Anna Sobkowicz - Academia.edu (original) (raw)

Papers by Anna Sobkowicz

Research paper thumbnail of Protein Engineered Variants of Hepatocyte Growth Factor/Scatter Factor Promote Proliferation of Primary Human Hepatocytes and in Rodent Liver

Research paper thumbnail of Potential Implication of Paxillin in Cancer Establishment Within the Bone Environment

Research paper thumbnail of Modulation of expression in BEAS-2B airway epithelial cells of α-l-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-l-fucosidase 2

Molecular and Cellular Biochemistry, Jan 28, 2014

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regu... more Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-c, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/ and IFN-c. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise a-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p \ 0.01) in response to IFN-c, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-c stimulation ([twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-c addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-c-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active a-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-c, a Th1 cytokine. Active a-L-fucosidase 2 was overexpressed in BEAS-2B cells.

Research paper thumbnail of Paxillin in the prostate cancer cell models

European Journal of Cancer

Research paper thumbnail of A role for activated leukocyte cell adhesion molecule (ALCAM) in the growth and matrix adhesion of prostate cancer cells

European Journal of Cancer

Research paper thumbnail of Expression profile of amphyphysin I in normal and breast cancer specimens

European Journal of Cancer

Research paper thumbnail of Exploring the chemical space of the lysine-binding pocket of the first kringle domain of Hepatocyte Growth Factor/Scatter Factor (HGF/SF) yields a new class of inhibitors of HGF/SF-MET binding

Chemical Science

The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the... more The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activ...

Research paper thumbnail of Modulation of expression in BEAS-2B airway epithelial cells of α-l-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-l-fucosidase 2

Molecular and Cellular Biochemistry, 2014

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regu... more Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-γ, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/and IFN-γ. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise α-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p < 0.01) in response to IFN-γ, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-γ stimulation (>twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-γ addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-γ-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active α-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-γ, a Th1 cytokine. Active α-L-fucosidase 2 was overexpressed in BEAS-2B cells.

Research paper thumbnail of Protein Engineered Variants of Hepatocyte Growth Factor/Scatter Factor Promote Proliferation of Primary Human Hepatocytes and in Rodent Liver

Gastroenterology, 2012

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects... more Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.

Research paper thumbnail of Protein Engineered Variants of Hepatocyte Growth Factor/Scatter Factor Promote Proliferation of Primary Human Hepatocytes and in Rodent Liver

Research paper thumbnail of Potential Implication of Paxillin in Cancer Establishment Within the Bone Environment

Research paper thumbnail of Modulation of expression in BEAS-2B airway epithelial cells of α-l-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-l-fucosidase 2

Molecular and Cellular Biochemistry, Jan 28, 2014

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regu... more Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-c, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/ and IFN-c. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise a-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p \ 0.01) in response to IFN-c, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-c stimulation ([twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-c addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-c-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active a-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-c, a Th1 cytokine. Active a-L-fucosidase 2 was overexpressed in BEAS-2B cells.

Research paper thumbnail of Paxillin in the prostate cancer cell models

European Journal of Cancer

Research paper thumbnail of A role for activated leukocyte cell adhesion molecule (ALCAM) in the growth and matrix adhesion of prostate cancer cells

European Journal of Cancer

Research paper thumbnail of Expression profile of amphyphysin I in normal and breast cancer specimens

European Journal of Cancer

Research paper thumbnail of Exploring the chemical space of the lysine-binding pocket of the first kringle domain of Hepatocyte Growth Factor/Scatter Factor (HGF/SF) yields a new class of inhibitors of HGF/SF-MET binding

Chemical Science

The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the... more The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activ...

Research paper thumbnail of Modulation of expression in BEAS-2B airway epithelial cells of α-l-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-l-fucosidase 2

Molecular and Cellular Biochemistry, 2014

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regu... more Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-γ, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/and IFN-γ. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise α-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p < 0.01) in response to IFN-γ, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-γ stimulation (>twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-γ addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-γ-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active α-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-γ, a Th1 cytokine. Active α-L-fucosidase 2 was overexpressed in BEAS-2B cells.

Research paper thumbnail of Protein Engineered Variants of Hepatocyte Growth Factor/Scatter Factor Promote Proliferation of Primary Human Hepatocytes and in Rodent Liver

Gastroenterology, 2012

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects... more Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.