Aspartic proteinases are a class of enzymes widely distributed among fungi, plants, vertebrates, and viruses. They are involved in numerous disease conditions, including hypertension, amyloid disease, malaria, and AIDS. 1 In HIV, the proteinase is essential for the maturation of the virus particle, and inhibitors have a proven therapeutic effect in the treatment of AIDS. There have also been significant recent advances in the development of orally active renin inhibitors which are in clinical trials. 2 Thus inhibitors to this class of enzyme with improved characteristics are therefore much sought after as potential therapeutic agents. The aspartic proteinase class of enzyme comprises two structurally similar domains, each contributing an aspartic acid residue to form a catalytic dyad that cleaves the substrate peptide bond. The two catalytic aspartate carboxyls are held coplanar by a network of hydrogen bonds involving the surrounding main chain and the conserved amino acid side-chain groups. Most aspartic proteinases are inhibited by pepstatin, a microbial peptide that contains the unusual amino acid statine, and a number of crystal structures of complexes containing this naturally occurring analogue have been reported. 3,4 Endothiapepsin, a member of the aspartic proteinase family, is composed of 330 amino acid residues, with roughly 170 residues in each domain. Endothiapepsin is derived from the fungus Endothia parasitica, and numerous structures of this enzyme bound to a range of renin inhibitors have been analyzed with the aim of developing improved compounds by structure-based design. 5 Crystallographic studies of all native aspartic proteinases have revealed a water molecule hydrogen bonded to the carboxyl groups of both aspartate residues. This water molecule is within hydrogen-bonding distance of all four carboxyl O atoms and has been implicated in catalysis. It has been suggested 6 that this water is partly displaced upon substrate binding and is polarized by one of the catalytic aspartate residues. It then initiates enzymatic catalysis by attacking the scissile bond carbonyl group of the substrate. The transition state of aspartic proteinase catalysis has been studied most extensively using enzyme-inhibitor complexes which possess a range of different transition state mimics. Most inhibitors mimic one or both hydroxyls in the putative transition state. The statine-based inhibitors contain one hydroxyl that occupies the same position as the water E-mail: coatesl@ornl.gov. † Oak Ridge National Laboratory. ‡ University of Toledo. § Los Alamos National Laboratory. ¶ University College London.Supporting Information Available: Data collection and refinement statistics for all three structures discussed within this paper, the atomic resolution, the room temperature X-ray, and room temperature neutron. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 1. NIH Public AccessResults from this study suggest that the transition state is stabilized by a negative charge on Asp...

Citation: Fu Q, Wang F, Wang H, et al. Next-generation sequencing-based molecular diagnosis of a Chinese patient cohort with autosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2013;54:4158-4166. DOI:10.1167/iovs.13-11672 PURPOSE. Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease; therefore, an accurate molecular diagnosis is essential for appropriate disease treatment and family planning. The prevalence of RP in China had been reported at 1 in 3800, resulting in an estimated total of 340,000 Chinese RP patients. However, genetic studies of Chinese RP patients have been very limited. To date, no comprehensive molecular diagnosis has been done for Chinese RP patients. With the emergence of next-generation sequencing (NGS), comprehensive molecular diagnosis of RP is now within reach. The purpose of this study was to perform the first NGS-based comprehensive molecular diagnosis for Chinese RP patients.METHODS. Thirty-one well-characterized autosomal recessive RP (arRP) families were recruited. For each family, the DNA sample from one affected member was sequenced using our custom capture panel, which includes 163 retinal disease genes. Variants were called, filtered, and annotated by our in-house automatic pipeline. RESULTS.Twelve arRP families were successfully molecular diagnosed, achieving a diagnostic rate of approximately 40%. Interestingly, approximately 63% of the pathogenic mutations we identified are novel, which is higher than that observed in a similar study on European descent (45%). Moreover, the clinical diagnoses of two families were refined based on the pathogenic mutations identified in the patients.CONCLUSIONS. We conclude that comprehensive molecular diagnosis can be vital for an accurate clinical diagnosis of RP. Applying this tool on patients from different ethnic groups is essential for enhancing our knowledge of the global spectrum of RP disease-causing mutations.