Investigation into the lipid activation of calpain and its role in cataract formation
Age-related cataract is the commonest cause of treatable blindness in the world today. It is an ever-increasing problem with prolonged life-expectancy and a burgeoning elderly population. Although successfully treated by modem, sophisticated cataract surgery, the resources needed to provide a surgical set-up and the prolonged training time required to produce a competent surgeon can challenge the available finances of developed economies and result in unacceptable waiting times while they may simply be unaffordable in developing economies and result in high prevalence of blindness. Surgical treatment, although effective in dramatically reversing blindness, is associated with a small risk of sight-threatening complications. Against this background, it is worth considering an alternative, simpler, medical means of treating cataracts that is easily administered and equally effective. Calpains are Ca2+dependent intracellular proteases and several of these enzymes are believed to participate in cataractogenesis. Calpain 2 is the major isoform of calpain involved in human cataractogenesis and its activation appears to be investigate the mechanism of lipid activation of calpains as an essential link towards understanding the molecular and cellular dynamics of cataractogenesis in vivo. A clear picture of the enzymatic events in cataractogenesis will form the basis of drug development to cause selective enzyme inhibition. In chapter 2, atomic absorption spectroscopy shows that the progressive uptake of extra-lenticular Ca2+ by porcine lenses correlates with increases in levels of lens opacification with 8.0 umoles Ca2+ (gm wet lens weight)-1 corresponding to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when a calpain inhibitor, SJA6017 (final concentration 0.8 uM), was included in the extra-lenticular medium, therefore suggesting that the observed porcine opacifications involve the Ca2+ mediated activation of calpains and that cataract could be retarded by the topical administration of calpain inhibitors. In chapter 3, DWIH (Depth weighted insertion hydrophobicity) analysis shows the small subunit of several calpain 2 isozymes to each posess a segment with the potential to form a lipid / membrane interactive a-helix (DWIH values circa 7.0). Extended hydrophobic moment analysis shows these segments to be potential oblique orientated a-helix formers (< uH > circa 0.5). This potential is confirmed by hydropathy plot and graphical analyses, which show each a-helix to possess a significant N -> C hydrophobicity gradient. It is suggested that the lipid / membrane activation of calpain 2 may involve oblique angled membrane penetration by an ahelical segment in domain V of the enzyme. In chapter 4, VP1, a peptide homologue of this domain V segment, is shown to be strongly haemolytic (half-maximal lyric dose = 1.45 mM). FTIR conformational analysis shows VP1 to adopt a-helical structure (20% - 65% of primary structure) in the presence of lipids. These levels are maximal in the presence of anionic lipid (65% of primary structure) and monolayer studies show the peptide to exhibit high levels of anionic lipid monolayer penetration with surface pressure changes (A SP) of 5-6 mN m-1 at 30 mN m-1, which are reduced by approximately 40% ± 15% in the presence of 100 mM NaCl. It is suggested that membrane penetration by the domain V a-helix of calpain 2 may proceed via electrostatic interactions and snorkelling, involving associations between an arginine residue located in the polar face of this a-helix and anionic membrane lipid. It has been suggested that lipid / membranes may modulate calpain 2 activity by lowering the enzyme's in vivo Ca2 requirements. In chapter 5, colorimetric assay of calpain 2 shows that the enzyme requires 4 mM Ca2+ for 100% proteolytic activity, as defined by this assay. In the presence of 1 mM Ca2+, negligible calpain 2 proteolysis is detected but at this level of Ca2+, in the presence of either Dimyristoyl phosphatidylinositol(DMPI), Dimyristoyl phosphatidylserine(DMPS), Dimyristoyl phosphatidylcholine(DMlPC) or Dimyristoyl phosphätidylethanolamine(DMPE), calpain 2 shows proteolytic activity which ranges between 37% and 77% of the protein's full enzymatic activity. The large subunit of calpain 2 (LS-calpain 2) is proteolytical]y active in the absence of the calpain 2 small subunit and it has been suggested that this latter subunit is dispensable for the lipid activation of calpain 2. LS-calpain 2 is assayed under conditions corresponding to those used here for calpain 2 assay and is shown to require the presence of 6 mM Ca 2 for 100% enzymatic activity. These Ca2+ levels are unaffected by the presence of either: DMPI, DMPS, DMPC or DMPE, and based on these combined results, it is suggested that the lipid activation of calpain 2 requires the presence of the small subunit. In addition, it is shown that when compared to zwitterionic lipid under corresponding conditions anionic lipid induces an approximate twofold enhancement of calpain 2 proteolytic activity (70% - 77% as compared to 37% - 49%) and a similar enhancement in its average rates of lipid monolayer interaction (ASP = 1.5 x 10 mN M-1 sec-1 at 10 mN M-1 as compared to ASP = 5.0 x 10-4 mN M-1 sec-1 at 10 mN M-1). It is suggested that calpain 2 may posess an electrostatically driven preference for anionic lipid, which contributes to lowering the enzyme's Ca2+ requirement for activation. In chapter 6, these combined data are discussed in relation to a model for the lipid activation of calpain 2 and proposals for future work are presented.