Lyndsay Davies

P53 apoptosis mediator PERP: localization, function and caspase activation in uveal melanoma

p53 apoptosis effector related to PMP‐22 (PERP) is a transcriptional target gene of p53 tumour suppressor that is specifically induced during apoptosis and not during cell cycle arrest. In primary uveal melanoma (UM), the most common intraocular malignancy in adults that has a reportedly unaffected signalling pathway upstream of and including p53, PERP expression is down‐regulated in the metastatic monosomy 3‐type tumours, compared with the less aggressive disomy 3‐type tumours. Here, we demonstrate experimentally, by the use of full‐length PERP‐green fluorescent protein (GFP) fusions and real‐time confocal microscopy, the intracellular targeting and plasma membrane localization of PERP in living UM cells and show that expression of PERP induces caspase‐mediated apoptosis in UM cells. Induction of PERP expression in GFP‐PERP‐transfected UM cells leads to increased levels of cleaved caspase‐8 forms, as well as to reduction of its full‐length substrate Bid, but not to detectable processing of caspase‐9. The levels of mature caspase‐8, ‐9 and ‐3 proteins significantly correlate with PERP expression levels in primary UMs. Transcriptional profiling of PERP and caspase‐8 in tumour specimens indicates that the positive association of PERP and caspase‐8 proteins is a consequence of post‐translational processing, most likely at the level of caspase‐8 cleavage, and not of increased transcription of pro‐caspase‐8. We conclude that PERP expression leads to activation of an extrinsic receptor‐mediated apoptotic pathway, with a possible subsequent engagement of the intrinsic apoptotic pathway. The findings underline the apoptotic pathway mediated by PERP as a critical mechanism employed by UM tumours to modulate susceptibility to apoptosis.

PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells

The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

An unsuspected ecdysteroid/steroid phosphatase activity in the key T-cell regulator, Sts-1: surprising relationship to insect ecdysteroid phosphate phosphatase.

The insect enzyme ecdysteroid phosphate phosphatase (EPP) mobilizes active ecdysteroids from an inactive phosphorylated pool. Previously assigned to a novel class, it is shown here that it resides in the large histidine phosphatase superfamily related to cofactor‐dependent phosphoglycerate mutase, a superfamily housing notably diverse catalytic activities. Molecular modeling reveals a plausible substrate‐binding mode for EPP. Analysis of genomic and transcript data for a number of insect species shows that EPP may exist in both the single domain form previously characterized and in a longer, multidomain form. This latter form bears a quite unexpected relationship in sequence and domain architecture to vertebrate proteins, including Sts‐1, characterized as a key regulator of T‐cell activity. Long form Drosophila melanogaster EPP, human Sts‐1, and a related protein from Caenorhabditis elegans have all been cloned, assayed, and shown to catalyse the hydrolysis of ecdysteroid and steroid phosphates. The surprising relationship described and explored here between EPP and Sts‐1 has implications for our understanding of the function(s) of both. Proteins 2007.