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Biomedical subjects

Sarah J Luchansky

Publications and source records attributed to Sarah J Luchansky.

8 recordsLinked to original sources
PubMed2006

Substrate recognition and catalysis by UCH-L1.

Deubiquitinating enzymes regulate essential cellular processes, and their dysregulation is implicated in multiple disease states. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has garnered attention for its links with Parkinson's disease and cancer; however, the mechanism of action of this enzyme in cells remains poorly understood. In order to advance our understanding of UCH-L1 function, we have been developing small molecule modulators of the enzyme for use as tools to probe its role in cells. In support of these efforts, an investigation of the mechanism of UCH-L1 catalysis was previously reported. Here, we extend this mechanistic evaluation and examine substrate recognition by UCH-L1. We developed a panel of ubiquitin fusions to test the contribution of specific residues of ubiquitin to binding and catalysis by the enzyme, and determined the activation parameters of selected variants to gain additional mechanistic insight. Ubiquitin side chains critical for establishing the Michaelis complex and enabling catalysis were identified, and features of this complex that differ between UCH-L1 and a homologue, UCH-L3, were revealed. These data provide dramatic examples of differences in substrate specificity between these enzymes. The implications of our experiments with UCH-L1 for selective inhibitor design and the relationship to disease are discussed.

Amino Acid Substitution↗
PubMed2006

Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1.

The ubiquitin C-terminal hydrolase UCH-L1 (PGP9.5) comprises >1% of total brain protein but is almost absent from other tissues [Wilkinson, K. D., et al. (1989) Science 246, 670-673]. Mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from Parkinson's disease [Leroy, E., et al. (1998) Nature 395, 451-452; Maraganore, D. M., et al. (1999) Neurology 53, 1858-1860]. Abnormal overexpression of UCH-L1 has been shown to correlate with several forms of cancer [Hibi, K., et al. (1998) Cancer Res. 58, 5690-5694]. Because the amino acid sequence of UCH-L1 is similar to that of other ubiquitin C-terminal hydrolases, including the ubiquitously expressed UCH-L3, which appear to be unconnected to neurodegenerative disease, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable importance. We have determined the three-dimensional structure of human UCH-L1 at 2.4-A resolution by x-ray crystallography. The overall fold resembles that of other ubiquitin hydrolases, including UCH-L3, but there are a number of significant differences. In particular, the geometry of the catalytic residues in the active site of UCH-L1 is distorted in such a way that the hydrolytic activity would appear to be impossible without substrate induced conformational rearrangements.

Amino Acid Sequence↗
PubMed2004

Metabolic functionalization of recombinant glycoproteins.

Glycoproteins are essential for cellular communication and are the most rapidly growing class of therapeutic agents. Chemical modification of glycoproteins has been employed to improve their in vivo efficacy or to label them for detection. Methods for the controlled derivatization of glycoproteins are presently limited by the repertoire of natural amino acid side chain and carbohydrate functionalities. Here, we use metabolic oligosaccharide engineering to introduce a bioorthogonal functional group, the azide, into cellular and recombinant glycoproteins for subsequent chemical elaboration via Staudinger ligation. As most therapeutic glycoproteins are sialylated and require this saccharide for optimal pharmacokinetics, we targeted sialic acid as a host for azides using N-azidoacetylmannosamine (ManNAz) as a biosynthetic precursor. Metabolic conversion of ManNAz to N-azidoacetylsialic acid (SiaNAz) within membrane-bound and secreted glycoproteins was quantified in a variety of cell types. SiaNAz was found to comprise between 4% and 41% of total sialosides, depending on the system. Metabolic labeling of recombinant interferon-beta and GlyCAM-Ig was achieved, demonstrating the utility of the method for functionalizing N-linked and O-linked glycoproteins of therapeutic interest. More generally, the generation of recombinant glycoproteins containing chemical handles within their glycans provides a means for studying their behavior and for improving their in vivo efficacy.

Animals↗
PubMed2002

GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism.

Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.

Base Sequence↗
PubMed2002

Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation.

The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.

Acetylglucosamine↗