Quantitative studies of caspase-3 catalyzed αII-spectrin breakdown

Abstract

Under various physiological and patho-physiological conditions, spectrin breakdown reactions generate several spectrin breakdown products (SBDPs) - in particular SBDP of 150 kDa (SBDP150) and of 120 kDa (SBDP120). Recently, numerous studies have shown that reactions leading to SBDPs are physiologically relevant, well regulated, and complex. Yet molecular studies on the mechanism of the SBDP formation are comparatively scarce. We have designed basic systems to allow us to follow the breakdown of αII-spectrin model proteins by caspase-3 in detail with gel electrophoresis, fluorescence and mass spectrometry methods. Amongst the predicted and reported sites, our results show that caspase-3 cleaves after residues D1185 and D1478, but not after residues D888, D1340 and D1475. We also found that the cleavage at these two sites are independent of each other. It may be possible to inhibit one site without affecting the other site. Cleavage after residue D1185 in intact αII-spectrin leads to SBDP150, and cleavage after D1478 site leads to SBDP120. Our results also show that the cleavage after the D1185 residue is unusually efficient, with a kcat/KM value of 40,000 M−1 sec−1, and the cleavage after the D1478 site is more similar to most of the other reported caspase-3 substrates, with a kcat/KM value of 3,000 M−1 sec−1. We believe that this study lays out a methodology and foundation to study caspase-3 catalyzed spectrin breakdown to provide quantitative information. Molecular understanding may lead to better understanding of brain injuries and more precise and specific biomarker development.

SBDPs and Tau proteins for diagnosis and hypothermia therapy in neonatal hypoxic ischemic encephalopathy

“…This process is irreversible. Thus, SBDPs do not only indicate brain injuries, but also contribute to the pathological mechanism of apoptosis and necrosis of brain cells (12,15). Under normal circumstances, Tau proteins have two forms, dephosphorylated form and phosphorylated, in a balanced state.…”

Mechanical behavior of actin and spectrin subjected to high strain rate: A molecular dynamics simulation study

“…Similar to that of actin, the mechanical behavior of spectrin can also be explained from the biochemical perspective [34] . Earlier atomic force microscopy (AFM) studies on spectrin tandem repeats and single spectrin have established that susceptibility to tensile loading leads to unfolding and stretching, which is impressively close to the observation in the current study [39] , [40] , [42] .…”

“…Studies on spectrin has been performed mostly from biological perspective [34] , and therefore, functionality has been the prime focus of such research. Furthermore, most of the studies have been limited to erythroid spectrins, leading to limited mechanical insight into axonal spectrin [35] .…”

Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration

“…[ 218 ] Moreover, αII-spectrin cleavage by caspase and caspase proteases produce signature cleavage products including SBDP120 and 145 kDa and 150 kDa αII-SBDP (SBDP145 and SBDP150) resulted primarily from caspase-3-mediated (apoptosis) and calpain-mediated (necrosis) proteolysis, respectively. [ 219 220 ] However, there is evidence that SBDP150 might be associated with activities of both calpain and caspase-3 proteases. [ 221 ] Moreover, a recent experimental study using in vitro primary rat cerebrocortical cell cultures under apoptotic, necrotic, and excitotoxic conditions together with an in vivo rat TBI model suggests that breakdown of βII-spectrin, another important neuronal cytoskeletal protein, by caspase-3 and calpain-mediated proteolysis, contributes to cell death following brain injuries and that protease-specific signature βII-SBDPs may serve as biomarkers indicative of neuronal cell death mechanism.…”