Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis.
Hurpin (headpin/PI13/serpinB13) is an intracellular, differentially spliced member of the serpin superfamily that has been linked to differentiation and apoptosis of human keratinocytes. It is transiently downregulated by UV light and overexpressed in psoriatic skin lesions. Although it has all of the features of an inhibitory serpin, a productive interaction between hurpin and a proteinase has not yet been reported. Here we demonstrate that hurpin is a potent and selective inhibitor of the archetypal lysosomal cysteine proteinase cathepsin L (catL). Recombinant hurpin inhibits human catL with a stoichiometry of inhibition (SI) of 1.7 and a rate constant k(assoc) of (4.6 +/- 0.14) x 10(5) M(-1) s(-1). It inefficiently inhibits catV and does not inhibit papain, catB, or catK. To investigate the inhibitory mechanism, we determined the P1-P1' bond in the reactive center loop cleaved by catL ((356)Thr-(357)Ser) and expressed variants in which the proximal hinge, P1 residue, or differentially spliced CD loop was mutated. The results of assays using these proteins suggest that inhibition of catL by hurpin occurs via the conventional serpin inhibitory mechanism and that the CD loop plays no role in the process. Finally, it was found that the majority of hurpin is cytosolic and that its overexpression in human keratinocytes confers resistance to UV-induced apoptosis. Given that lysosomal disruption, release of catL, and catL-mediated caspase activation are known to occur in response to cellular stress, we propose that a physiological role of hurpin is to protect epithelial cells from ectopic catL.
Welss
Thomas
T
Department of Dermatology, Heinrich Heine University, D-40225 Düsseldorf, Germany.
Sun
Jiuru
J
Irving
James A
JA
Blum
Rainer
R
Smith
A Ian
AI
Whisstock
James C
JC
Pike
Robert N
RN
von Mikecz
Anna
A
Ruzicka
Thomas
T
Bird
Phillip I
PI
Abts
Harry F
HF
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014466:Ultraviolet Rays; D015003:Yeasts / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; C530652:SERPINB13 protein, human; D015843:Serpins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L