Proteolysis of poly(ADPribose) polymerase by a pyrophosphate- and nucleotide-stimulated system dependent on two different classes of proteinase.
We have identified a system in human lymphocytes which proteolytically cleaves poly(ADPribose) polymerase to specific fragments of molecular weight 96 000, 79 000 and 62 000-60 000. This proteolytic processing is dependent on two different classes of proteinase. One of these proteinases is a serine proteinase, since the processing is inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor and diisopropylfluorophosphate, the other is a cathepsin D-like proteinase, since processing is also inhibited by pepstatin A. The processing that occurs in permeabilized cells can be simulated in vitro by treating purified poly(ADPribose) polymerase with trypsin, but not by treating the polymerase with cathepsin D. Since processing at the cellular level is blocked by inhibitors of either of the two proteinases, but only trypsin could cleave the purified polymerase, this suggests that in the cell the action of the cathepsin D-like proteinase is a prerequisite for cleavage of poly(ADPribose) polymerase by the serine proteinase. Thus, a pathway involving sequential action of these proteinases may exist. Proteolysis in permeabilized human lymphocytes is stimulated by nucleotides containing a pyrophosphate group, such as 5',5'''-P1,P4-tetraphosphate and ATP, or by pyrophosphate itself. In contrast, nucleotides containing only a single phosphate, such as AMP and cyclic AMP, or inorganic sodium phosphate, do not show this stimulation of proteolysis. These results suggest that a pyrophosphate linkage is the minimum molecular requirement for stimulation of proteolytic processing of poly(ADPribose) polymerase. Proteolytic processing of poly(ADPribose) polymerase is independent of ADPribosylation. Following proteolysis, specific fragments of the polymerase, particularly the 62 000-60 000 molecular weight fragment(s), are still capable of being ADPribosylated.
D000227:Adenine Nucleotides / Q000494:pharmacology; D002402:Cathepsin D / Q000378:metabolism; D015226:Dinucleoside Phosphates; D011756:Diphosphates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D014357:Trypsin / Q000378:metabolism
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; D011756:Diphosphates; C020733:diadenosine tetraphosphate; D011065:Poly(ADP-ribose) Polymerases; D010447:Peptide Hydrolases; D014357:Trypsin; D002402:Cathepsin D