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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

Polymerisation kinetics.(a,b) Non-denaturing PAGE of WT and E289A NS incubated at 52 °C for different time intervals (as reported in the figure). (c,d) Band density from non-denaturing PAGE as in panels (a,b). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments. The values of dimer, trimer, tetramer and pentamer populations are scaled by a factor of 5 for easier visualisation.
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f4: Polymerisation kinetics.(a,b) Non-denaturing PAGE of WT and E289A NS incubated at 52 °C for different time intervals (as reported in the figure). (c,d) Band density from non-denaturing PAGE as in panels (a,b). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments. The values of dimer, trimer, tetramer and pentamer populations are scaled by a factor of 5 for easier visualisation.

Mentions: The polymerisation of NS can be induced by moderate thermal stress. We incubated both WT and E289 NS at 52 °C for different time intervals and monitored the progress of polymerisation by non-denaturing poly-acrylamide gel electrophoresis (PAGE) (Fig. 4). The formation of small-size polymers produced the typical ladder-like pattern presented in Fig. 4a,b29. At the same time we observed the formation of latent NS, which run faster than native monomeric NS, due to its reduced hydrodynamic radius23. Both WT and E289A NS exhibited analogous polymerisation kinetics. A closer inspection by densitometry analysis (Fig. 4c,d) revealed that the polymerisation kinetics of E289A NS was slightly faster when compared to WT NS.


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)

Polymerisation kinetics.(a,b) Non-denaturing PAGE of WT and E289A NS incubated at 52 °C for different time intervals (as reported in the figure). (c,d) Band density from non-denaturing PAGE as in panels (a,b). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments. The values of dimer, trimer, tetramer and pentamer populations are scaled by a factor of 5 for easier visualisation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Polymerisation kinetics.(a,b) Non-denaturing PAGE of WT and E289A NS incubated at 52 °C for different time intervals (as reported in the figure). (c,d) Band density from non-denaturing PAGE as in panels (a,b). Points are normalised by the total density value of each lane. The points are averages over triplicate experiments. The values of dimer, trimer, tetramer and pentamer populations are scaled by a factor of 5 for easier visualisation.
Mentions: The polymerisation of NS can be induced by moderate thermal stress. We incubated both WT and E289 NS at 52 °C for different time intervals and monitored the progress of polymerisation by non-denaturing poly-acrylamide gel electrophoresis (PAGE) (Fig. 4). The formation of small-size polymers produced the typical ladder-like pattern presented in Fig. 4a,b29. At the same time we observed the formation of latent NS, which run faster than native monomeric NS, due to its reduced hydrodynamic radius23. Both WT and E289A NS exhibited analogous polymerisation kinetics. A closer inspection by densitometry analysis (Fig. 4c,d) revealed that the polymerisation kinetics of E289A NS was slightly faster when compared to WT NS.

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