Structural Modeling of a Novel CAPN5 Mutation that Causes Uveitis and Neovascular Retinal Detachment

CAPN5 mutations have been linked to autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV), a blinding autoimmune eye disease. Here, we link a new CAPN5 mutation to ADNIV and model the three-dimensional structure of the resulting mutant protein. In our study, a kindred with inflammatory vitreoretinopathy was evaluated by clinical eye examinations, DNA sequencing, and protein structural modeling to investigate the disease-causing mutation. Two daughters of an affected mother demonstrated symptoms of stage III ADNIV, with posterior uveitis, cystoid macular edema, intraocular fibrosis, retinal neovascularization, retinal degeneration, and cataract. The women also harbored a novel guanine to thymine (c.750G>T, p.Lys250Asn) missense mutation in exon 6 of CAPN5, a gene that encodes a calcium-activated cysteine protease, calpain-5. Modeling based on the structures of all known calpains revealed the mutation falls within a calcium-sensitive flexible gating loop that controls access to the catalytic groove. Three-dimensional modeling placed the new mutation in a region adjacent to two previously identified disease-causing mutations, all three of which likely disrupt hydrogen bonding within the gating loop, yielding a CAPN5 with altered enzymatic activity. This is the third case of a CAPN5 mutation leading to inherited uveitis and neovascular vitreoretinopathy, suggesting patients with ADNIV features should be tested for CAPN5 mutations. Structural modeling of novel variants can be used to support mechanistic consequences of the disease-causing variants

PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

The PLUNC ("Palate, lung, nasal epithelium clone") protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family--the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP)--are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways.Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model.Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen

Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay

The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9 mg/ml; SPLUNC1 concentrations ranged from 34.7 ng/ml to 13.8 μg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7 ng/ml to 5.3 μg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies

Missense mutations that cause Van der Woude syndrome and popliteal pterygium syndrome affect the DNA-binding and transcriptional activation functions of IRF6

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype–phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype–phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity

Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor

AbstractResistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.</jats:p

The structure of LsrB from <i>Yersinia pestis</i> complexed with autoinducer-2

The crystal structure of LsrB from Yersinia pestis complexed with autoinducer-2 (AI-2; space group P212121, unit-cell parameters a = 40.61, b = 61.03, c = 125.23 Å) has been solved by molecular replacement using the structure of LsrB from Salmonella typhimurium (PDB entry 1tjy) and refined to R = 0.180 (R free = 0.213) at 1.75 Å resolution. The electron density for bound AI-2 and the stereochemistry of the AI-2-binding site are consistent with bound AI-2 adopting the (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran conformation, just as has been observed in the crystal structures of the Salmonella typhimurium and Sinorhizobium meliloti LsrB–AI-2 complexes.</jats:p

Crystal structure of a complex of NOD1 CARD and ubiquitin.

The Caspase Recruitment Domain (CARD) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance

A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

Eukaryotic proliferating cell nuclear antigen (PCNA), an essential accessory factor in DNA replication and repair, is a ring-shaped homotrimer. A novel nontrimeric structure of E113G-mutant PCNA protein is reported, which shows that this protein forms alternate subunit interactions. It is concluded that the charged side chain of Glu113 promotes normal trimer formation by destabilizing these alternate subunit interactions