Structural and functional investigations of two lysosomal transporters implicated in developmental disorders

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PROJECT SUMMARY Lysosomal storage diseases (LSDs) comprise a group of over 70 inherited autosomal recessive disorders that are caused by malfunctions in lysosomal proteins, leading to a buildup of specific substrates in the lysosome. These relatively rare disorders tend to manifest in infants, although adult forms also exist. Cystinosis and Infantile Sialic acid Storage Disease (ISSD) are two of the more common LSDs. Symptoms of Cystinosis include renal and vision problems, while symptoms of ISSD include problems with the central nervous system and early death. Both of these genetic disorders also cause severe developmental delay. Cystinosis is caused by mutations in the protein Cystinosin, a lysosomal H+:cystine symporter, while ISSD is caused by mutations in the protein Sialin, a lysosomal H+:sialic acid symporter and neurotransmitter uniporter. Structural and functional data will help shed light on the exact molecular mechanism by which disease mutations affect the function of these proteins. In my preliminary studies, I generated a structurally specific monoclonal antibody that recognizes the luminal domain of Cystinosin, Fab3H5. Using this antibody, I solved three structures of human full-length Cystinosin: an outward- facing apo conformation at 3.4-Å resolution at pH 7.5, an inward-facing apo conformation at 3.2-Å resolution at pH 5.0, and a cystine-bound conformation at 3.4-Å resolution. These structures revealed the residues involved in the cytosolic and luminal gates that mediate the transport of its substrate cystine, as well as the residues that interact with cystine in the binding pocket of Cystinosin. Preliminary electrophysiological data for this protein further identified mutations D205N and D305N, which abolished transport, and mutations Q284A and D346N, which maintained a transport activity similar to the wild-type protein. In order to fully define the molecular mechanism of transport for Cystinosin, I propose to solve the structure of these Cystinosin mutants and test them with electrophysiological assays, building a more complete picture of the binding and transport mechanism. With reference to Sialin and ISSD, my preliminary work has yielded a monoclonal antibody; however, the 3D reconstruction of Sialin with this antibody did not yield visible secondary structures. Therefore, I will first optimize the antibody screening process. This will allow me to isolate a structurally specific monoclonal antibody that I can use for structural studies. I will then solve the structure of Sialin using the same general approach that has been successful with Cystinosin. Since Sialin is also a H+:sialic acid symporter, different pH environments will be used to capture different conformations. Sialin also binds and transports various substrates. Therefore, I will also solve the substrate-bound structures of Sialin, thus providing more information about the binding pocket(s) of the substrates. Important mechanistic and substrate binding residues will then be tested via electrophysiology (for sialic acid) and radiolabeled substrate uptake assays (for neurotransmitters). These proposed studies will reveal the underlying molecular mechanisms behind the LSDs Cystinosis and ISSD, paving the way for further translational research and therapeutic design.