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The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop.

Noto R, Randazzo L, Raccosta S, Caccia S, Moriconi C, Miranda E, Martorana V, Manno M - Sci Rep (2015)

Bottom Line: Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli.The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively.Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.

ABSTRACT
Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

No MeSH data available.


Related in: MedlinePlus

Inhibitory activity.(a,b) SDS-Page of 50 μM WT NS and 50 μM E289A NS incubated with 22 μM tPA at 23 °C for different time intervals (as reported in the figure); the first lanes are the Molecular Weight markers (M), the second lanes are the native NS alone (NS). An arrow indicates the different species: double chain (dc) tPA/NS Complex, single chain (sc) tPA/NS Complex, single chain (sc) tPA, native NS, cleaved NS. The dc-Complex originates from the residual dc-tPA, and it looses the not bonded chain upon denaturation in reducing conditions. Analogously, the two chain of dc-tPA are separated upon denaturation with SDS and run with different velocity in the gel lanes (below 37 kDa). (c) Progress curves of hydrolysis of IPR-pNA (0.2 mM) by tPA (1 nM) in the presence of 0 NS (black line), 15 nM E289A NS (dotted violet line), 15 nM WT NS (solid violet line), 45 nM E289A NS (dotted blue line), 45 nM WT NS (solid blue line). (d) Band densities from SDS PAGE as in panel (a) for WT NS (solid lines) and panel (b) for E289A NS (dashed lines): NS/tPA Complex (blue lines), cleaved NS (green lines), native NS (brown lines). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments.
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f5: Inhibitory activity.(a,b) SDS-Page of 50 μM WT NS and 50 μM E289A NS incubated with 22 μM tPA at 23 °C for different time intervals (as reported in the figure); the first lanes are the Molecular Weight markers (M), the second lanes are the native NS alone (NS). An arrow indicates the different species: double chain (dc) tPA/NS Complex, single chain (sc) tPA/NS Complex, single chain (sc) tPA, native NS, cleaved NS. The dc-Complex originates from the residual dc-tPA, and it looses the not bonded chain upon denaturation in reducing conditions. Analogously, the two chain of dc-tPA are separated upon denaturation with SDS and run with different velocity in the gel lanes (below 37 kDa). (c) Progress curves of hydrolysis of IPR-pNA (0.2 mM) by tPA (1 nM) in the presence of 0 NS (black line), 15 nM E289A NS (dotted violet line), 15 nM WT NS (solid violet line), 45 nM E289A NS (dotted blue line), 45 nM WT NS (solid blue line). (d) Band densities from SDS PAGE as in panel (a) for WT NS (solid lines) and panel (b) for E289A NS (dashed lines): NS/tPA Complex (blue lines), cleaved NS (green lines), native NS (brown lines). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments.

Mentions: The ability of E289A NS to form a complex with tPA was tested by mixing NS and tPA and incubating for different time intervals. The formation of the NS/tPA complex was monitored by SDS-PAGE (Fig. 5a,b). We clearly observed the rapid formation of the complex, and since the complex is known to be fragile at late stages1030, we also observed the appearance of protein bands corresponding to cleaved NS and to non-complexed tPA, as well as the reduction in intensity of the complex. The densities of the gel bands in Fig. 5d did not display any significant difference in the ability to form the complex, while they showed a slower reduction of native NS for E289A NS with respect to the WT NS equivalent, in agreement with the chromogenic assay presented in Fig. 5c, which reports the progress of tPA inhibition by NS against a chromogenic substrate, IPR-pNA. The inhibitory activity of E289A NS was lower than that of WT NS. At later stages, our results suggest that tPA released after complex formation was still active, meaning that the protease is only partially translocated. Alternatively, this may suggest a disruption of the tPA-NS covalent bond and subsequent recovery of the integrity of the active site of tPA.


The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop.

Noto R, Randazzo L, Raccosta S, Caccia S, Moriconi C, Miranda E, Martorana V, Manno M - Sci Rep (2015)

Inhibitory activity.(a,b) SDS-Page of 50 μM WT NS and 50 μM E289A NS incubated with 22 μM tPA at 23 °C for different time intervals (as reported in the figure); the first lanes are the Molecular Weight markers (M), the second lanes are the native NS alone (NS). An arrow indicates the different species: double chain (dc) tPA/NS Complex, single chain (sc) tPA/NS Complex, single chain (sc) tPA, native NS, cleaved NS. The dc-Complex originates from the residual dc-tPA, and it looses the not bonded chain upon denaturation in reducing conditions. Analogously, the two chain of dc-tPA are separated upon denaturation with SDS and run with different velocity in the gel lanes (below 37 kDa). (c) Progress curves of hydrolysis of IPR-pNA (0.2 mM) by tPA (1 nM) in the presence of 0 NS (black line), 15 nM E289A NS (dotted violet line), 15 nM WT NS (solid violet line), 45 nM E289A NS (dotted blue line), 45 nM WT NS (solid blue line). (d) Band densities from SDS PAGE as in panel (a) for WT NS (solid lines) and panel (b) for E289A NS (dashed lines): NS/tPA Complex (blue lines), cleaved NS (green lines), native NS (brown lines). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556959&req=5

f5: Inhibitory activity.(a,b) SDS-Page of 50 μM WT NS and 50 μM E289A NS incubated with 22 μM tPA at 23 °C for different time intervals (as reported in the figure); the first lanes are the Molecular Weight markers (M), the second lanes are the native NS alone (NS). An arrow indicates the different species: double chain (dc) tPA/NS Complex, single chain (sc) tPA/NS Complex, single chain (sc) tPA, native NS, cleaved NS. The dc-Complex originates from the residual dc-tPA, and it looses the not bonded chain upon denaturation in reducing conditions. Analogously, the two chain of dc-tPA are separated upon denaturation with SDS and run with different velocity in the gel lanes (below 37 kDa). (c) Progress curves of hydrolysis of IPR-pNA (0.2 mM) by tPA (1 nM) in the presence of 0 NS (black line), 15 nM E289A NS (dotted violet line), 15 nM WT NS (solid violet line), 45 nM E289A NS (dotted blue line), 45 nM WT NS (solid blue line). (d) Band densities from SDS PAGE as in panel (a) for WT NS (solid lines) and panel (b) for E289A NS (dashed lines): NS/tPA Complex (blue lines), cleaved NS (green lines), native NS (brown lines). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments.
Mentions: The ability of E289A NS to form a complex with tPA was tested by mixing NS and tPA and incubating for different time intervals. The formation of the NS/tPA complex was monitored by SDS-PAGE (Fig. 5a,b). We clearly observed the rapid formation of the complex, and since the complex is known to be fragile at late stages1030, we also observed the appearance of protein bands corresponding to cleaved NS and to non-complexed tPA, as well as the reduction in intensity of the complex. The densities of the gel bands in Fig. 5d did not display any significant difference in the ability to form the complex, while they showed a slower reduction of native NS for E289A NS with respect to the WT NS equivalent, in agreement with the chromogenic assay presented in Fig. 5c, which reports the progress of tPA inhibition by NS against a chromogenic substrate, IPR-pNA. The inhibitory activity of E289A NS was lower than that of WT NS. At later stages, our results suggest that tPA released after complex formation was still active, meaning that the protease is only partially translocated. Alternatively, this may suggest a disruption of the tPA-NS covalent bond and subsequent recovery of the integrity of the active site of tPA.

Bottom Line: Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli.The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively.Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.

ABSTRACT
Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

No MeSH data available.


Related in: MedlinePlus