Determination of Intracellular Prolyl/Glycyl Proteases in Intact Living Human Cells and Protoporphyrin IX Production as a Reporter System

Abstract

The determination of enzyme activity or inhibition in intact living cells is a problem in the development of inhibitors for intracellular proteases. The production of fluorescent protoporphyrin IX (PpIX) from the nonfluorescent (N)-Gly/Pro-5-aminolevulinic acid (ALA) substrates was used to evaluate the prolyl/glycyl-specific dipeptidylpeptidase IV (DPPIV)-like and prolyloligopeptidase (POP)-like activities of human cells. The results demonstrated that whereas POP-like activity could be attributed to the actual POP, the DPPIV-like activity could be related to actual DPPIV only in one colon cell line. In the other breast and colon cell lines, DPPIV-like activity was intracellular and displayed by other prolyl-specific aminopeptidases. Our experiments also demonstrated the involvement of glycyl-specific proteases in the processing of ALA precursors. These observations have important consequences for the development and evaluation of selective inhibitors for these enzymes.

Dipeptidyl Peptidase IV and Its Inhibitors: Therapeutics for Type 2 Diabetes and What Else?

“…Thus, DPP IV truncation has important effects on these peptide functions, involving for example (more detailed reviews in refs and ) the proinflammatory cytokines (IL-2, IL-6, IL-1β, IL-10, IL-12, IFN-γ, MIP1α, IL-3, TNF-α), the chemokines (SDF-1α, MIP-1β/CCL4, RANTES/CCL5), or other inflammation-modulating peptides (substance P, neurotensin, bradykinin, leptin). Consequently, DPP IV inhibitors have received some interest in this context as relevant therapeutics for the treatment of diseases of the colon, joints, and lung or for neurodegeneration. ,− However, it is not always clear which of these families of related prolyl-specific proteases are important in immune diseases. For example, DPP IV inhibitors were able to induce apoptosis even in DPP IV-negative lymphocytes, suggesting the inhibition of other enzymes with similar activity.…”

“…In support of this hypothesis, we have previously shown that detection of DPP IV mRNA does not always correlate with protein expression and enzymatic activity. 12 The 3D-structure of DPP IV (X-ray crystal structure) and the crystal structure of DPP IV bound to its inhibitors have been determined. 13−17 The enzyme is a homodimer (the crystallized porcine enzyme may be a symmetric tetramer, depending on the gylcosylation of the β-propeller 13 ).…”

Regulation of Fibroblast Activation Protein-α Expression: Focus on Intracellular Protein Interactions

“…Human tissue and cell expression of FAP-α mRNA and protein (adapted from refs and ). (A) Detection of FAP-α protein (brown precipitate) in human normal colon (left) and colon cancer (right) tissues.…”

“…For the RT-PCR experiments, loading was comparable for all samples as determined by GAPDH transcripts (not shown). Unless otherwise stated, the cells, cell culture conditions, methods, and reagents used were as described previously. − The anti-FAP-α ab53066 (Abcam) antibody was used for the dot-blots and anti-FAP-α MABS1001 (Vitatex) antibody for Western blotting.…”

Fibroblast activation protein-α in fibrogenic disorders and cancer: more than a prolyl-specific peptidase?

“…DPP IV, known as CD26, is a marker of T-cell activation in immune and inflammatory diseases [26]. DPP IV activity has been linked to prostate, colon and lung carcinoma or glioblastoma tumor cells [27][28][29][30]. DPP IV expression, as a receptor for tumor-associated fibronectin on endothelial cells favors tumor cell adhesion and metastasis, independently of its enzymatic activity [31].…”