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Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment.

Przygodzka P, Ramstedt B, Tengel T, Larsson G, Wilczynska M - BMC Cell Biol. (2010)

Bottom Line: On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation.Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important.The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

ABSTRACT

Background: Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function.

Results: We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal.

Conclusions: We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.

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C395S bomapin mutant, representing reduced form of bomapin, does not enhance proliferation of K562 cells. (A) SDS-PAGE analysis (followed by Coomassie blue staining) of the E. coli expressed and purified recombinant C395S bomapin mutant (lane 1) and the mutant protein incubated with a 4-fold molar excess of trypsin (lane 2). (B) Cellular localization of C395S-bomapin-EGFP in stably transfected K562 cells. (C) Proliferation of the stably transfected multiclonal K562 cells expressing bomapin-EGFP, C395S-bomapin-EGFP, and EGFP alone, as measured by manual cell counting. Higher proliferation of K562 cells expressing wt bomapin, compared to Fig. 2B, is due to higher generation number of the cells that were used in this experiment.
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Figure 3: C395S bomapin mutant, representing reduced form of bomapin, does not enhance proliferation of K562 cells. (A) SDS-PAGE analysis (followed by Coomassie blue staining) of the E. coli expressed and purified recombinant C395S bomapin mutant (lane 1) and the mutant protein incubated with a 4-fold molar excess of trypsin (lane 2). (B) Cellular localization of C395S-bomapin-EGFP in stably transfected K562 cells. (C) Proliferation of the stably transfected multiclonal K562 cells expressing bomapin-EGFP, C395S-bomapin-EGFP, and EGFP alone, as measured by manual cell counting. Higher proliferation of K562 cells expressing wt bomapin, compared to Fig. 2B, is due to higher generation number of the cells that were used in this experiment.

Mentions: As majority of natural bomapin was found in the conformation where the CD-loop was linked to C-terminal part of the protein via a disulfide bond, it was of interest whether the oxidized form of bomapin is important for the observed bomapin effect on cell proliferation. Therefore, we created a single-cysteine bomapin mutant (C395S) lacking the disulfide bond, which represents the reduced form of bomapin. Expressed in E. coli, this mutant was active as inhibitor and formed an SDS-stable complex with trypsin (Figure 3A). The C395S-bomapin-EGFP fusion expressed in K562 cells had nuclear localization (Figure 3B), as it was shown for wt bomapin (Figure 2A). Expression level of the C395S mutant in K562 cells was also similar to the wild type bomapin (Table 1). However, proliferation of the cells that were expressing the C395S-bomapin-EGFP mutant was identical to that of the control cells expressing EGFP (Figure 3C). This strongly suggests that it is the oxidized form of bomapin that is important for the enhancement of cell proliferation.


Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment.

Przygodzka P, Ramstedt B, Tengel T, Larsson G, Wilczynska M - BMC Cell Biol. (2010)

C395S bomapin mutant, representing reduced form of bomapin, does not enhance proliferation of K562 cells. (A) SDS-PAGE analysis (followed by Coomassie blue staining) of the E. coli expressed and purified recombinant C395S bomapin mutant (lane 1) and the mutant protein incubated with a 4-fold molar excess of trypsin (lane 2). (B) Cellular localization of C395S-bomapin-EGFP in stably transfected K562 cells. (C) Proliferation of the stably transfected multiclonal K562 cells expressing bomapin-EGFP, C395S-bomapin-EGFP, and EGFP alone, as measured by manual cell counting. Higher proliferation of K562 cells expressing wt bomapin, compared to Fig. 2B, is due to higher generation number of the cells that were used in this experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874763&req=5

Figure 3: C395S bomapin mutant, representing reduced form of bomapin, does not enhance proliferation of K562 cells. (A) SDS-PAGE analysis (followed by Coomassie blue staining) of the E. coli expressed and purified recombinant C395S bomapin mutant (lane 1) and the mutant protein incubated with a 4-fold molar excess of trypsin (lane 2). (B) Cellular localization of C395S-bomapin-EGFP in stably transfected K562 cells. (C) Proliferation of the stably transfected multiclonal K562 cells expressing bomapin-EGFP, C395S-bomapin-EGFP, and EGFP alone, as measured by manual cell counting. Higher proliferation of K562 cells expressing wt bomapin, compared to Fig. 2B, is due to higher generation number of the cells that were used in this experiment.
Mentions: As majority of natural bomapin was found in the conformation where the CD-loop was linked to C-terminal part of the protein via a disulfide bond, it was of interest whether the oxidized form of bomapin is important for the observed bomapin effect on cell proliferation. Therefore, we created a single-cysteine bomapin mutant (C395S) lacking the disulfide bond, which represents the reduced form of bomapin. Expressed in E. coli, this mutant was active as inhibitor and formed an SDS-stable complex with trypsin (Figure 3A). The C395S-bomapin-EGFP fusion expressed in K562 cells had nuclear localization (Figure 3B), as it was shown for wt bomapin (Figure 2A). Expression level of the C395S mutant in K562 cells was also similar to the wild type bomapin (Table 1). However, proliferation of the cells that were expressing the C395S-bomapin-EGFP mutant was identical to that of the control cells expressing EGFP (Figure 3C). This strongly suggests that it is the oxidized form of bomapin that is important for the enhancement of cell proliferation.

Bottom Line: On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation.Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important.The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

ABSTRACT

Background: Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function.

Results: We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal.

Conclusions: We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.

Show MeSH
Related in: MedlinePlus