Human Delta1-pyrroline-5-carboxylate synthase: function and regulation

Abstract

Mammalian Delta(1)-pyrroline-5-carboxylate synthase (P5CS) is a bifunctional ATP- and NAD(P)H-dependent mitochondrial enzyme that catalyzes the coupled phosphorylation and reduction-conversion of L: -glutamate to P5C, a pivotal step in the biosynthesis of L: -proline, L: -ornithine and L: -arginine. Previously, we reported cloning and characterization of two P5CS transcript variants generated by exon sliding that encode two protein isoforms differing only by a two amino acid-insert at the N-terminus of the gamma-glutamyl kinase active site. The short form (P5CS.short) is highly expressed in the gut and is inhibited by ornithine. In contrast, the long form (P5CS.long) is expressed ubiquitously and is insensitive to ornithine. Interestingly, we found that all the established human cell lines we have studied expressed P5CS.long but not P5CS.short. In addition, expression of P5CS.long can be modulated by hormones: downregulation by hydrocortisone and dexamethasone and upregulation by estradiol, for example. Using a quantitative proteomic approach, we showed that P5CS.long is upregulated by p53 in p53-induced apoptosis in DLD-1 colorectal cancer cells. Functional genomic analysis confirmed that there are two p53-binding consensus sequences in the promoter region and in the intron 1 of the human P5CS gene. Interestingly, overexpression of P5CS by adenoviruses harboring P5CS.long or P5CS.short in various cell types has no effect on cell growth or survival. It would be of importance to further investigate the role of P5CS as a p53 downstream effector and how P5CS.short expression is regulated by hormones and factors of alternative splicing in cells isolated from model animals.

Fig1

Model for the regulation of mammalian P5CS and P5CR by ornithine and proline in arginine and proline biosynthesis, respectively. The pathway for arginine (Arg) biosynthesis in the small intestinal mucosa is shown above. In this tissue, particularly in the neonates, the short isoform of P5C synthase (P5CS.short), which converts glutamate (Glu) to Δ¹-pyrroline-5-carboxylate (P5C), predominates and is subject to feedback inhibition by ornithine (Orn). In peripheral tissues the pathway serves to synthesize proline (Pro). In these tissues, the long isoform of P5C synthase predominates and is insensitive to Orn. The pathway appears to be regulated by Pro inhibition of P5C reductase (P5CR).

Fig 2

A Expression of P5CS.long in established human cell lines. RT-PCR analysis showed that P5CS.long, no P5CS.short, is expressed in established human cell lines. B Regulation of P5CS.long expression by hormones. Semi-quantitative RT-PCR was conducted using total RNA isolated from human small intestine CCL-6 cells treated with indicated compounds for 24 hrs.

Fig 3

Expression of human P5CS is upregulated by p53 as revealed by quantitative proteomic analysis. MALDI-TOF tandem mass spectrometry was used to profile the regulated proteins by p53. A An example of spectrum analysis of a peptide showed significant signal increase and 2-Da shift in the labeled isotopic peaks, indicating upregulation of the corresponding protein by p53. This peptide corresponded to codons 748–759 of human P5CS. B Four peptides derived from P5CS were identified and shown to be upregulated by p53. The identified peptides were located on a schematically presented P5CS protein, and corresponding codons and sequence of each peptide were as indicated.

Fig 4

P53 induces overexpression of human P5CS. A Semi-quantitative RT-PCR and B Immunoblot analysis confirmed that expression of P5CS is inducible by p53 at both RNA and protein levels 22 hrs after p53 overexpression in DLD-1.p53 cells. β-tubulin and α–actin served as loading control for RT-PCR and immunoblot analysis, respectively.

Fig 5

Overexpression of human P5CS failed to induce cell death or growth arrest in human cells. DLD-1 cells were infected with AD-p53, AD-P5CS.long or AD-P5CS.short. Attached cells were counted 36 hrs after infection. AD-p53 clearly induced apoptosis whereas P5CS.long and P5CS.short did not.