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J W Cuozzo

Publications and source records attributed to J W Cuozzo.

9 recordsLinked to original sources
PubMed2001

A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation.

Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway.

Amino Acid Sequence↗
PubMed2000

Pathways for protein disulphide bond formation.

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.

Animals↗
PubMed1999

Competition between glutathione and protein thiols for disulphide-bond formation.

It has long been assumed that the oxidized form of glutathione, the tripeptide glutamate-cysteine-glycine, is a source of oxidizing equivalents needed for the formation of disulphide bonds in proteins within the endoplasmic reticulum (ER), although the in vivo function of glutathione in the ER has never been studied directly. Here we show that the major pathway for oxidation in the yeast ER, defined by the protein Ero1, is responsible for the oxidation of both glutathione and protein thiols. However, mutation and overexpression studies show that glutathione competes with protein thiols for the oxidizing machinery. Thus, contrary to expectation, cellular glutathione contributes net reducing equivalents to the ER; these reducing equivalents can buffer the ER against transient hyperoxidizing conditions.

Disulfides↗
PubMed1998

Lysine-based structure responsible for selective mannose phosphorylation of cathepsin D and cathepsin L defines a common structural motif for lysosomal enzyme targeting.

Previous studies have shown that lysine residues on the surface of cathepsins and other lysosomal proteins are a shared component of the recognition structure involved in mannose phosphorylation. In this study, the involvement of specific lysine residues in mannose phosphorylation of cathepsin D was explored by site-directed mutagenesis. Mutation of two lysine residues in the mature portion of the protein, Lys-203 and Lys-293, cooperated to inhibit mannose phosphorylation by 70%. Other positively charged residues could not substitute for lysine at these positions, and comparison of thermal denaturation curves for the wild type and mutant proteins indicated that the inhibition could not be explained by alterations in protein folding. Structural comparisons of the two lysine residues with those required for phosphorylation of cathepsin L, using models generated from recently acquired crystal structures, revealed several relevant similarities. On both molecules, the lysine residues were positioned approximately 34 A apart (34.06 A for cathepsin D and 33.80 A for cathepsin L). When the lysine pairs were superimposed, N-linked glycosylation sites on the two proteins were found to be oriented so that oligosaccharides extending out from the sites could share a common region of space. Further similarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeting structure based on a specific arrangement of lysine residues with respect to each other and to glycosylation sites on the surface of lysosomal proteins.

Animals↗
PubMed1995

Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation.

The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.

3T3 Cells↗
PubMed1994

Lysine is a common determinant for mannose phosphorylation of lysosomal proteins.

The phosphorylation of lysosomal enzymes on high mannose residues is the first step in the targeting of these enzymes to lysosomes in a wide range of mammalian cells. Phosphorylated lysosomal enzymes bind to mannose 6-phosphate receptors, which divert them from the secretory pathway and direct them toward the lysosome. We have been investigating the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase by using the precursor form of the lysosomal cysteine protease, cathepsin L, as a model lysosomal enzyme in an in vitro assay for mannose phosphorylation. Cathepsin L was found to be phosphorylated in vitro with the same efficiency as other lysosomal enzymes and to contain a conformationally sensitive protein signal that is recognized by phosphotransferase. Biochemical modification of lysine residues on cathepsin L with sulfo-N-hydroxysuccinimide acetate prevented the enzyme from being phosphorylated, indicating that lysine is an important component of the signal. The modification itself did not cause any major conformational changes in cathepsin L. When the same modification was performed on a number of other lysosomal enzymes, phosphorylation was also inhibited. Thus, we conclude that lysine residues are important features of lysosomal enzyme phosphotransferase recognition sites in general, and we discuss the implications of this finding in the ongoing efforts to define the phosphotransferase recognition site.

Animals↗