title: Matrix metalloproteinase-1 induces cleavage of exogenous alphaB-crystallin transduced by a cell-penetrating peptide.
abstract: Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.
authors.affiliation: Department of Oral Histology and Developmental Biology & Program of Cell and Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, Korea.
authors.forename: Seung Won
authors.initials: SW
authors.lastname: Yang
authors.forename: Seung-Min
authors.initials: SM
authors.lastname: Lee
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Choi
authors.forename: Kyung Hye
authors.initials: KH
authors.lastname: Lee
authors.forename: Soo Hyuk
authors.initials: SH
authors.lastname: Kim
authors.forename: Min-Jeong
authors.initials: MJ
authors.lastname: Shin
authors.forename: Ye Sun
authors.initials: YS
authors.lastname: Han
authors.forename: Seok-Min
authors.initials: SM
authors.lastname: Kang
authors.forename: Ji Hyung
authors.initials: JH
authors.lastname: Chung
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology; D019610:Cytoprotection; D016503:Drug Delivery Systems; D057075:Enzyme Assays; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism*; D032446:Myoblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology; D038203:alpha-Crystallin B Chain / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology
chemical_list: D057846:Cell-Penetrating Peptides; D011993:Recombinant Fusion Proteins; D038203:alpha-Crystallin B Chain; D054322:tat Gene Products, Human Immunodeficiency Virus; D020781:Matrix Metalloproteinase 1