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Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

Gawlik K, Shiryaev SA, Zhu W, Motamedchaboki K, Desjardins R, Day R, Remacle AG, Stec B, Strongin AY - PLoS ONE (2009)

Bottom Line: Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms.In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.

Methodology/principal findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.

Conclusions/significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

Show MeSH

Related in: MedlinePlus

Constructs and the purification of soluble furin.(A) Furin constructs. The constructs were truncated at position Ala574 and C-terminally linked to a Gly3-Ser-Gly3 linker followed by a His6 tag. To generate insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), Asp-Asp-Asp-Asp-Lys (D4K) and His-His-His-His-His-His (H6) sequences (enlarged) were inserted between positions Pro113 and Thr114 of the furin sequence. The hinge motion Lys117 and the essential Asp153 were mutated to generate the K117P and inert D153N constructs. SP, PRO, CAT, P, Cys-rich, TM and CT, signal peptide, prodomain, catalytic domain, P domain, cysteine-rich region, transmembrane domain anchor and cytoplasmic tail, respectively. The primary Arg-Thr-Lys-Arg107↓Asp108 autocatalytic cleavage site is shown by an arrow. The numbers show the molecular mass of the furin constructs. (B) Intracellular and secreted furin in Sf9 cells. Cells (2×106/ml) were placed in wells of a 6-well plate and infected with the respective recombinant baculoviruses with a 1–2 multiplicity of infection. After a 36–38 h incubation at 27°C the cells and the 1 ml medium samples were collected. Cells were then lysed in 1 ml 50 mM Tris-HCl, pH 8.0, containing 1% SDS, 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethyl sulfonylfluoride and protease inhibitor cocktail. Both the cell lysate and medium samples (1 µl each) were analyzed by Western blotting with the MON-148 antibody against the catalytic domain of furin. C, Purification of furin by metal-chelating chromatography. Upper panel - Impurities were removed using a linear gradient of 0–25 mM imidazole; furin was eluted with 500 mM imidazole. In the chromatogram, the furin-containing fractions are marked with a solid line. Inset - SDS-PAGE of the 500 mM imidazole fraction. Lower panel - SDS-PAGE (1 µg protein/lane) followed by Coomassie staining (CS) and Western blotting (WB; 100 ng protein/lane) of the purified furin samples.
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pone-0005031-g002: Constructs and the purification of soluble furin.(A) Furin constructs. The constructs were truncated at position Ala574 and C-terminally linked to a Gly3-Ser-Gly3 linker followed by a His6 tag. To generate insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), Asp-Asp-Asp-Asp-Lys (D4K) and His-His-His-His-His-His (H6) sequences (enlarged) were inserted between positions Pro113 and Thr114 of the furin sequence. The hinge motion Lys117 and the essential Asp153 were mutated to generate the K117P and inert D153N constructs. SP, PRO, CAT, P, Cys-rich, TM and CT, signal peptide, prodomain, catalytic domain, P domain, cysteine-rich region, transmembrane domain anchor and cytoplasmic tail, respectively. The primary Arg-Thr-Lys-Arg107↓Asp108 autocatalytic cleavage site is shown by an arrow. The numbers show the molecular mass of the furin constructs. (B) Intracellular and secreted furin in Sf9 cells. Cells (2×106/ml) were placed in wells of a 6-well plate and infected with the respective recombinant baculoviruses with a 1–2 multiplicity of infection. After a 36–38 h incubation at 27°C the cells and the 1 ml medium samples were collected. Cells were then lysed in 1 ml 50 mM Tris-HCl, pH 8.0, containing 1% SDS, 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethyl sulfonylfluoride and protease inhibitor cocktail. Both the cell lysate and medium samples (1 µl each) were analyzed by Western blotting with the MON-148 antibody against the catalytic domain of furin. C, Purification of furin by metal-chelating chromatography. Upper panel - Impurities were removed using a linear gradient of 0–25 mM imidazole; furin was eluted with 500 mM imidazole. In the chromatogram, the furin-containing fractions are marked with a solid line. Inset - SDS-PAGE of the 500 mM imidazole fraction. Lower panel - SDS-PAGE (1 µg protein/lane) followed by Coomassie staining (CS) and Western blotting (WB; 100 ng protein/lane) of the purified furin samples.

Mentions: To support our modeling studies experimentally and to determine the requirements for the activation of furin, WT and mutant constructs were designed. To obtain the soluble constructs, which could be readily isolated from the medium by metal-chelating chromatography, the human furin sequence was truncated at Ala574 and the truncation was then linked to a flexible 7-residue linker followed by a C-terminal His6 tag. To increase the distance from the N-terminus to the critical Lys117 in the N-terminal region of furin, we constructed the insertion mutants. In the insertion mutants, a single Gly residue and the Gly5 and Gly6 peptide sequences were inserted between positions Pro113 and Thr114 of the furin sequence (G, G5 and G6 mutants, respectively). To confirm that the observed effects were not unique for the Gly-containing sequences, both the positively charged His6 and the negatively charged Asp-Asp-Asp-Asp-Lys sequences were also inserted between Pro113 and Thr114 (H6 and D4K mutants, respectively). To determine the role of the hinge motion Lys117, the K117P mutant was also isolated. As a control, we constructed the catalytically inert D153N mutant with the substitution of the essential active site Asp153 (Figure 2A).


Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

Gawlik K, Shiryaev SA, Zhu W, Motamedchaboki K, Desjardins R, Day R, Remacle AG, Stec B, Strongin AY - PLoS ONE (2009)

Constructs and the purification of soluble furin.(A) Furin constructs. The constructs were truncated at position Ala574 and C-terminally linked to a Gly3-Ser-Gly3 linker followed by a His6 tag. To generate insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), Asp-Asp-Asp-Asp-Lys (D4K) and His-His-His-His-His-His (H6) sequences (enlarged) were inserted between positions Pro113 and Thr114 of the furin sequence. The hinge motion Lys117 and the essential Asp153 were mutated to generate the K117P and inert D153N constructs. SP, PRO, CAT, P, Cys-rich, TM and CT, signal peptide, prodomain, catalytic domain, P domain, cysteine-rich region, transmembrane domain anchor and cytoplasmic tail, respectively. The primary Arg-Thr-Lys-Arg107↓Asp108 autocatalytic cleavage site is shown by an arrow. The numbers show the molecular mass of the furin constructs. (B) Intracellular and secreted furin in Sf9 cells. Cells (2×106/ml) were placed in wells of a 6-well plate and infected with the respective recombinant baculoviruses with a 1–2 multiplicity of infection. After a 36–38 h incubation at 27°C the cells and the 1 ml medium samples were collected. Cells were then lysed in 1 ml 50 mM Tris-HCl, pH 8.0, containing 1% SDS, 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethyl sulfonylfluoride and protease inhibitor cocktail. Both the cell lysate and medium samples (1 µl each) were analyzed by Western blotting with the MON-148 antibody against the catalytic domain of furin. C, Purification of furin by metal-chelating chromatography. Upper panel - Impurities were removed using a linear gradient of 0–25 mM imidazole; furin was eluted with 500 mM imidazole. In the chromatogram, the furin-containing fractions are marked with a solid line. Inset - SDS-PAGE of the 500 mM imidazole fraction. Lower panel - SDS-PAGE (1 µg protein/lane) followed by Coomassie staining (CS) and Western blotting (WB; 100 ng protein/lane) of the purified furin samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662429&req=5

pone-0005031-g002: Constructs and the purification of soluble furin.(A) Furin constructs. The constructs were truncated at position Ala574 and C-terminally linked to a Gly3-Ser-Gly3 linker followed by a His6 tag. To generate insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), Asp-Asp-Asp-Asp-Lys (D4K) and His-His-His-His-His-His (H6) sequences (enlarged) were inserted between positions Pro113 and Thr114 of the furin sequence. The hinge motion Lys117 and the essential Asp153 were mutated to generate the K117P and inert D153N constructs. SP, PRO, CAT, P, Cys-rich, TM and CT, signal peptide, prodomain, catalytic domain, P domain, cysteine-rich region, transmembrane domain anchor and cytoplasmic tail, respectively. The primary Arg-Thr-Lys-Arg107↓Asp108 autocatalytic cleavage site is shown by an arrow. The numbers show the molecular mass of the furin constructs. (B) Intracellular and secreted furin in Sf9 cells. Cells (2×106/ml) were placed in wells of a 6-well plate and infected with the respective recombinant baculoviruses with a 1–2 multiplicity of infection. After a 36–38 h incubation at 27°C the cells and the 1 ml medium samples were collected. Cells were then lysed in 1 ml 50 mM Tris-HCl, pH 8.0, containing 1% SDS, 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethyl sulfonylfluoride and protease inhibitor cocktail. Both the cell lysate and medium samples (1 µl each) were analyzed by Western blotting with the MON-148 antibody against the catalytic domain of furin. C, Purification of furin by metal-chelating chromatography. Upper panel - Impurities were removed using a linear gradient of 0–25 mM imidazole; furin was eluted with 500 mM imidazole. In the chromatogram, the furin-containing fractions are marked with a solid line. Inset - SDS-PAGE of the 500 mM imidazole fraction. Lower panel - SDS-PAGE (1 µg protein/lane) followed by Coomassie staining (CS) and Western blotting (WB; 100 ng protein/lane) of the purified furin samples.
Mentions: To support our modeling studies experimentally and to determine the requirements for the activation of furin, WT and mutant constructs were designed. To obtain the soluble constructs, which could be readily isolated from the medium by metal-chelating chromatography, the human furin sequence was truncated at Ala574 and the truncation was then linked to a flexible 7-residue linker followed by a C-terminal His6 tag. To increase the distance from the N-terminus to the critical Lys117 in the N-terminal region of furin, we constructed the insertion mutants. In the insertion mutants, a single Gly residue and the Gly5 and Gly6 peptide sequences were inserted between positions Pro113 and Thr114 of the furin sequence (G, G5 and G6 mutants, respectively). To confirm that the observed effects were not unique for the Gly-containing sequences, both the positively charged His6 and the negatively charged Asp-Asp-Asp-Asp-Lys sequences were also inserted between Pro113 and Thr114 (H6 and D4K mutants, respectively). To determine the role of the hinge motion Lys117, the K117P mutant was also isolated. As a control, we constructed the catalytically inert D153N mutant with the substitution of the essential active site Asp153 (Figure 2A).

Bottom Line: Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms.In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.

Methodology/principal findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.

Conclusions/significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

Show MeSH
Related in: MedlinePlus