Crystal Structure of Human UG
Zhang in collaboration with N. Pattabiraman (NCI-Frederick) and L. Miele (University of Illinois, Chicago IL)
During the past year, we resolved the crystal structure of human UG (hUG) at 1.8 Å resolution. Molecular modeling studies revealed that the hUG dimer forms a large hydrophobic cavity (C1) and that each monomer forms a smaller cavity (C2 and C3, respectively). The largest cavity appears to be capable of harboring hydrophobic compounds such as progesterone, retinol, and polychlorinated biphenyls. Our results confirm previous reports that UG binds hydrophobic ligands.

UG in the Treatment of Inflammatory Diseases
Zhang, Chowdhury, Choi, Pilon-Clayton
Under a collaborative research and development agreement (CRADA) with Claragen, Inc., we have completed a phase I clinical trial with recombinant human UG to treat the first cohort of neonatal respiratory distress patients. The study has now entered the second phase to determine if the treatment prevents the development of bronchopulmonary dysplasia (BPD), a chronic inflammatory disease with high mortality. A U.S. patent has been granted for the use of recombinant human UG as a potential therapeutic agent for the treatment of inflammatory and fibrotic disorders, and another application for the use of recombinant UG for the treatment of IgA-nephropathy has been filed.

Figure 6

(a) Ribbon diagram of the crystal structure of recombinant human UG (rhUG) at 1.8Å resolution. Four cysteine residues forming two disulfide bridges are shown as Corey-Pauling-Koltun representations. (b) Molecular surface representation of the crystal structure of rhUG dimer. C1 is the largest hydrophobic cavity in dimeric rhUG, and C2 and C3 , respectively, represent small cavities in monomers.

Lysosomal Ceroid Depletion by Drugs
Zhang, Levin, Butler in collaboration with K. Wisniewski (NY State Institute for Basic Research, Staten Island NY)
Lysosomal accumulation of palmitoylated proteins (constituents of ceroid) due to the lack of palmitoyl-protein thioesterase activity causes INCL. Recognizing that nucleophilic attacks disrupt thioester linkages, we tested whether nucleophilic drugs such as cysteamine, N-acetylcysteine, and hydroxylamine can disrupt thioester linkages in palmitoylated proteins in INCL cells. We found that the drugs are effective in disrupting the thioester linkages not only in a model thioester compound, palmitoyl-CoA, but also in palmitoylated proteins in INCL cells. Further, phosphocysteamine caused the depletion of ceroid deposits in lysosomes of intact cells.

Figure 7

(a) Structure of three drugs with nucleophilic activity: phosphocysteamine, N-acetylcysteine, and hydroxylamine with (b) thioesterase activity demonstrated by using [14C]Palmitoyl-CoA. Note free palmitate release (arrow). (c) Cleavage of thioester linkages in palmitoylated proteins in lymphoblasts from normal and INCL patients. Even lanes are phosphocysteamine treated, and odd lanes are untreated cells.

Figure 8

Depletion of lysosomal ceroid from fibroblasts and lymphoblasts of INCL patients. Note the depletion of dense cytoplasmic granules within three weeks of treatment with phosphocysteamine. Upper panel: a, untreated; b, two weeks; and c, three weeks of treatment. Lower panel: d, untreated lymphoblasts; e-h, treated lymphoblasts.