Active Site Residues of Human β-Glucuronidase

Islam, M. Rafiq; Tomatsu, Shunji; Shah, Gul N.; Grubb, Jeffrey H.; Jain, Sanjeev; Sly, William S.

doi:10.1074/jbc.274.33.23451

Cited by 75 publications

(43 citation statements)

References 31 publications

(27 reference statements)

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“…Expression of the cDNA containing the E540A missense mutation in COS cells or insect cells produced an inactive protein with normal turnover and stability (43). These studies suggested that a transgene expressing human GUS E540A on the MPS VII (gus mps/mps ) background might provide a murine MPS VII model that retains the MPS VII phenotype, but has the added desirable feature of being immune tolerant to human GUS.…”

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confidence: 93%

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Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

Sly

Vogler

Grubb

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of ␤-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gus mps/mps ) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human ␤-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gus mps/mps ) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gus mps/mps ) mouse. However, they are now tolerant to immune challenge with human ␤-glucuronidase. This ''tolerant MPS VII mouse model'' should be useful for preclinical trials evaluating the effectiveness of enzyme and͞or gene therapy with the human gene products likely to be administered to human patients with MPS VII.

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confidence: 93%

“…In prior work, we characterized the structure and active site of human GUS, and identified residue E540 as the active site nucleophile (42,43). Expression of the cDNA containing the E540A missense mutation in COS cells or insect cells produced an inactive protein with normal turnover and stability (43).…”

mentioning

confidence: 99%

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

Sly

Vogler

Grubb

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Briefly, 200 bp of PKD1 promoter DNA (0.5 g) with the vector or CA-Mekk1 expression plasmids (0.2 g of DNA) with 80 pmol of scrambled siRNA, human p53 siRNA, or Mekk1 siRNA duplex in FBS and antibiotic-free medium was incubated, added to cells, and incubated overnight at 37°C. The medium was then replaced with fresh DMEM containing 2% serum and antibiotics, and the cells were harvested at 40 h. M-1 cells grown in DMEM/F-12 supplemented with 5% FBS, antibiotic, 1.5 mM Hepes, and 24 mM NaHCO 3 at 37°C were transfected with pEGFPN3 vector or kinase-dead CA-Mekk1 (DC) in the same vector using Lipofectamine and selected for neomycin (G418) resistance at a concentration of 400 g/ml as before (35). Mekk1 Ϫ/Ϫ and Mekk1 ϩ/ϩ mouse embryonic fibroblast cells were cultured in DMEM with 4.5 g/liter of glucose containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and antibiotic (100 IU/ml of penicillin, 100 g/ml of streptomycin).…”

Section: Methodsmentioning

confidence: 99%

MAP/ERK Kinase Kinase 1 (MEKK1) Mediates Transcriptional Repression by Interacting with Polycystic Kidney Disease-1 (PKD1) Promoter-bound p53 Tumor Suppressor Protein

Islam

Jiménez

Pelham

et al. 2010

Journal of Biological Chemistry

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Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNF␣, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

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“…The active site of human β-glucuronidase has been based on the experimentally determined active site in E. coli β-galactosidase and corresponds to the amino acid residues Glu451, Glu540 and Tyr504. The nature of the active site residues have been discussed previously (Islam et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Studies of Human β-Glucuronidase

Noorbatcha¹

2010

American Journal of Applied Sciences

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Problem statement:The enzyme β-glucuronidase is being used as a reporter molecule in the area of genetic engineering, as a component of prodrug therapy in cancer treatment and in the scouring process of cotton fabrics. However, a detailed understanding of the factors responsible for the stability and the activity of this enzyme is still not available. Molecular Dynamics (MD) simulations provide an estimate of equilibrium and dynamic properties of enzyme systems that cannot be calculated analytically. With this perspective, molecular dynamics simulations of human β-glucuronidase (GUS) have been carried out to determine the behavior of this enzyme in vacuum and solvent environments at a defined temperature. Approach: CHARMM force field along with distance dependent dielectric model was used to represent the solvent environment in the MD simulations. The parameters employed in various stages of MD simulations had been selected based on repeated trials under various conditions as a method of choosing the optimum parameters for each stage. Results: It was found that simulations in vacuum caused the backbone of GUS to have smaller fluctuations from their mean values compared with the fluctuation in implicit solvent simulations, due to the fact that vacuum environment did not provide for the electrostatic interactions affecting the backbone of GUS that may otherwise exist in a solvent environment. Conclusion: Inclusion of solvent effects in MD simulations is crucial in understanding structural flexibility and stability of β-glucuronidase. Implicit solvent method can provide a realistic inclusion of backbone flexibility and structural compactness of GUS, which will have profound influence on the stability and activity of the enzymes, with a marginal increase in computational time.

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Active Site Residues of Human β-Glucuronidase

Cited by 75 publications

References 31 publications

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

MAP/ERK Kinase Kinase 1 (MEKK1) Mediates Transcriptional Repression by Interacting with Polycystic Kidney Disease-1 (PKD1) Promoter-bound p53 Tumor Suppressor Protein

Molecular Dynamics Studies of Human β-Glucuronidase

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