatase inhibitor micr... more In this review we present recent studies on the effects of the protein phosphatase inhibitor microcystin on mammalian cells. Whereas high concentrations of microcystin promote liver cell death induced by ROS signalling without the involvement of typical apoptotic proteins, intermediate doses activate classic apoptotic pathways. Low concentrations however, increase liver cell survival and proliferation, and can cause primary liver cancer.
Thrombosis Research, 1999
We previously have demonstrated that human platelets have functionally active platelet-derived gr... more We previously have demonstrated that human platelets have functionally active platelet-derived growth factor α-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether