PEPD

PEPD encodes prolidase, a ubiquitous cytosolic metalloenzyme that hydrolyzes oligopeptides bearing a C-terminal proline or hydroxyproline, a reaction central to collagen turnover (PMID:16470701). Its catalytic apparatus depends on a 'pita bread' fold homologous to E. coli methionine aminopeptidase, and structure-function mapping of patient and engineered variants has identified residues critical for both catalytic competence and structural integrity, including an exon 14-encoded region required for activity, the alpha2-domain residue Tyr231, and Leu192 (PMID:2010534, PMID:15309682, PMID:33877262). Loss-of-function alleles—missense, in-frame deletions, splice-site, and nonsense mutations—abolish prolidase activity and cause prolidase deficiency, with accumulation of proline-containing dipeptides such as Gly-Pro (PMID:15309682, PMID:8198124), and in at least one case associate the enzymatic defect with hyper-IgE immune dysregulation (PMID:16681595). Beyond catalysis, PEPD protein has a non-enzymatic role: it binds directly to the proline-rich domain of p53, sequestering the majority of nuclear and cytoplasmic p53 under basal conditions, blocking its phosphorylation and MDM2-driven mitochondrial translocation, and releasing it for activation upon ROS-generating stress (PMID:29233996). Extracellularly, secreted PEPD acts as an EGFR ligand that drives fibro-inflammatory signalling through PI3K/Akt/mTOR, FAK, Grb2, and ERK1/2 and increases collagen biosynthesis (PMID:33477899); loss of its intracellular enzymatic function together with extracellular EGFR-driven signalling produces adipose tissue fibrosis and insulin resistance in vivo (PMID:35478031).

1. 1991 Medium

   Establishing which region of prolidase is required for catalysis answered how specific lesions destroy enzyme function in prolidase deficiency.

   Evidence mRNA/genomic analysis and transfection of an exon 14-skipping mutant cDNA into prolidase-deficient fibroblasts with activity readout

   PMID:2010534

   Open questions at the time

   • No structural model of how the exon 14 region contributes to the active site
   • Single mutant studied; broader domain map incomplete
2. 1994 Medium

   Cataloguing distinct loss-of-function alleles established that severe prolidase deficiency arises from multiple independent mutational mechanisms rather than a single founder lesion.

   Evidence RT-PCR, SSCP, sequencing and inverse PCR across patients identifying missense, deletion, and splice-acceptor mutations

   PMID:8198124

   Open questions at the time

   • Enzymatic consequence of each allele not biochemically reconstituted
   • Genotype-phenotype severity correlation not resolved
3. 2004 Medium

   Mapping Y231del to the alpha2 domain of the 'pita bread' fold connected a clinical mutation to a structural model and to substrate accumulation, defining functional regions of the enzyme.

   Evidence cDNA sequencing, transient expression in deficient fibroblasts, activity assay, and capillary electrophoresis detecting Gly-Pro accumulation

   PMID:15309682

   Open questions at the time

   • Catalytic mechanism at atomic resolution not directly demonstrated here
   • Link between dipeptide accumulation and tissue pathology not established
4. 2006 Low

   Defining prolidase as a cytosolic C-terminal proline/hydroxyproline peptidase and linking a nonsense allele to near-absent activity grounded the enzyme's basic biochemical identity.

   Evidence Genomic sequencing and patient-cell enzymatic activity assays (p.R265X)

   PMID:16470701

   Open questions at the time

   • Activity assay in patient cells without biochemical reconstitution or mutagenesis rescue
   • Substrate specificity range not exhaustively mapped
5. 2006 Low

   Associating a PEPD missense mutation with hyper-IgE syndrome extended the phenotypic consequences of loss-of-function beyond classic prolidase deficiency to immune dysregulation.

   Evidence PCR/RFLP genotyping and family segregation in a single case

   PMID:16681595

   Open questions at the time

   • Single case without functional enzymatic or mechanistic validation
   • Mechanism linking prolidase loss to elevated IgE unknown
6. 2017 High

   Discovery that PEPD binds the p53 proline-rich domain independent of catalysis revealed a moonlighting role as a basal p53 reservoir and stress-gated regulator, distinct from its peptidase function.

   Evidence Reciprocal Co-IP, domain mapping, enzymatic-null mutagenesis, genetic elimination causing tumor regression, and ROS-inhibitor rescue

   PMID:29233996

   Open questions at the time

   • Structural basis of the PEPD–p53 interface not resolved
   • Precise ROS-dependent release mechanism not defined
   • Generality across cell types and in vivo relevance not established
7. 2021 Medium

   Demonstrating extracellular PEPD as an EGFR ligand that activates PI3K/Akt/mTOR, FAK/Grb2/ERK and integrin/IGF-1R signalling defined a secreted, receptor-mediated pro-fibrotic function separate from intracellular catalysis.

   Evidence Recombinant PEPD treatment of fibroblasts with Western blot pathway analysis, proliferation/migration assays, and collagen biosynthesis measurement

   PMID:33477899

   Open questions at the time

   • No mutagenesis of the EGFR-binding site or genetic rescue
   • Direct PEPD–EGFR binding affinity and stoichiometry not measured
   • Single lab, in vitro only
8. 2021 Medium

   Crystallographic comparison of variants pinpointed which residues compromise the enzyme via destabilization versus dynamics, refining the structure-function basis of deficiency-causing mutations.

   Evidence Site-directed mutagenesis, protein purification, and X-ray crystallography of wild-type and variant prolidase (R470H, L192P, Y231del)

   PMID:33877262

   Open questions at the time

   • Single lab with limited functional follow-up beyond structural comparison
   • Catalytic activity of each variant not quantitatively correlated with structure
9. 2022 High

   In vivo dissection of PEPD's intracellular enzymatic versus extracellular EGFR-ligand activities established that loss of catalysis and secreted-PEPD signalling jointly drive adipose tissue fibrosis and insulin resistance.

   Evidence Mouse genetic ablation and pharmacological inhibition with fibrosis phenotyping, systemic PEPD measurement, and EGFR pathway analysis in macrophages and adipocytes

   PMID:35478031

   Open questions at the time

   • Relative contribution of intracellular vs extracellular roles to the phenotype not fully separated
   • Source of secreted PEPD and regulation of its secretion unknown

• How PEPD's three activities—peptidase catalysis, non-enzymatic p53 sequestration, and extracellular EGFR ligation—are coordinated within a single cell or tissue, and whether they are mechanistically interdependent, remains unresolved.
• No structural model of the PEPD–EGFR or PEPD–p53 interfaces
• Trigger and route of PEPD secretion uncharacterized
• Interplay between collagen-turnover catalysis and signalling roles unknown