title: Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells.
abstract: Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Brisbane, Queensland 4059, Australia.
authors.forename: Washington Y
authors.initials: WY
authors.lastname: Sanchez
authors.forename: Simon J
authors.initials: SJ
authors.lastname: de Veer
authors.forename: Joakim E
authors.initials: JE
authors.lastname: Swedberg
authors.forename: Eui-Ju
authors.initials: EJ
authors.lastname: Hong
authors.forename: Janet C
authors.initials: JC
authors.lastname: Reid
authors.forename: Terry P
authors.initials: TP
authors.lastname: Walsh
authors.forename: John D
authors.initials: JD
authors.lastname: Hooper
authors.forename: Geoffrey L
authors.initials: GL
authors.lastname: Hammond
authors.forename: Judith A
authors.initials: JA
authors.lastname: Clements
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Harris
mesh_terms: D000728:Androgens / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D013196:Dihydrotestosterone / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007610:Kallikreins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009369:Neoplasms / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012738:Sex Hormone-Binding Globulin / Q000378:metabolism*; D013256:Steroids / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D000728:Androgens; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin; D013256:Steroids; D013196:Dihydrotestosterone; D005982:Glutathione Transferase; D010447:Peptide Hydrolases; C425336:KLK14 protein, human; D007610:Kallikreins; C120801:kallikrein 4