Cysteine protease activated by expression of HIV-1 protease in transgenic mice. MIP26 (aquaporin-0) cleavage and cataract formation in vivo and ex vivo.
Transgenic mice, homozygous for HIV-1 protease expression in the eye lens, display degradation of some lens crystallins and cytoskeletal proteins prior to cataract formation on postnatal days 23-25. Alterations to the internal lens hydration state also occur; therefore, the status of the aquaporin protein MIP26 was examined over postnatal days 16-25 to determine if it was altered during cataractogenesis. The MIP was identical in transgenic and control lenses until day 21. By postnatal day 25 (frank cataract), in the lenses obtained from transgenic animals, the 26-kDa band was absent and there was a concurrent increase in the proportion of MIP23. Immunoblotting demonstrated cleavage at the C terminus. Lenses were also maintained in an organ culture system to demonstrate that the cataractogenic process is inherent to the isolated lens and to determine the contribution of cysteine protease action. Organ culture experiments revealed a similar progression to nuclear cataract formation as seen in vivo. Two-dimensional gel analysis of the soluble lens crystallin fraction of organ cultured lenses revealed the same cleavage pattern as occurs in vivo. Organ culture of transgenic lenses with E64, a cysteine protease inhibitor, dramatically delayed cataractogenesis and prevented proteolytic cleavage of both MIP26 and crystallins. HIV-1 protease, while the trigger of cataract formation, does not appear to be the protease responsible for cleavage of MIP or lens crystallins. These results suggest that activation of endogenous cysteine protease activity is involved in the cleavage of these proteins and occurs downstream of HIV-1 protease action.
Mitton
K P
KP
Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. kpmitton@helix.nih.gov
Kamiya
T
T
Tumminia
S J
SJ
Russell
P
P
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D020346:Aquaporins; D002386:Cataract / Q000209:etiology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008970:Molecular Weight; D009924:Organ Culture Techniques; D010750:Phosphoproteins / Q000378:metabolism*
D000954:Antigens, Surface; D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; C030328:aquaporin 0; D003546:Cysteine Endopeptidases; D016333:HIV Protease