Elsevier

Experimental Neurology

Volume 202, Issue 2, December 2006, Pages 506-513
Experimental Neurology

Inhibition of calpain-mediated cell death by a novel peptide inhibitor

https://doi.org/10.1016/j.expneurol.2006.07.016Get rights and content

Abstract

Calpains are calcium- and thiol-dependent proteases whose overactivation and degradation of various substrates have been implicated in a number of diseases and conditions such as cardiovascular dysfunction and ischemic stroke. With increasing evidence for calpain's role in cellular damage, the development of calpain inhibitors continues to be an important objective. Previously, we identified a highly specific calcium-dependent, calpain interacting peptide L-S-E-A-L, that showed homology to domains A and C of the only known endogenous inhibitor of calpains, calpastatin. This suggested that LSEAL had a calpain inhibitory function and synthetic LSEAL inhibited calpain I and II proteolysis of two calpain substrates, tau and alpha-synuclein. In the present study, we demonstrate that synthetic LSEAL is membrane permeable and is a potent inhibitor in two established models of calpain-mediated cell death using primary rat cortical neurons and SH-SY5Y neuroblastoma cells. In addition, we show that LSEAL inhibits calpain activity towards protein substrates as detected by an antibody to a calpain-specific breakdown product of spectrin. Taken together, these results suggest that LSEAL may represent a novel calpastatin mimetic with the potential for benefit in conditions of increased calpain activity such as stroke, traumatic brain injury or heart attack.

Introduction

Calpains are a family of thiol proteases that are widely expressed with both ubiquitous (calpain I and II) and tissue-specific isoforms. Calpains are typically expressed as a heterodimer and consist of an 80-kDa catalytic subunit and a 30-kDa regulatory subunit with a few exceptions observed in tissue-specific calpain isoforms. Moreover, calpain's proteolytic activity is both calcium- and redox-dependent requiring both increased calcium concentration and a reducing environment in order for proteolytic processing of its substrates (Goll et al., 2003, Guttmann et al., 1997, Guttmann and Johnson, 1998, Marcum et al., 2005, McCollum et al., 2004a, McCollum et al., 2004b, Melloni and Pontremoli, 1989, Murachi et al., 1980). Calpain activity is generally limited and typically results in the processing of a protein whereby large enzymatically active domains of a substrate or substrates remain intact. This type of action suggests that calpain cleavage events may play a more modulatory role in biological processes. Previous studies have shown that increased calpain proteolytic activity likely contributes to the cytoskeletal disruption and cell death that occurs in models of both focal and global ischemia (Bartus et al., 1994, Bartus et al., 1995, Blomgren et al., 1995, Hong et al., 1994, Markgraf et al., 1998, Minger et al., 1998, Neumar et al., 2001, Rami and Krieglstein, 1993, Robinson, 1996, Sun and Cheng, 1999, Zalewska, 1996). In fact, in a temporary focal cerebral ischemia/reperfusion rat model of stroke, the addition of the calpain-selective inhibitor, benzyloxycarbonyl-Val-Phe-CHO resulted in reduced infarct volume 24 h after ischemia (Hong et al., 1994). However, these inhibitors lack specificity for the calpains and also target other thiol-proteases such as the caspases and cathepsins (Wang, 1990, Wang, 2000, Wang and Yuen, 1997). In addition, as the active site cysteine is easily targeted by oxidants such as hydrogen peroxide or nitric oxide (Guttmann and Johnson, 1998, Michetti et al., 1995, Rackoff et al., 2001) current compounds that react to the thiol moiety may not react to calpains under conditions of oxidative stress. Therefore, the development of potent, specific and cell permeable calpain inhibitors is paramount in order to translate the tremendous efforts of defining calpain's role in disease into a useful therapeutic tool.

It is known that calpains become active in the presence of calcium due to conformational changes induced by calcium binding to EF hand motifs in the C-terminus of both the large and small subunits (Moldoveanu et al., 2002). The binding of calcium results in a change of configuration that exposes a cysteine in calpain's active-site to substrate. This allows for the catalytic triad (Cys-His-Asn) to be properly aligned and for peptide bond cleavage to occur. However, it has also been shown that bovine calpain irreversibly inactivated with Z-LLY-FMK can still bind the substrate casein, suggesting that a site of substrate interaction different from the active-site is present within the calpain protein that is calcium-dependent (Dutt et al., 1998). This suggested that other domains of the calpain protein could be targeted for potential modulatory action by pharmacological agents. Using this knowledge, we previously identified peptides using a phage-display based methodological approach which interacted with calpain in a calcium-dependent manner at sites other than the active site (Guttmann et al., 2005). Analysis of the identified peptides indicated a strong homology to calpastatin domains A and C.

Calpastatin is the natural endogenous inhibitor of calpain activity. The amino acid sequence of calpastatin contains repetitive peptide domains each of which is capable of binding and inactivating one molecule of calpain (Maki et al., 1987). Because calpastatin is the most specific calpain inhibitor known, a goal of synthetic calpain inhibitor development would be to mimic calpastatin. We previously identified a lead peptide, LSEAL, that appears to represent a new class of calpastatin mimetics which may be useful to future research as a potential therapeutic agent by specifically targeting the calpains (Guttmann et al., 2005). The next stage in the development of these compounds is to test them for effectiveness in a cellular context in model systems of selective calpain activation. To explore this possibility, we tested LSEAL for membrane permeability as well as its ability to inhibit calpain-mediated cell death in both rat cortical neurons and the human neuroblastoma cell line SH-SY5Y. We also determined the extent to which LSEAL directly inhibited calpain activity towards a known substrate, spectrin. The results presented here demonstrate that LSEAL is membrane permeable and a potent inhibitor of calpain activity and calpain-mediated cell death. These results suggest that this family of peptide compounds warrant continued testing for the potential treatment of conditions such as ischemic stroke.

Section snippets

Peptide synthesis

Peptides were synthesized using standard Fmoc chemistry and purified by reverse-phase HPLC by Cell Essentials Inc., Boston MA. The N-terminus was acetylated and the C-terminus amidated. In all experiments shown, LSEAL was solubilized to 100 mM in DMSO and further dilutions made in water. LSEAL was also tested for solubility and was found to be soluble to 100 mM with water pH 10.0 with no adverse effects on its ability to inhibit calpain activity (data not shown).

Permeation studies

The EpiAirway™ tissues used for

LSEAL is membrane permeable

To determine whether LSEAL is membrane permeable, we utilized the EpiAirway system which consists of normal, human-derived tracheal/bronchial epithelial cells that are cultured to form a pseudo-stratified, ciliated, columnar epithelium. This system is an established model for the assessment of nasal drug absorption and represents a reasonable approach to determine drug permeation across human epithelial membranes (Chemuturi et al., 2005). Using this method, we show that LSEAL penetrates the

Discussion

Inhibition of calpain is a reasonable target for the treatment of multiple disorders including stroke and heart attack (Huang and Wang, 2001, Robinson, 1996, Sun and Cheng, 1999, Vanderklish and Bahr, 2000). In a previous study, we demonstrated that small peptide molecules that appear to represent critical binding domains of calpastatin (domains A and C), inhibited calpain activity in a calcium-dependent manner, in vitro. The lead compound tested was the amino acid sequence LSEAL. This prompted

Acknowledgments

The authors would like to thank Drs. Jeff Keller and Harry Levine for materials and technical assistance and MatTek Corporation for kindly providing the EpiAirway™ tissue samples. Supported by grants from the NIH (NS047635–RPG) and the AHA (0335233N–RPG).

References (41)

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