Premium
Matrix metalloproteinase‐1 induces cleavage of exogenous alphab‐crystallin transduced by a cell‐penetrating peptide
Author(s) -
Yang Seung Won,
Lee SeungMin,
Choi Eun Young,
Lee Kyung Hye,
Kim Soo Hyuk,
Shin MinJeong,
Han Ye Sun,
Kang SeokMin,
Chung Ji Hyung
Publication year - 2011
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.23167
Subject(s) - cleavage (geology) , transfection , crystallin , microbiology and biotechnology , chemistry , recombinant dna , peptide , cleavage stimulation factor , matrix metalloproteinase , fusion protein , cell culture , heat shock protein , biochemistry , biology , cleavage factor , gene , messenger rna , paleontology , genetics , fracture (geology)
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. J. Cell. Biochem. 112: 2454–2462, 2011. © 2011 Wiley‐Liss, Inc.