Abstract
Human immunodeficiency virus is an epidemic totaling over 37 million cases worldwide. The development of efficient treatment would save and improve millions of lives. Current treatment involves antiretrovirals which can block viral transformation but do not cure infection. A promising direction is to inhibit the viral protein Nef, which plays a key role in viral pathogenesis. Nef manipulates the trafficking and cellular distribution of several immune proteins and thereby enables infected cells to evade immune-mediated killing. A main target of Nef is the CD4 receptor. Here, Nef works by hijacking clathrin-adaptor protein complex (AP2) inducing the endocytosis of cell surface CD4. This downregulation benefits the virus on multiple fronts, including but not limited to facilitating new virion budding, ensuring proper processing of viral Env protein, and evading antibody-dependent cellular cytotoxicity (ADCC) that is otherwise induced by the exposure of Env epitopes resulted from the Env-CD4 interaction. Developing Nef inhibitors has been difficult largely due to the lack of in-depth understanding of its molecular actions. A recent crystal structure solved in our lab revealed the molecular details of the Nef-mediated, clathrin AP2-dependent CD4 downregulation. The structure inspired us to design cyclic peptides, which mimic CD4-binding, as potential inhibitors of Nef. To this end, I have designed, created, and produced a protein construct, which has subsequently enabled our collaborators to use the Macro-cyclic organo-peptide hybrids, or the MOrPHs methodology to develop cyclic peptide inhibitors. Carboxylic acid reductase (CAR) enzymes are commonly found in bacteria. This class of enzymes has recently come under scrutiny for its use in biomanufacturing. Bioengineering has shown it is possible to use these enzymes in production of industrially important biochemical precursors. The advantage of these enzymes is the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The potential for applications of carboxylic acid reductases include use in the fragrance and flavor industries—key intermediates here could be produced by CARs in cascade reactions. Most recently, CARs have been used in chemo-enzymatic reaction sequences and in-vivo cascade pathways. Although the scope of available substrates is broad, it may be further expanded to include novel small-molecule substrates to produce a greater diversity of the end-products. To broaden the substrate specificity, our collaborators in the Niu Lab at University of Nebraska have employed a platform to target residues which have been found to be key in substrate binding, both native and nonnative. The nonnative substrates studied in this research are small carbon molecules, which can be used in bio-cascades to produce valuable intermediates and end-products. To better understand the binding occurring between the nonnative substrate and the active site of the enzyme, the structures of the mutated enzyme in complex with the nonnative substrate and a wild-type enzyme were determined using x-ray crystallography. These will enhance the understanding of binding between the enzyme and nonnative substrate, and aid future enzyme engineering efforts.