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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 47�54 |
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doi:10.1111/j.1745-7270.2008.00369.x |
Preliminary computational modeling of
nitric oxide synthase 3 interactions with caveolin-1: influence of exon 7
Glu298Asp polymorphism
Mandar S. Joshi and John
Anthony Bauer*
Center for
Cardiovascular Medicine, The Research Institute at Nationwide Children's
Hospital, Columbus Ohio 43205, USA
Received: July 18,
2007�������
Accepted: September
13, 2007
*Corresponding
author: Tel, 1-614-722-4984; Fax, 1-614-355-3444; E-mail, [email protected]
The significance
of endothelial nitric oxide synthase 3 (NOS3) activity has been recognized for
many years, however� it was only recently that the complicated regulation of
this constitutively expressed enzyme in endothelial cells was identified. A
critical component of the NOS3 regulatory cycle in endothelial cells is its
intracellular localization to caveolae. The caveolar coordination of NOS3, more
specifically its interaction� with caveolin-1 (Cav-1), plays a major role in
normal� endothelial NOS3 activity and vascular bioavailability of nitric oxide.
We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism
caused diminished shear-dependent NOS activation, was less extensively
associated� with caveolae, and had a decreased degree of interaction� with
Cav-1. Here, we carried out preliminary investigations� to identify possible
mechanisms of the genotype�-dependent endothelial cell responses we observed in
our previous investigations. Through this approach we tested the hypothesis
that computer simulations could provide� insights regarding the contribution of
this single nucleotide polymorphism to regulation of the NOS3 isoform. We
observed� that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3
associated with Cav-1 was significantly lower. Additionally, we have shown,
using a theoretical computational� model, that mutation of an amino acid at
position� 298 might affect the protein-protein interactions and localization�
of the NOS3 protein. These alterations might also affect the protein function
and explain the enhanced disease� risk associated with the presence of
Glu298Asp polymorphism� in the NOS3 protein.
Keywords �������NOS3; caveolin-1; gene polymorphism; computational modeling;
protein-protein interaction
The vascular endothelium is highly metabolically active and plays a key role in vascular homeostasis through the release of a variety of substances, including the vaso-regulator� nitric oxide (NO) [1]. The actions of NO can be altered severely under various conditions of stress, such as hypoxia, oxidants, reactive nitrogen species, and shear stress [2,3]. Diminished NO availability has been associated� with several settings of endothelial impairment, including acute hypoxic or hyperoxic organ injury and chronic forms of vascular disease [3-8]. NO synthase 3 (NOS3) is important� for maintenance of systemic blood pressure, vascular remodeling, angiogenesis, and wound healing [9-12].
A critical component of the NOS3 regulatory cycle in endothelial cells is its intracellular localization to caveolae [13-15]. Caveolae are 50-100 nm flask-shaped, cholesterol�- and glycosphingolipid-rich membrane microdomains in the plasma membrane that provide intracellular� coordination of many membrane-associated proteins and putatively function as regulators of cholesterol� transport, endocytosis, and signal transduction [16,17]. In endothelial cells, caveolin-1 (Cav-1), a 22-kDa protein that coats the cytoplasmic surface of this specialized microdomain, is the major protein constituent of caveolae and is thought to hold or "store" NOS3 in an inactive state, wherein phosphorylation causes dissociation and NOS3 catalytic activation [18]. The caveolar coordination of NOS3, more specifically its interaction with Cav-1, plays a major role in normal endothelial NOS3 activity and vascular� bioavailability of NO and is a key controller of shear-dependent NO release [19].
Several specific allelic variations of the NOS3 gene are known to date, with many of these having been linked to increased cardiovascular disease risk [20-27]. Out of a total of eight variations in the human NOS3 gene, only one codes for a variant form of the enzyme; a single nucleotide� polymorphism (SNP) (G>T) at position 894, leading to the sequence change Glu298Asp. Using a "candidate" gene approach, in which patient genotypes are related to the incidence of a disease and/or clinical outcome to test for statistically significant associations (parametric and/or non-parametric linkage analyses), many reports have indicated� that the NOS3 Glu298Asp polymorphism is associated� with increased occurrence of cardiovascular disease, including coronary artery disease [25,28,29], myocardial infarction [30,31], hypertension [32,33], and stroke [34]. Although the exact biological consequences of this specific variation are not identified, prior studies by others using isolated NOS3 enzyme derived from transfected� COS7 cells indicated no difference in purified enzyme catalytic activity between wild-type (Glu/Glu) and variant (Asp/Asp) NOS3 [35].
Given that caveolar associations have been recognized as important for normal NOS3 performance in endothelial cells, and the established importance of this variation for disease risk in humans, we recently investigated the impact� of the NOS3 Glu298Asp polymorphism on endothelial� cell responses to shear stress and caveolar localization. We found initial evidence that the altered enzyme, when in its native endothelial cell environment, has a diminished shear-dependent NOS activation, is less extensively associated with caveolae, and has a decreased degree of interaction with Cav-1 [36]. Our preliminary findings using protein biochemistry techniques thus suggested� that the Glu298Asp variation might influence protein-protein interactions. In this project we carried out preliminary computer modeling to further investigate possible� mechanisms of the genotype-dependent endothelial� cell responses we observed in our previous investigations [36]. Through this approach we tested the hypothesis that computer simulations could provide insights regarding the contribution of this SNP to regulation of the NOS3 isoform.
Materials and Methods
NOS3 genotyping
Human umbilical vein endothelial cells were screened for NOS3 genotype using polymerase chain reaction-based DNA amplification followed by restriction enzyme digestion� as described previously [30,36].
Cell lysis and
immunoprecipitation
After washing three times with ice-cold phosphate-buffered� saline,
cells were lysed in NP-40 lysis buffer containing 1% NP-40, 50 mM Tris, pH 7.5,
150 mM NaCl, 5 mM EDTA, 0.5 mM Na3VO4, 50 mM
NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 1 mM phenylmethylsulphonyl fluoride. The cell
extracts were used for immunoprecipitation reactions. The protein G immuno�precipitation
kit from Sigma (St. Louis, USA) was used. The cell extracts were incubated
overnight at 4 �C, with NOS3 polyclonal antibody (pAb) on protein G agarose�
beads. The beads were washed thoroughly after incubation� and the sample was
prepared for sodium dodecyl sulfate-polyacrylamide� gel electrophoresis. The
blots were probed for Cav-1 protein. The amount of Cav-1 associated with NOS3
was calculated as a ratio of Cav-1 band optical density� to NOS3 band optical
density. The optical density analysis was carried out using LabWorks image
analysis software (version 4.6; UVP-Media Cybernetics, Upland, USA).
Western blotting
Samples prepared as described above were separated on 4%-20% Tris-glycine gel. Standard Western blotting protocols� were used as described previously [36].
Protein structures and
computational modeling
The amino acid sequence of human NOS3 (accession No. AAH69465) was retrieved from the National Center for Biotechnology Information protein sequence database (http://www.ncbi.nlm.nih.gov/). Computational modeling was carried out using the DeepView software Swiss-PdbViewer [37]. The NOS3 protein amino acid sequence was loaded into DeepView software and the 3-D structure was generated. Using the mutation feature in the software, variation of the amino acid at position 298 (Asp) was done in chain B of the protein. Chain A of the protein had the wild-type amino acid Glu at position 298. Visualization of the protein structure was carried out in the secondary structure� form. Specific amino acid residues required for enzyme function were identified in the protein 3-D structure� and amino acid distances were calculated using the distance� measurement feature of the PdbViewer software. Using the amino-acid visualization tool in the software, specific amino acids of interest were selected one at a time and distance measurements were carried out from amino acid 298. All the measurements from amino acid 298 were done from the side chain carbon atom. All distances were measured� in angstrom units () and are expressed here as absolute values and percentage variation.
Confocal microscopy
Cells were grown on a collagen-coated slide to confluence. They were fixed with formalin and washed with Tris-buffered� saline. Double labeling was done using rabbit anti-NOS3 pAb (Transduction Biolabs, Bedford, USA) and rabbit anti-Cav-1 pAb (Upstate Biotechnology, Lake Placid, USA). Chicken anti-rabbit (Alexa Fluor 488; Molecular Probes, Carlsbad, USA) labeled antibody was used as a secondary for NOS3 primary antibody, which showed green fluorescence. Goat anti-rabbit F(ab)2 fragment (Alexa Fluor 633; Molecular Probes) labeled antibody was used as a secondary for Cav-1 primary antibody, which showed red fluorescence. Confocal microscopic observations were done using the LSM Meta 510 laser confocal microscope.
Statistical analysis
All data are expressed as mean�SEM. Each cell line was treated as an individual sample. GraphPad Prism software (version 4.0; San Diego, USA) was used to fit the data and carry out the statistical analysis. A simultaneous comparison� of groups was handled by one-way anova, with Student�-Newman-Keuls post-hoc analysis. For analyzing the combination� effects, two-way anova was used to test differences between the genotypes and various treatments. In all cases, significance was defined as P<0.05.
Results
Identification of key amino
acid residues
Using the National Center for Biotechnology Information sequence database and previous reports in the literature, key amino acids essential for NOS3 enzymatic activity were identified. These amino acid residues are listed in Table 1, and were used for theoretical distance calculations� in the computational modeling studies.
Basal Cav-1/NOS3 association
is lower in the variant genotypes
Initially, immunoprecipitation studies were carried out and
quantified as described in "Materials and Methods". The ratio of
Cav-1/NOS3 band optical density was 2.53�0.15 in Glu/Glu, but it was
significantly lower in Glu/Asp (1.57�0.31) and Asp/Asp (1.36�0.23) variant
genotypes (P<0.05) (Fig. 1). These results show that
under basal conditions the NOS3/Cav-1 association is altered in the Asp variant
genotypes.
Basal NOS3 membrane
localization diminished in the Asp variant genotypes
We also carried out confocal microscopy under basal conditions and
probed for co-localization of NOS3 and Cav-1. We observed greater co-localization
of NOS3 and Cav-1 at the cell membrane in the wild-type cells. These studies
showed that under basal conditions, the Glu/Glu wild-type cells have a greater
NOS3 localization at the caveolar regions on the cell membrane as compared to
the Glu/Asp and Asp/Asp variant endothelial cells (Fig. 2).
Altered protein structural
geometry in the variant amino acid chain
The 3-D protein structure was constructed using DeepView software [37]. The important substrate and co-factor binding sites were highlighted and shown in Fig. 3. Using the software, the amino acid at position 298 in chain B was mutated from Glu to Asp. Individual amino acid distances from various substrate and co-factor binding sites were calculated for chain A (Glu at position 298) and chain B (Glu at position 298) based on this 3-D structure (Fig. 4). The absolute distances are shown in Table 2 and represented as angstrom units () as well as the percentage� difference. Variation of the amino acid residue at position 298 from Glu to Asp in chain B resulted in altered protein geometry. The distance between the important sites and altered residues were consistently lower in chain B compared� to chain A. The percentage differences were greatest in the Cav-1 binding region when amino acid variation occurred at position 298.
Discussion
Theoretical protein modeling serves as an important tool for structure elucidation and prediction of protein interactions� and structural features [38]. Since the identification� of the NOS family of enzymes, many structural� features have been elucidated. Based on previous� reports we compiled a list of the important sites that are necessary for NOS3 protein function [39-41]. NOS3 is regulated in a complex manner and requires many post-translational modifications for proper functioning. Myristoylation and palmitoylation at the N-terminal are important for proper localization and targeting of the protein� to organelles in the cell [42]. NOS3 enzyme function� is also dependent on substrate (L-arginine) and co-factor (tetrahydrobiopterine, Zn) availability as well as their interactions� with other known regulatory proteins (Cav-1 and Hsp90) [13].
Given the importance of tight
regulation of NOS3 protein� and its important interactions with Cav-1 protein,
we carried� out NOS3 immunoprecipitation in endothelial cells that were
previously genotyped for the presence of Glu298Asp polymorphism. We observed
that in the Glu/Asp and Asp/Asp variant genotypes, the amount of NOS3
associated� with Cav-1 was significantly lower. These observations� were
recapitulated by the confocal microscopic� observations. Moreover, using a
caveolar membrane-enrichment method we also showed that the amount of NOS3
present in the caveolar membrane is significantly lower in the variant
genotypes. These data indicate that NOS3 is not localized properly to the
caveolar membrane and its interaction with Cav-1 protein is altered� in the
Glu/Asp and Asp/Asp variants.
NOS3 is regulated in a cyclic manner and under static conditions, and it is bound to membrane caveolae through its interactions with Cav-1. On stimulation, this interaction� is dissociated and NOS3 enters the cytosol where it interacts� with other proteins and co-factors to produce NO. Thus, Cav-1-bound NOS3 represents the available pool of NOS3 that can readily produce NO in the cell.
The 3-D protein structures might provide valuable insights� into the molecular basis of protein function. Combining� sequence information with the 3-D structure might give detailed information regarding the structural relevance of variations and protein function [43]. Traditional� methods like X-ray crystallography and nuclear magnetic resonance spectroscopy are time-consuming and require a pure form of the protein. We used the Swiss-Prot DeepView software to carry out theoretical structural� modeling of the NOS3 protein [37,43,44]. After resolving the structure and marking key sites on the protein, we observed that the amino acid at position 298 is not close to the active site pocket of NOS3. The important site for NOS3 function is the dimerization site that has all the substrate� and co-factor binding regions. Thus it is unlikely� that the presence of the mutated amino acid (Asp) at position� 298 will alter the enzymatic function of NOS3. We carried out computational modeling studies of the protein. Theoretical distance calculations revealed that variation of the amino acid at position 298 might affect protein geometry. The variation of an amino acid in one chain or both chains might introduce a structural variation� in the protein structure and also affect the interactions of the protein. It is unlikely that this change in protein amino acid distances will affect the enzymatic pocket of the protein, but it might alter the protein localization and trafficking. It is not completely understood whether an amino acid distance change of 3-10 � will actually make a difference in protein structure, but some reports suggest� that a change of distance by 1 � might affect the electrostatic� energies to a great extent [45]. We do not know the electrostatic energies in this modeling study, but it would be interesting to study these in light of the variation at position 298. Although this is not the most suitable method to characterize protein structural alterations, it does provide a good "starting point". The differences observed by using this method provide some insights into the structural and functional implications of a gene SNP. These observations provide preliminary evidence� that the changes in protein structure might affect� its interactions with substrate or co-factor.�
In conclusion, we have shown, using a
theoretical computational� model, that variation of an amino acid at position 298
might affect the protein-protein interactions and localization of the NOS3
protein. These alterations might also affect the protein function and might
explain the enhanced disease risk associated with the presence of Glu298Asp
polymorphism in the NOS3 protein.
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