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Computational and functional characterization of Angiogenin mutations, and correlation with amyotrophic lateral sclerosis.

Padhi AK, Banerjee K, Gomes J, Banerjee M - PLoS ONE (2014)

Bottom Line: We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins.Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation.Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.

View Article: PubMed Central - PubMed

Affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India.

ABSTRACT
The Angiogenin (ANG) gene is frequently mutated in patients suffering from the neurodegenerative disease--amyotrophic lateral sclerosis (ALS). Most of the ALS-causing mutations in Angiogenin affect either its ribonucleolytic or nuclear translocation activity. Here we report the functional characterization of two previously uncharacterized missense mutations in Angiogenin--D22G and L35P. We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins. Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation. Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.

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Purification, secondary structure depiction and ribonucleolytic activity of wild-type Angiogenin-GST and mutants.A) Coomassie stained SDS-PAGE gel showing wild-type Angiogenin-GST (lane 2), D22G (lane 3) and L35P (lane 4) proteins after Ni-NTA affinity purification. Lane 1 contains molecular weight markers. The Angiogenin proteins (14.1 kDa) are all tagged with Glutathione S-transferase (GST, ∼26 kDa) for soluble expression in E. coli. B) Plot showing CD spectra for wild-type (black), D22G (green), and L35P (brown) Angiogenin-GST proteins. Samples were diluted in PBS to yield a concentration of 0.4 mg/ml; three spectra were recorded, averaged and plotted after subtracting the buffer baseline for each sample. C) Ribonucleolytic activity of wild-type (black), D22G (green) and L35P (red) Angiogenin-GST proteins measured using yeast tRNA as substrate. The proteins, at concentrations of 0.05 to 0.5 mg/ml, were incubated with yeast tRNA (2 mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by addition of ice-cold perchloric acid, and the absorbance of the supernatents was measured at 260 nm; data were collected from three independent experiments for each protein concentration. Student’s t-test of three independent experiments shows that the difference between wild-type and each of the three mutant protein is significant (n = 3; p<0.05). D) The loss of ribonucleolytic activity of D22G and L35P mutants compared to wild-type Angiogenin-GST. The amount of protein required to generate 1.0 optical density (OD) is compared with wild-type Angiogenin-GST to generate same OD unit for mutants.
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pone-0111963-g003: Purification, secondary structure depiction and ribonucleolytic activity of wild-type Angiogenin-GST and mutants.A) Coomassie stained SDS-PAGE gel showing wild-type Angiogenin-GST (lane 2), D22G (lane 3) and L35P (lane 4) proteins after Ni-NTA affinity purification. Lane 1 contains molecular weight markers. The Angiogenin proteins (14.1 kDa) are all tagged with Glutathione S-transferase (GST, ∼26 kDa) for soluble expression in E. coli. B) Plot showing CD spectra for wild-type (black), D22G (green), and L35P (brown) Angiogenin-GST proteins. Samples were diluted in PBS to yield a concentration of 0.4 mg/ml; three spectra were recorded, averaged and plotted after subtracting the buffer baseline for each sample. C) Ribonucleolytic activity of wild-type (black), D22G (green) and L35P (red) Angiogenin-GST proteins measured using yeast tRNA as substrate. The proteins, at concentrations of 0.05 to 0.5 mg/ml, were incubated with yeast tRNA (2 mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by addition of ice-cold perchloric acid, and the absorbance of the supernatents was measured at 260 nm; data were collected from three independent experiments for each protein concentration. Student’s t-test of three independent experiments shows that the difference between wild-type and each of the three mutant protein is significant (n = 3; p<0.05). D) The loss of ribonucleolytic activity of D22G and L35P mutants compared to wild-type Angiogenin-GST. The amount of protein required to generate 1.0 optical density (OD) is compared with wild-type Angiogenin-GST to generate same OD unit for mutants.

Mentions: In order to confirm our predictions, we generated GST-tagged constructs of wild-type Angiogenin, D22G and L35P mutants, with C-terminus His-tags. While the GST tag was required to generate Angiogenin in the soluble form in a bacterial expression system, the His-tags were included for ease of purification. The proteins were purified to >95% homogeneity through Ni-NTA affinity and size exclusion chromatography (Figure 3A), where they eluted as dimers with approximate molecular weights of 80 kDa (see Figure S1). Initial characterization of the purified proteins was carried out through circular dichroic spectroscopy. It was observed that the far-UV CD spectra of the mutants were largely similar to that of wild-type Angiogenin-GST (Figure 3B), indicating that the missense mutations did not significantly affect the secondary structure, overall stability or folding of Angiogenin-GST. The root mean square deviation (RMSD) profiles for D22G and L35P-Angiogenin also support our CD measurements, suggesting that the structural stability of the mutants was similar to that of wild-type Angiogenin (Figures 2G and 3B). In addition, MD simulation was carried out for wild-type Angiogenin conjugated with GST and a hexa-histidine His-tag, and it was observed that for the 50 ns duration of simulation, presence of the GST moiety did not affect the structural integrity or folding of Angiogenin, including its functional sites – the catalytic triad and nuclear localization signal residues (see Figure S2). This indicated that the presence of GST- or His-tag will probably not interfere with the biochemical activity of Angiogenin.


Computational and functional characterization of Angiogenin mutations, and correlation with amyotrophic lateral sclerosis.

Padhi AK, Banerjee K, Gomes J, Banerjee M - PLoS ONE (2014)

Purification, secondary structure depiction and ribonucleolytic activity of wild-type Angiogenin-GST and mutants.A) Coomassie stained SDS-PAGE gel showing wild-type Angiogenin-GST (lane 2), D22G (lane 3) and L35P (lane 4) proteins after Ni-NTA affinity purification. Lane 1 contains molecular weight markers. The Angiogenin proteins (14.1 kDa) are all tagged with Glutathione S-transferase (GST, ∼26 kDa) for soluble expression in E. coli. B) Plot showing CD spectra for wild-type (black), D22G (green), and L35P (brown) Angiogenin-GST proteins. Samples were diluted in PBS to yield a concentration of 0.4 mg/ml; three spectra were recorded, averaged and plotted after subtracting the buffer baseline for each sample. C) Ribonucleolytic activity of wild-type (black), D22G (green) and L35P (red) Angiogenin-GST proteins measured using yeast tRNA as substrate. The proteins, at concentrations of 0.05 to 0.5 mg/ml, were incubated with yeast tRNA (2 mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by addition of ice-cold perchloric acid, and the absorbance of the supernatents was measured at 260 nm; data were collected from three independent experiments for each protein concentration. Student’s t-test of three independent experiments shows that the difference between wild-type and each of the three mutant protein is significant (n = 3; p<0.05). D) The loss of ribonucleolytic activity of D22G and L35P mutants compared to wild-type Angiogenin-GST. The amount of protein required to generate 1.0 optical density (OD) is compared with wild-type Angiogenin-GST to generate same OD unit for mutants.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4221194&req=5

pone-0111963-g003: Purification, secondary structure depiction and ribonucleolytic activity of wild-type Angiogenin-GST and mutants.A) Coomassie stained SDS-PAGE gel showing wild-type Angiogenin-GST (lane 2), D22G (lane 3) and L35P (lane 4) proteins after Ni-NTA affinity purification. Lane 1 contains molecular weight markers. The Angiogenin proteins (14.1 kDa) are all tagged with Glutathione S-transferase (GST, ∼26 kDa) for soluble expression in E. coli. B) Plot showing CD spectra for wild-type (black), D22G (green), and L35P (brown) Angiogenin-GST proteins. Samples were diluted in PBS to yield a concentration of 0.4 mg/ml; three spectra were recorded, averaged and plotted after subtracting the buffer baseline for each sample. C) Ribonucleolytic activity of wild-type (black), D22G (green) and L35P (red) Angiogenin-GST proteins measured using yeast tRNA as substrate. The proteins, at concentrations of 0.05 to 0.5 mg/ml, were incubated with yeast tRNA (2 mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by addition of ice-cold perchloric acid, and the absorbance of the supernatents was measured at 260 nm; data were collected from three independent experiments for each protein concentration. Student’s t-test of three independent experiments shows that the difference between wild-type and each of the three mutant protein is significant (n = 3; p<0.05). D) The loss of ribonucleolytic activity of D22G and L35P mutants compared to wild-type Angiogenin-GST. The amount of protein required to generate 1.0 optical density (OD) is compared with wild-type Angiogenin-GST to generate same OD unit for mutants.
Mentions: In order to confirm our predictions, we generated GST-tagged constructs of wild-type Angiogenin, D22G and L35P mutants, with C-terminus His-tags. While the GST tag was required to generate Angiogenin in the soluble form in a bacterial expression system, the His-tags were included for ease of purification. The proteins were purified to >95% homogeneity through Ni-NTA affinity and size exclusion chromatography (Figure 3A), where they eluted as dimers with approximate molecular weights of 80 kDa (see Figure S1). Initial characterization of the purified proteins was carried out through circular dichroic spectroscopy. It was observed that the far-UV CD spectra of the mutants were largely similar to that of wild-type Angiogenin-GST (Figure 3B), indicating that the missense mutations did not significantly affect the secondary structure, overall stability or folding of Angiogenin-GST. The root mean square deviation (RMSD) profiles for D22G and L35P-Angiogenin also support our CD measurements, suggesting that the structural stability of the mutants was similar to that of wild-type Angiogenin (Figures 2G and 3B). In addition, MD simulation was carried out for wild-type Angiogenin conjugated with GST and a hexa-histidine His-tag, and it was observed that for the 50 ns duration of simulation, presence of the GST moiety did not affect the structural integrity or folding of Angiogenin, including its functional sites – the catalytic triad and nuclear localization signal residues (see Figure S2). This indicated that the presence of GST- or His-tag will probably not interfere with the biochemical activity of Angiogenin.

Bottom Line: We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins.Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation.Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.

View Article: PubMed Central - PubMed

Affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India.

ABSTRACT
The Angiogenin (ANG) gene is frequently mutated in patients suffering from the neurodegenerative disease--amyotrophic lateral sclerosis (ALS). Most of the ALS-causing mutations in Angiogenin affect either its ribonucleolytic or nuclear translocation activity. Here we report the functional characterization of two previously uncharacterized missense mutations in Angiogenin--D22G and L35P. We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins. Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation. Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.

Show MeSH
Related in: MedlinePlus