Identification of a Survival-Promoting Peptide in Medium Conditioned by Oxidatively Stressed Cell Lines of Nervous System Origin

Abstract

A survival-promoting peptide has been purified from medium conditioned by Y79 human retinoblastoma cells and a mouse hippocampal cell line (HN 33.1) exposed to H 2 O 2 . A 30 residue synthetic peptide was made on the basis of N-terminal sequences obtained during purification, and it was found to exhibit gel mobility and staining properties similar to the purified molecules. The peptide maintains cells and their processes in vitro for the HN 33.1 cell line treated with H 2 O 2 , and in vivo for cortical neurons after lesions of the cerebral cortex. It has weak homology with a fragment of a putative bacterial antigen and, like that molecule, binds IgG. The peptide also contains a motif reminiscent of a critical sequence in the catalytic region of calcineurin-type phosphatases; surprisingly, like several members of this family, the peptide catalyzes the hydrolysis of para-nitrophenylphosphate in the presence of Mn 2ϩ . Application of the peptide to one side of bilateral cerebral cortex lesions centered on area 2 in rats results in an increase in IgG immunoreactivity in the vicinity of the lesions 7 d after surgery. Microglia immunopositive for IgG and ED-1 are, however, dramatically reduced around the lesions in the treated hemisphere. Furthermore, pyramidal neurons that would normally shrink, die, or disintegrate were maintained, as determined by MAP2 immunocytochemistry and Nissl staining. These survival effects were often found in both hemispheres. The results suggest that this peptide operates by diffusion to regulate the immune response and thereby rescue neurons that would usually degenerate after cortical lesions. The phosphatase activity of this molecule also suggests the potential for direct neuron survivalpromoting effects.

Dermcidin expression in hepatic cells improves survival without N-glycosylation, but requires asparagine residues

“…Proteolysis-inducing factor shares all but three amino acids with the predicted dermcidin protein and mismatches may be the result of sequencing artefact resulting in the conversion of cysteine to serine (Todorov et al, 1996). Similarly, only the two unidentifiable amino acids of Y-P30 (Cunningham et al, 1998) Asparagine and serine residues, which are the only potential N-and O-glycosylation sites, respectively, are underlined and can be seen to lie in the Y-P30 sequence, with the exception of the four serine residues of DCD-1. The Y-P30 and DCD-1 sequences were derived as described (Cunningham et al, 1998;Schittek et al, 2001).…”

“…Similarly, only the two unidentifiable amino acids of Y-P30 (Cunningham et al, 1998) Asparagine and serine residues, which are the only potential N-and O-glycosylation sites, respectively, are underlined and can be seen to lie in the Y-P30 sequence, with the exception of the four serine residues of DCD-1. The Y-P30 and DCD-1 sequences were derived as described (Cunningham et al, 1998;Schittek et al, 2001). The signal peptide sequence and N-and O-glycosylation sites were derived by computer modelling using SignalP, Net-N-Glyc and Net-O-Glyc, respectively.…”

“…The amino-acid sequence of DCD-1 does not overlap with that of PIF. A second product is diffusible survival evasion peptide (DSEP, or Y-P30), a neuronal survival peptide identified in the medium of oxidatively stressed neuronal cell lines (Cunningham et al, 1998). Y-P30 is a product of the same region of dermcidin as the core peptide of PIF ( Figure 1B).…”

“…The unglycosylated PIF amino-acid sequence is predicted to be identical to that of Y-P30 (Todorov et al, 1996;Cunningham et al, 1998), and it is now believed that dermcidin may play a role in carcinogenesis via the action of this peptide. Dermcidin has been identified as a candidate oncogene in breast cancer and stable transfection has been shown to stimulate cell growth and inhibit menadione-induced cell death, as assessed by cell counting (Porter et al, 2003).…”

“…Stable transfection of dermcidin has also been demonstrated to improve the survival of neuronal cells subjected to oxidative stress with H 2 O 2 (Cunningham et al, 2002), and it is feasible that these effects are due to the activity of Y-P30. The mechanism by which this peptide improves survival appears to involve a central, calcineurin-like phosphatase domain (Cunningham et al, 1998). This domain contains one of the two possible Nglycosylation sites of PIF and lies just seven amino acids from the other.…”