Calmodulin stimulates the degradation of brain spectrin by calpain

Brain spectrin has been shown to be a preferential substrate of calcium-dependent proteases (Baudry, Bundman, Smith, and Lynch: Science 212:937-938, 1981) and a major calmodulin-binding protein (Kakiuchi, Sobue, and Fujita: FEBS Lett. 132:144-148, 1981). Since calmodulin, spectrin, and a proteolytically derived spectrin fragment are all components of isolated postsynaptic density preparations (Grab, Berzins, Cohen, and Siekevitz: J. Biol. Chem. 254:8690-8696, 1979; Carlin, Bartelt, and Siekevitz: J. Cell Biol. 96:443-448, 1983), we investigated the functional role of calmodulin binding to brain spectrin with respect to its susceptibility to digestion by proteases. We report that calmodulin's interaction with brain spectrin results in a marked acceleration of the rate of spectrin degradation by calcium-dependent proteases (calpains I and II), but not by chymotrypsin. The cleavage of erythrocyte spectrin (which lacks a high-affinity calmodulin binding site) by calpain I is unaffected by the presence of calmodulin. The stimulatory effect of calmodulin is blocked by trifluoperazine, a calmodulin antagonist, which by itself does not modify brain spectrin proteolysis by calcium-dependent proteases. These results suggest a novel role for calmodulin in neuronal function--namely, a synergistic interaction with calcium-dependent proteases in the regulation of cytoskeletal integrity.     

慢性常压缺氧和缺氧伴CO_2潴留大鼠红细胞变形能力与膜收缩蛋白的研究

本文测定了慢性常压缺氧（Ⅱ组）、缺氧伴ＣＯ－２潴留（Ⅲ组）及对照组（Ⅰ组）大鼠红细胞变形能力与红细胞膜收缩蛋白二聚体（ＳＰ－Ｄ）和四聚体（ＳＰ－Ｔ）的相对含量。结果表明，Ⅱ、Ⅲ组红细胞变形指数（ＤＩ）和ＳＰ－Ｄ、ＳＰ－Ｔ相对含量与Ⅰ组均有显著差异，且Ⅱ、Ⅲ组的ＤＩ与ＳＰ－Ｄ／ＳＰ－Ｔ比值呈显著负相关。提示膜收缩蛋白的异常，可能是导致慢性缺氧和伴ＣＯ－２潴留大鼠红细胞变形能力降低的重要因素之一。     

冠心病病人红细胞变形能力与血浆、红细胞膜硒浓度的关系

目的：从硒与红细胞膜收缩蛋白的关系探讨冠心病病人红细胞变形能力变化的机制。方法：检测１３５例冠心病病人（不稳定性心绞痛４８例，急性心肌梗死５０例，陈旧性心肌梗死３７例）和６６例健康人血浆硒，红细胞膜硒和脂质过氧化物浓度、收缩蛋白二聚体（下简称二聚体）、收缩蛋白四聚体（下简称四聚体）及红细胞变形能力的变化。结果：冠心病病人红细胞滤过指数明显增高，血浆、红细胞膜硒浓度明显降低，与对照组比较有极显著性差异（Ｐ＜０．００１）；冠心病病人红细胞膜脂质过氧化物、二聚体、二聚体与四聚体比值增高，四聚体明显减少，与对照组比较有极显著性差异（Ｐ＜０．００１）。急性心肌梗死病人上述变化均较不稳定性心绞痛和陈旧性心肌梗死病人更明显。冠心病病人红细胞滤过指数与血浆、红细胞膜硒浓度呈负相关（ｒ＝－０．５２４和－０．６６１，Ｐ＜０．００１），红细胞膜硒浓度与脂质过氧化物、二聚体、二聚体与四聚体比值呈负相关（ｒ＝－０．５２１、－０．５７９和－０．５８６，Ｐ＜０．００１），与四聚体呈正相关（ｒ＝０．５５０，Ｐ＜０．００１）。结论：冠心病病人硒缺乏引起的膜骨架结构蛋白异常是红细胞变形能力降低的原因之一。     

Protofilament and Hexagon: A Three-Dimensional Mechanical Model for the Junctional Complex in the Erythrocyte Membrane Skeleton

It is a long-standing mystery why erythrocyte actin filaments in the junctional complex (JC) are uniformly 37 nm and the membrane skeleton consists of hexagons. We have previously proposed that a molecular ruler formed by E-tropomodulin and tropomyosin 5 or 5b functions to generate protofilaments of 12 G actin under mechanical stress. Here, we illustrate that intrinsic properties of actin filaments, e.g., turns, chemical bonds, and dimensions of the helix, also favor fragmentation into protofilaments under mechanical stress. We further construct a mechanical model in that a pair of G actin is wrapped around by a split and spectrin, which may spin to two potential positions, and stabilize to one when the tail end is restricted. A reinforced protofilament may function as a mechanical axis to anchor three (top, middle, and bottom) pairs of Sp. Each Sp pair may wrap around the protofilament with a wide dihedral angle (166.2°) and a minimal axial distance (2.75 nm). Such three Sp pairs may spiral down (right handed) the protofilament from the pointed end with a dihedral angle of 55.4° in between the Sp pairs. This first three-dimensional model of JC may explain the hexagonal geometry of the erythrocyte membrane skeleton. © 2003 Biomedical Engineering Society. PAC2003: 8716Ka, 8716Dg, 8714Ee, 8716Ac     

Evidence of calpain/cdk5 pathway inhibition by lithium in 3-nitropropionic acid toxicity in vivo and in vitro

Lithium reduced striatal neurodegeneration induced in rats by 3-nitropropionic acid inhibiting calpain activation. Lithium prevented an increase in cdk5 activity, as shown by the levels of the co-activator p35. Myocite enhancer factor 2 (MEF2), a downstream substrate for cdk5 with pro-survival activity, showed increased phosphorylation. In primary cultures of neurons treated with 3-NP, lithium also reduced protease activity mediated by calpain, cdk5 activation and cellular death. These observations indicate that lithium has a neuroprotective effect. Lithium treatment also reduced the intracellular increase in calcium induced by 3-NP. The finding that lithium mediates the modulation of the calpain/cdk5 pathway further supports its use in the treatment of neurodegenerative diseases.     

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase

Decreases in the expression of connexin 43 and the integrity of gap junctions in cardiac muscle, induced by the constitutive activation of the c-Jun N-terminal kinase (JNK) signaling pathway, have been linked to conduction defects and sudden cardiac failure in mice [Petrich BG, Gong X , Lerner DL , Wang X , Brown JH , Saffitz JE , Wang Y. c-Jun N-terminal kinase activation mediates downregulation of connexin 43 in cardiomyocytes. Circ Res. 91 (2002) 640-647; B.G. Petrich, B.C. Eloff, D.L. Lerner, A. Kovacs, J.E. Saffitz, D.S. Rosenbaum, Y. Wang, Targeted activation of c-Jun N-terminal kinase in vivo induces restrictive cardiomyopathy and conduction defects. J. Biol. Chem. 2004;279: 15330-15338]. We examined the membrane cytoskeletal protein, alphaII-spectrin, which associates with connexin 43, to learn if changes in its association with connexin 43 are linked to the instability of gap junctions. Several forms of alphaII-spectrin are expressed in the heart, including one, termed alphaII-SH3i, which contains a 20-amino-acid sequence next to the SH3 domain of repeat 10. In adult mouse heart, antibodies to all forms of alphaII-spectrin labeled the sarcolemma, transverse ("t-") tubules and intercalated disks of cardiomyocytes. In contrast, antibodies specific for alphaII-SH3i labeled only gap junctions and transverse tubules. In transgenic hearts, in which the JNK pathway was constitutively activated, alphaII-SH3i was lost specifically from gap junctions but not from t-tubules while other isoforms of alphaII-spectrin were retained at intercalated disks. Immunoprecipitations confirmed the decreased association of alphaII-SH3i with connexin 43 in transgenic hearts compared to controls. Furthermore, activation of JNK in neonatal myocytes blocked the formation of gap junctions by exogenously expressed Cx43-GFP fusion protein. Similarly, overexpression of the SH3i fragment in the context of repeats 9-11 of alphaII-spectrin specifically caused the accumulation of Cx43-GFP in the perinuclear region and inhibited its accumulation at gap junctions. These results support a critical role for the alphaII-SH3i isoform of spectrin in intracellular targeting of Cx43 to gap junctions and implicates alphaII-SH3i as a potential target for stress signaling pathways that modulate intercellular communication.     

Mechanism of hemolysis of G-6-PD deficient red cells: changes in membrane lipids and polypeptides

Summary A study of red cell membrane polypeptide and lipid profiles in G-6-PD deficient subjects has been made. High membrane spectrin and lipid content was demonstrated in the red cells of drug sensitive G-6-PD deficient individuals, while it was normal in non-sensitive G-6-PD deficient subjects. An inverse relationship was observed between GSH level and spectrin content in the former group. Possible mechanism of increased spectrin content in relation to hemolysis is discussed.     

Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis

Summary In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.This work was supported by an Australian Government Research Grant