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Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

Fan F, Geurts AM, Pabbidi MR, Smith SV, Harder DR, Jacob H, Roman RJ - PLoS ONE (2014)

Bottom Line: We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background.The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats.These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America.

ABSTRACT
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

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Identification of the Zn-finger target site and deletion in the Dusp5 KO strain.Panel A presents a schematic model of the Dusp5 protein. The Dusp5 Zinc-finger construct targets amino acids (AA) 92–96 in the N-terminal regulatory rhodanese domain (AA5-140) resulting in the introduction of a premature stop codon at AA121 that is predicted to produce a truncated protein. The Dusp5 antibody used in these studies targets AA286-384 in the C-terminal phosphatase catalytic domain (AA178-314). Panel B presents a comparison of I-TASSER predicted structure and the folding of the Dusp5 protein in FHH (155R) and FHH.1BN (155G) rats. The upper panels show the predicted structure of the Dusp5 protein in both strains based on the complete AA sequence. The putative catalytic triad (Asp232/Ser268/Cys263) is shown in a “stick figure” form and the 3-letter AA codes are labeled in black. The rest of the protein is represented as ribbon running along the backbone. Secondary structural elements are depicted by color with helices, beta sheets and coils represented in red, cyan and white, respectively. The putative catalytic triad is magnified and shown in “stick figure” form in the lower panel and the 3-letter AA codes are labeled with in black. Only residues 174–320 of Dusp5 protein are presented in order to enhance the view of the putative catalytic triad. There are significant structural differences both in the overall folding of the Dusp5 protein that impact on the structure of the active site/catalytic triad region between the strains. This may account for the observed differences in the activity of the Dusp5 protein in FHH versus FHH.1BN rats.
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pone-0112878-g002: Identification of the Zn-finger target site and deletion in the Dusp5 KO strain.Panel A presents a schematic model of the Dusp5 protein. The Dusp5 Zinc-finger construct targets amino acids (AA) 92–96 in the N-terminal regulatory rhodanese domain (AA5-140) resulting in the introduction of a premature stop codon at AA121 that is predicted to produce a truncated protein. The Dusp5 antibody used in these studies targets AA286-384 in the C-terminal phosphatase catalytic domain (AA178-314). Panel B presents a comparison of I-TASSER predicted structure and the folding of the Dusp5 protein in FHH (155R) and FHH.1BN (155G) rats. The upper panels show the predicted structure of the Dusp5 protein in both strains based on the complete AA sequence. The putative catalytic triad (Asp232/Ser268/Cys263) is shown in a “stick figure” form and the 3-letter AA codes are labeled in black. The rest of the protein is represented as ribbon running along the backbone. Secondary structural elements are depicted by color with helices, beta sheets and coils represented in red, cyan and white, respectively. The putative catalytic triad is magnified and shown in “stick figure” form in the lower panel and the 3-letter AA codes are labeled with in black. Only residues 174–320 of Dusp5 protein are presented in order to enhance the view of the putative catalytic triad. There are significant structural differences both in the overall folding of the Dusp5 protein that impact on the structure of the active site/catalytic triad region between the strains. This may account for the observed differences in the activity of the Dusp5 protein in FHH versus FHH.1BN rats.

Mentions: The results of the comparative sequence analysis are presented in Figure 1. We identified 17 SNPs in the Dusp5 gene in FHH versus the BN reference sequence. Most of the SNPs were in introns, however, there were four SNPs in the Dusp5 mRNA including a C107T SNP in the 5′-UTR, a G330T SNP in exon 1, a C627T and a G637A in exon 2. All of these SNPs were verified in our FHH and FHH.1BN strains by sequencing cDNAs derived from mRNA extracted from the isolated cerebral vessels. The C107T SNP altered a CpG site and the C627T SNP altered one of six CpGs in exon 2 that were previously identified as methylation sites by bisulfite modification. [34]. Moreover, the G637A SNP caused a G155R mutation (Figure 2A) that is predicted using I-TASSER modeling package [35]–[37] may alter the folding of the protein and the active site conformation of the Dusp5 protein in FHH versus FHH.1BN rats as shown in Figure 2B.


Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

Fan F, Geurts AM, Pabbidi MR, Smith SV, Harder DR, Jacob H, Roman RJ - PLoS ONE (2014)

Identification of the Zn-finger target site and deletion in the Dusp5 KO strain.Panel A presents a schematic model of the Dusp5 protein. The Dusp5 Zinc-finger construct targets amino acids (AA) 92–96 in the N-terminal regulatory rhodanese domain (AA5-140) resulting in the introduction of a premature stop codon at AA121 that is predicted to produce a truncated protein. The Dusp5 antibody used in these studies targets AA286-384 in the C-terminal phosphatase catalytic domain (AA178-314). Panel B presents a comparison of I-TASSER predicted structure and the folding of the Dusp5 protein in FHH (155R) and FHH.1BN (155G) rats. The upper panels show the predicted structure of the Dusp5 protein in both strains based on the complete AA sequence. The putative catalytic triad (Asp232/Ser268/Cys263) is shown in a “stick figure” form and the 3-letter AA codes are labeled in black. The rest of the protein is represented as ribbon running along the backbone. Secondary structural elements are depicted by color with helices, beta sheets and coils represented in red, cyan and white, respectively. The putative catalytic triad is magnified and shown in “stick figure” form in the lower panel and the 3-letter AA codes are labeled with in black. Only residues 174–320 of Dusp5 protein are presented in order to enhance the view of the putative catalytic triad. There are significant structural differences both in the overall folding of the Dusp5 protein that impact on the structure of the active site/catalytic triad region between the strains. This may account for the observed differences in the activity of the Dusp5 protein in FHH versus FHH.1BN rats.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232417&req=5

pone-0112878-g002: Identification of the Zn-finger target site and deletion in the Dusp5 KO strain.Panel A presents a schematic model of the Dusp5 protein. The Dusp5 Zinc-finger construct targets amino acids (AA) 92–96 in the N-terminal regulatory rhodanese domain (AA5-140) resulting in the introduction of a premature stop codon at AA121 that is predicted to produce a truncated protein. The Dusp5 antibody used in these studies targets AA286-384 in the C-terminal phosphatase catalytic domain (AA178-314). Panel B presents a comparison of I-TASSER predicted structure and the folding of the Dusp5 protein in FHH (155R) and FHH.1BN (155G) rats. The upper panels show the predicted structure of the Dusp5 protein in both strains based on the complete AA sequence. The putative catalytic triad (Asp232/Ser268/Cys263) is shown in a “stick figure” form and the 3-letter AA codes are labeled in black. The rest of the protein is represented as ribbon running along the backbone. Secondary structural elements are depicted by color with helices, beta sheets and coils represented in red, cyan and white, respectively. The putative catalytic triad is magnified and shown in “stick figure” form in the lower panel and the 3-letter AA codes are labeled with in black. Only residues 174–320 of Dusp5 protein are presented in order to enhance the view of the putative catalytic triad. There are significant structural differences both in the overall folding of the Dusp5 protein that impact on the structure of the active site/catalytic triad region between the strains. This may account for the observed differences in the activity of the Dusp5 protein in FHH versus FHH.1BN rats.
Mentions: The results of the comparative sequence analysis are presented in Figure 1. We identified 17 SNPs in the Dusp5 gene in FHH versus the BN reference sequence. Most of the SNPs were in introns, however, there were four SNPs in the Dusp5 mRNA including a C107T SNP in the 5′-UTR, a G330T SNP in exon 1, a C627T and a G637A in exon 2. All of these SNPs were verified in our FHH and FHH.1BN strains by sequencing cDNAs derived from mRNA extracted from the isolated cerebral vessels. The C107T SNP altered a CpG site and the C627T SNP altered one of six CpGs in exon 2 that were previously identified as methylation sites by bisulfite modification. [34]. Moreover, the G637A SNP caused a G155R mutation (Figure 2A) that is predicted using I-TASSER modeling package [35]–[37] may alter the folding of the protein and the active site conformation of the Dusp5 protein in FHH versus FHH.1BN rats as shown in Figure 2B.

Bottom Line: We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background.The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats.These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America.

ABSTRACT
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

Show MeSH
Related in: MedlinePlus