'Il destino della scienza'.
Explore the source record for details and available documents.
Financing, Organized↗
Biomedical subjects
L Fricker
Publications and source records attributed to L Fricker.
Induced and spontaneous mutations at Ser202 of carboxypeptidase E. Effect on enzyme expression, activity, and intracellular routing.
Carboxypeptidase E (CPE) is involved in peptide processing in the brain and various neuroendocrine tissues. In mice homozygous for the Cpefat mutation, the virtual absence of CPE activity in islets of Langerhans and pituitary was associated with a missense mutation effecting a Ser202 to Pro shift (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). To examine the importance of Ser202 in CPE function, several mutations in this position were generated (Pro202, Ala202, Gly202, and Phe202). When the mutant proteins were expressed in a Baculovirus system, both Phe202 and Pro202CPE were enzymatically inactive, were unable to bind to a substrate affinity column, and were not secreted from Sf9 cells. In contrast, Ala202CPE or Gly202CPE exhibited enzymatic properties similar to those of wild-type CPE and were secreted from Sf9 cells. When expressed in AtT-20 cells, a mouse pituitary-derived cell line, CPE with Pro202 and Phe202 were not secreted. Pulse-chase analysis with [35S]Met indicated that Pro202CPE was degraded in AtT-20 cells within several hours. This degradative process was blocked by incubation at 15 degrees C but not by brefeldin A or by lysosomotrophic drugs. Pulse-chase analysis using dispersed pituitary cells from C57BLKS/Lt-Cpefat/Cpefat mutant mice shows similar results; Pro202-CPE produced in these cells was not secreted but rather was degraded within 5 h. Immunofluorescence analysis of epitope-tagged CPE revealed Ser202CPE to be present primarily in secretory vesicles, whereas Pro202CPE was localized to the endoplasmic reticulum and not the secretory vesicle-like structures. These results support the previous finding that Cpefat/Cpefat mice are defective in CPE activity because of the point mutation producing the Ser202 to Pro substitution. Furthermore, these results are consistent with a model that Ser202 is important for the intracellular folding of CPE.
Amino Acid Sequence↗
Processing of procarboxypeptidase E into carboxypeptidase E occurs in secretory vesicles.
Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m-chlorophenylhydrazone, or 20 degrees C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.
Amino Acid Sequence↗
Ring-hydroxylated analogues of lucanthone as antitumor agents.
A series of ring-alkoxylated and ring-hydroxylated analogues of lucanthone was prepared and tested for antitumor activity. The most biologically interesting members of this group were the 7-hydroxylucanthone derivatives, 50 and 51, which gave T/C values in the NCI P-388 antitumor screen of 188 and 265, respectively. The apparent association constants and delta Tm values for a number of analogue-DNA complexes were determined to ascertain whether there was any quantitative correlation with biological activity. The most that can be said is that intercalation may be a necessary but far from sufficient condition for antitumor activity.
Animals↗