Figure 1.
NOX5 regulation and activity in RCSC.
(A) Ionomycin stimulated superoxide production (NOX5) was inhibited by SOD and DPI. Harvested RCS cells suspended in Hank's complete with Ca2+ were treated with DPI or SOD, as described under Methods. The cells were then stimulated by 100 nM ionomycin or 1 µl DMSO (control). Superoxide production was assayed by chemiluminescence using the Enhanced Diogenes Assay. The area under the curve (AUC) was calculated as a measure of total superoxide production. The values were expressed as RLU/105 cells over the first 30 seconds of the chemiluminescence output. The data represent the mean ± SEM. Asterisks indicate statistical significance: *p<0.05; **p<0.01. (B) RCSC were transfected with either 40 nM non target control siRNA (siNTC) or 40 nM siRNA against NOX5 (siNOX5). After 72 h, the cells were harvested and NOX5 activity was assayed for superoxide production in siNOX5 or siNTC transfectd RCSC with or without ionomycin stimulation. Superoxide production was expressed as RLU/105 cells over the first 30 seconds of the chemiluminescence output. AUC for siNTC with ionomycin (Solid boxes) versus AUC for siNTC without ionomycin (hollow boxes) was statistically significant,*p<0.05.
Figure 2.
Expression of NOX5 in RCSC determined by RT-PCR and western blot.
(A) NOX5 expression detected by RT-PCR using human NOX5-specific PCR primers. The amplified product was confirmed as a NOX5 sequence by nucleotide sequencing (n = 6). (B) Knockdown of the steady state levels of NOX5 mRNA. Values obtained by densitometry were analyzed and ratios were calculated. Amount of knockdown of NOX5 mRNA steady pools are depicted as vertical bars. (1) siNTC (40 nM), (2) siNOX5 (5 nM), (3) siNOX5 (10 nM), (4) siNOX5 (20 nM), (5) siNOX5 (40 nM). (C) Western blot analysis detected by NOX5 specific antibody. RCS cells were grown, harvested, lysed, and fractionated by centrifugation at 29,000× g. The pellets were suspended in lysis buffer and subjected to IP for enriching the NOX5 followed by Western blot analysis. Controls included NOX5 encoding HUVEC and anti NOX5 blocking peptide (NOX5 BP). (D) Knockdown of NOX5 determined by Western blotting. RCSC were transfected with 40 nM non target control siRNA (siNTC) or siRNA against NOX5 (siNOX5). After 72 h, the cells were harvested and total protein lysates were used for Western blot analysis by NOX5 specific antibody. β-Actin was used as loading control.
Figure 3.
Phylogenetic analysis of NOX5.
Phylogenetic analysis of NOX5 gene based on the schematic phylogeny of organisms created using multiple alignments of the putative amino acid sequence of rabbit NOX5 and deduced amino acid sequences retrieved from the GenBank database. The Tree was reconstructed following NCBI BLAST by the neighbor-joining method. Our rabbit NOX5 sequence (JF723383) is shown as the unknown query.
Figure 4.
Sequence alignment of rabbit NOX5 with human NOX5 isoforms.
Amino acid sequence alignment of rabbit NOX5 (RabbitC (insilico cloned), RabbitP (predicted) RabbitPCS (predicted partial) and XP_002722428.1 (predicted NCBI database)) with NOX5 isoforms from human AF325189_1 (isoforms beta), AF325200_1 (isoforms delta), NP_078781.3 (isoforms 1), NP_001171708.1 (isoforms 2), and NP_0011709.1 (isoforms 3) shows rabbit homolog is longer by 18 amino acids.
Figure 5.
Relationship between rabbit NOX5, Nox/Duox family members, and Calcineurin structure.
(5A) Cladogram of sequence relationships among Nox/Duox family members of four species; amino acid alignment of NOX5 from rabbit and human NOX (1–5), human DUOX and Duox from rats and mice was used to construct the cladogram illustrating sequence relationships. Conceptual protein sequences within the regions homologous to putative rabbit NOX5 protein sequence were aligned. Distances show rabbit NOX5 was closer to the human NOX5 than rodent Duox2. (5B) T-coffee analysis of NOX5 amino acid sequence aligning to structure of chain B and D of human Calcineurin ‘B’ subunit structure. 2p6b B confers calcium subunit and may have role in the calmodulin activation of calcineurin.
Figure 6.
Functional domains and domain architecture based on putative NOX5 sequence.
Based on the alignments, the essential functional residues retrieved from the conserved domain architecture retrieval tool (CDART) showed 10 sequences (coelaomata), 88 sequences (Bilateria), 3 sequences (Deuterostomia), 3 sequences (Magnoliphyta), 65 sequences (cellular organisms), and 120 sequences (embryophyta), in addition to several sequences from other organisms predicted to possess domain architectures similar to that of EF hand (cd00051), Ferric reduct superfamily (pfam01794) and NOX_DuoX_1 (cd06186). Figure illustrating few sequences from different species arranged in a bead_on_string_style showing the presence of predicted EF hand (cd00051), Ferric_reduct super family (pfam01794) and NOX_DuoX_1 (cd06186) domains when rabbit NOX5 was used as query sequence for the domain prediction.
Figure 7.
Graphic representations of conserved domains present in the putative rabbit NOX5 sequence.
(A) Motif search predicted the presence of at least 3 putative EFh motifs. (B) Predicted EF hands between amino acid 57–92; 93–128 and 137–172 in N-terminal region. (C) Amino acid sequences alignment in the N terminal region showing conserved EFh sequences in rabbit NOX5 and human NOX5 isoforms. (D) NOX/Duox like FAD/NADP binding domain (cd0186) and part of the pfam08030 super family that includes cd06205 and corresponds to Ferrodoxin reductase (FNR), a FAD and NADP binding protein in C terminal region. (E) Conserved sequence of C terminal region highlighting the conserved NOX domain in rabbit and human NOX5 isoforms.