Calcium and Proteases

2.30 Calcium and Proteases R G Schnellmann and M D Covington, Medical University of South Carolina, Charleston, SC, USA ª 2010 Elsevier Ltd. All rights reserved.

2.30.1 2.30.2 2.30.2.1 2.30.2.2 2.30.3 2.30.3.1 2.30.3.2 2.30.3.3 2.30.3.4 2.30.3.5 2.30.3.6 2.30.3.6.1 2.30.4 2.30.4.1 2.30.4.2 2.30.4.3 2.30.4.4 2.30.4.5 2.30.4.6 2.30.4.7 2.30.4.8 2.30.4.9 2.30.4.10 2.30.4.11 2.30.4.12 2.30.4.13 2.30.4.14 2.30.4.15 2.30.5 References

Introduction Components of Intracellular Ca2þ Homeostasis Ca2þ-Binding Proteins Ca2þ Channels Components of Intracellular Ca2þ Homeostasis – Ca2þ Channels Voltage-Operated Ca2þ Channels Voltage-Independent Ca2þ Channels Ca2þ Pumps The Plasma Membrane Ca2þ-ATPase The Sarcoplasmic/Endoplasmic Reticulum Ca2þ-ATPase Disruption of Ca2þ Homeostasis Renal cell Ca2þ regulation Calpains The Calpain Family of Proteases Calpain Structure Calpain Activation Physiological and Pathological Roles of Calpain Physiological Roles of Typical Calpains Calpain in Cell Cycle Calpain in Regulation of Gene Expression Calpain in Apoptosis Calpain in Signal Transduction Calpain in Cell Motility Pathological Roles of Typical Calpains Calpains in Ischemia Calpains in Cancer Mitochondrial Calpains Phospholipase A2 Conclusion

Abbreviations AIF BKA cAMP cPLA2 CsA ER FFA GFP GTP IP3 IP4 LPLA2

apoptosis-inducing factor bongkrekic acid cyclic adenosine monophosphate cytosolic/Ca2þ-dependent PLA2 cyclosporin A endoplasmic reticulum free fatty acid green fluorescent protein guanosine triphosphate inositol 1,4,5 trisphosphate inositol 1,3,4,5 trisphosphate lysosomal PLA2

MALDI-TOF MPT NMDA PAF-AH PKA PKC PL PLA2 ROC ROS RR

588 589 589 592 592 592 593 594 594 594 596 597 598 598 598 601 602 603 603 603 603 604 604 604 605 605 606 606 607 607

matrix-assisted laser desorption/ ionization time-of-flight mitochondrial permeability transition N-methyl-D-aspartate platelet-activating factor acetylhydrolase protein kinase A protein kinase C phospholipid phospholipase A2 receptor-operated calcium channel reactive oxygen species ruthenium red

587

588 Alterations in Cell Signaling

SERCA siRNA SMOC

sarco(endo)plasmic reticulum Ca2þATPase small interfering RNA second messenger-operated Ca2þ channel

2.30.1 Introduction Calcium ions (Ca2þ) are key second messengers in a variety of eukaryotic cell signaling pathways, and they function in the regulation of diverse cellular processes. The importance of Ca2þ in muscle contraction was first observed at the end of the nineteenth century, and since the mid-1970s, there has been an explosion in our understanding of the conceptual complexity and diversity of Ca2þ functions, as well as in the development of experimental tools to investigate the biomedical functions of Ca2þ. While it has become clear that Ca2þ stimulates many cellular processes, such as muscle contraction, cellular proliferation, gene expression, secretion of hormones and neurotransmitters, exocytosis, and chemotaxis, it has also been realized that Ca2þ is very toxic. Thus, the free intracellular Ca2þ concentration ([Ca2þ]i) must be highly regulated to achieve a proper balance between Ca2þ-mediated cell function and Ca2þ-mediated cell death. The intracellular cytoplasmic concentration of Ca2þ is low in quiescent cells at approximately 108–107 mol l1, whereas the extracellular Ca2þ concentration is 103 mol l1. In addition to the Ca2þ concentration gradient across the plasma membrane, Ca2þ is also sequestered within intracellular organelles, such as the endoplasmic reticulum (ER) in nonexcitable cells and the sarcoplasmic reticulum (SR) in excitable cells. Consequently, a steep electrochemical gradient on the order of 104 exists for Ca2þ between the cytoplasm and the exterior of the cell as well as the interior of certain intracellular organelles. It is the maintenance of this electrochemical gradient that both facilitates the second messenger role of Ca2þ and prevents Ca2þ-mediated cell injury. Upon stimulation by Ca2þ-elevating agonists, the average [Ca2þ]i can rise to the low micromolar range. Such Ca2þ signals have a complex temporal and spatial organization, and elevations in Ca2þ are frequently presented to the cytoplasm in a pulsatile manner in the form of baseline Ca2þ spikes or oscillations. The generation, propagation, and termination of the Ca2þ signal involve an elaborate system of channels, pumps, and exchangers that are localized within the plasma

SOCC sPLA2 SR VOCC

store-operated Ca2þ channel secretory PLA2 sarcoplasmic reticulum voltage-operated Ca2þ channel

membrane and the ER or SR (Figure 1) (Schafer and Heizmann 1996). Specific Ca2þ channels localized in the plasma membrane or in the membranes of internal organelles open in response to electrical or hormonal stimuli allowing Ca2þ to enter the cytoplasm by flowing down its concentration gradient. The Ca2þ-ATPase and Naþ/Ca2þ exchanger in the plasma membrane or the Ca2þ-ATPase in the ER terminates the Ca2þ signal by pumping Ca2þ into the extracellular fluid or by sequestering Ca2þ in intracellular stores, respectively. The continuous inflow of Ca2þ through the plasma membrane or leakage of Ca2þ from internal organelles into the cytoplasm is balanced by the highaffinity (but relatively low-capacity) Ca2þ-ATPase located in the plasma membrane, the high-affinity (high-capacity) Ca2þ-ATPase located in the SR/ER, and the low-affinity Naþ/Ca2þ exchanger located in the plasma membrane (Table 1) (Khanna et al. 1988). Additionally, other intracellular organelles and Ca2þ-binding proteins serve to buffer the cytoplasmic Ca2þ concentration, albeit with lower affinity. For example, the inner mitochondrial membrane contains a low-affinity uniport carrier that allows the electrogenic entry of Ca2þ as a result of the negative transmembrane potential. In addition, the nucleus reportedly sequesters Ca2þ through a mechanism that is coupled to ATP hydrolysis, suggesting that the nuclear membrane contains an additional Ca2þATPase (Nicotera et al. 1989). The importance of intracellular calcium homeostasis is appreciated when one considers the number of subcellular compartments that function in regulating the [Ca2þ]i as well as the diversity of cellular processes that are controlled by Ca2þ signaling pathways. Each of these subcellular compartments can be targeted by chemicals or drugs, which can elicit an imbalance in intracellular Ca2þ homeostasis resulting in toxicity manifested as impaired cellular function or cell death. This chapter provides a basic understanding of the Ca2þ messenger system, Ca2þ-activated proteases, such as calpains and phospholipase A2 (PLA2), and the importance of Ca2þactivated proteases in cell injury.

Calcium and Proteases

CaBPs:

Ca2+ Agonist

Ca2+ channels

589

Neurite extension Chemotactic activity Thymic hormone activity Extracellular martrix component

Receptor Secretion?

Extracellular PLC

G

PtdIns(4,5)P2

Ca2+– ATPase

Na+–Ca2+ channels

Cell membrane Ca2+

Ins(1,4,5)P3

CaBPs:

Intracellular

Ins(1,4,5)P3 receptor

Ca2+ buffering and transport Activation of enzymes Polymerization of cytoskeleton Cell cycle progression

Ca2+ Ca2+

Ca2+ 2+ Endoplasmic Ca -ATPase

Mitochondria CaBPs:

reticulum

Ca2+

Transcription apoptosis nucleus

Figure 1 Signal transduction by Ca2þ-binding proteins (CaBPs). Influx of Ca2þ upon stimulation either from intracellular stores or through different types of Ca2þ channels leads to an increase in intracellular Ca2þ concentration. This allows CaBPs to bind Ca2þ, undergo a conformational change and associate with different target proteins, thereby shaping the biological effects of the Ca2þ signal. Ca2þ-binding proteins are mainly found in the cytoplasm, but were discovered recently in other compartments of the cell, such as the nucleus or the mitochondria. Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate.

Table 1 Membranous calcium-binding proteins

Organelle

Tissue

Km (Ca2þ) (mol l1)

Heart Heart Heart

0.5 2–20 0.1–0.5

0.5 15–30 20–30

Naþ/Ca2þ exchanger Ca2þ uniporter Cadherin-E Cadherin-N

Plasma membrane Plasma membrane Sarcoplasmic reticulum Mitochondria Mitochondria Plasma membrane Plasma membrane

13 15–30

0.2–0.3 <0.5

Thrombospondin

Plasma membrane

Heart Heart Epithelium Neuronal tissue and muscle Platelets, and so on

100

12

Protein 2þ

Ca -ATPase Naþ/Ca2þ exchanger Ca2þ-ATPase

2.30.2 Components of Intracellular Ca2þ Homeostasis 2.30.2.1

Ca2þ-Binding Proteins

The elevation of [Ca2þ]i itself does not usually trigger a cellular response, but rather it mediates a response through Ca2þ receptors or Ca2þ-binding

Vmax of transport (nmol Ca2þ per mg protein)

proteins. Many Ca2þ-binding proteins exist in cells and they can be classified into two general categories – Ca2þ sensors and Ca2þ buffers (Table 2) (Ikura 1996). Calmodulin is a ubiquitous, evolutionarily conserved, and well-characterized Ca2þ-binding sensor protein, to which is ascribed a role in enabling cells to

590 Alterations in Cell Signaling Table 2 Some members of the EF-hand Ca2þ-binding protein family and their functions Protein

Function

Ca2þ sensors Calmodulin Troponin C -Actinin Myosin light chains Calpain (large subunit) Calcyclin Calcineurin

Activates several enzymes and proteins; mediates many Ca2þ-dependent processes (see Table 1) Modulates muscle contraction Actin-bundling protein Regulates muscle contraction Ca2þ-activated protease Exocytosis Protein dephosphorylation, protein phosphatase 2B regulatory subunit

Ca2þ buffers Calbindin-D28K, calbindin-D9K Calretinin Parvalbumin

Ca2þ buffering and transport Ca2þ buffering and transport Ca2þ buffering and transport

detect elevated [Ca2þ]i and thereby transducing this signal into a variety of cellular processes. The molecular mechanism attributed to the transduction of this Ca2þ-mediated activation of calmodulin lies in the conformational change induced by Ca2þ binding. Calmodulin, as well as several other Ca2þ sensor and Ca2þ buffer proteins, belongs to the EF-hand protein family, characterized by a helix–loop–helix structural motif first described for the crystal structure of parvalbumin by Kretsinger and Nockolds (1973). At quiescent cell Ca2þ concentrations (107– 108 mol l1), Ca2þ-binding EF-hand proteins are inactive, but undergo rapid and activating conformational changes as [Ca2þ]i rises in response to Ca2þelevating stimuli. Calmodulin is an acidic, heat-stable, 16.7-kDa protein that binds four calcium ions, cooperatively, with an association constant of about 106 mol l1, which allows calmodulin to serve as a molecular switch linked to agonist-stimulated Ca2þ response coupling (James et al. 1995; Klee et al. 1980; Lu and means 1993; Wang et al. 1985). Upon binding Ca2þ, calmodulin rapidly undergoes a conformational

change, which exposes hydrophobic regions and thereby enables it to interact and stimulate the activity of multiple enzymes including the Ca2þ/ calmodulin-dependent protein kinases of which there are now believed to be at least six members of the Ca2þ/CAM (calmodium) kinase family, PLA2, phosphodiesterase, and guanylate cyclase to name a few (Table 3). Through its actions on these target enzymes, Ca2þ-activated calmodulin is involved in the regulation of many cellular processes including cell cycle progression, exocytosis, and ion transport. Several pharmacological inhibitors of calmodulin exist including phenothiazines, for example, trifluoperazine and chlorpromazine, and napthalenesulfonamides (Hidaka and Ishikawa 1992). Many of these classes of inhibitory drugs exhibit functional selectivity with respect to impairing calmodulindependent processes, and thus may serve as powerful tools as molecular probes of calmodulin-dependent pathways in various cell functions. Calmodulin may be a target for some of the toxic effects of lead, since Pb2þ has been shown to be capable of occupying the

Table 3 Calmodulin-dependent enzymes and cellular processes Cyclic nucleotide metabolism Adenylate cyclase Cyclic nucleotide phosphodiesterase Guanylate cyclase Cellular Ca2þ homeostasis Plasma membrane Ca2þ-ATPase Phospholamban kinase Metabolic pathways Phosphorylase kinase NAD kinase

Contraction, motility, cytoskeleton Myosin light chain kinase Tubulin Caldesmon Fodrin, spectrin Signal transduction Phospholipase A2 Ca2þ/CAM-dependent protein kinases Calpain Nitric oxide synthetase

Calcium and Proteases

Ca2þ-binding sites of calmodulin as well as an allosteric-potentiating binding site (Chao et al. 1984; Goldstein and Ar 1983; Mills and Johnson 1985). Calmodulin is the primary intracellular receptor for Ca2þ and is involved in regulating numerous enzymes. Many of these enzymes are Ca2þ-binding proteins themselves, which are regulated, in some way, upon interaction with calmodulin. An example of such a Ca2þ/calmodulin-regulated enzyme is the cysteine protease calpain. Calpain is an important Ca2þ-binding protein in that it functions in regulating apoptosis in some cell types (Squier and Miller 1984). Additionally, calcineurin, a Ca2þ/calmodulindependent protein phosphatase, is an EF-hand Ca2þ sensor protein that also contains a calmodulin-binding site (Clipstone and Crabtree 1992; Klee et al. 1988). Thus, calmodulin is often the primary decoder of elevated intracellular Ca2þ concentration and it acts as a molecular switch for activation of many Ca2þ-dependent signaling enzymes. The analysis of

591

Ca2þ/calmodulin, and Ca2þ-binding proteins has aided our understanding of cellular processes associated with disease. For example, cyclosporine A (CsA) and FK506, structurally distinct inhibitors of T lymphocyte activation, are drugs that are used in organ transplantation. Both drugs bind to and inhibit the immunophilins, enzymes that catalyze cis/transpeptidylprolyl isomerase reactions. The ability of immunosuppressants to inhibit isomerase activity lies in the fact that the immunosuppressive immunophilin–drug complex binds specifically to and inhibits calcineurin. Therefore, the mechanism of immunosuppression by cyclosporin indirectly involves the Ca2þ messenger system (Figure 2) (Chang et al. 1991). In addition to its importance in T-cell activation, the Ca2þ-induced activation of calcineurin is also important in regulating apoptosis, because immunosuppressant drugs block apoptosis (Fruman et al. 1992). A number of pathological states may result from the alterations of the activities of

T-cell activation

Second messengers G protein, DG, IP3, Ca2+ Cyclophilin

FKBP FK506 FK506

CsA

RAPA CsA

Inactive complex X

CsA

Transduction protein X

Transduction protein Y

Inactive complex Y

or Inactive complex X FK506

RAPA

RAPA IL-2 gene expression

Ca2+-

Ca2+-

dependent events

independent events

Figure 2 Hypothetical role of immunophilins in immunosuppressive action. Stippling denotes immunosuppressive immunophilin complex. Proteins X and Y denote the unknown physiological targets for immunophilins. IP3, 1,4,5 inositol trisphosphate; IL-2, interleukin-2; RAPA, rapamycin; CsA, cyclosporine A; DG, diacylglycerol; FKBP, FK506-binding protein. Schafer, B. W.; Heizmann, C. W. Trends Biochem. Sci. 1996, 21 (4), 134–140.

592 Alterations in Cell Signaling Table 4 Medical aspects of Ca2þ signaling Pathological process

Tissue

Ischemia Manic depression Spreading depression Arrhythmias Hypertension Atherosclerosis Immunosuppression Transformation and cancer Malignant hyperthermia

Brain and heart Brain Brain Heart Smooth muscle contraction Smooth muscle contraction Lymphocytes Fibroblasts, lymphocytes

Oculocerebrorenal syndrome Sepsis

Skeletal muscle ryanodine receptor Brain Smooth muscle

Ca2þ-binding proteins, which exemplifies the biomedical importance of intracellular Ca2þ homeostasis (Table 4) (Berridge 1994). 2.30.2.2

Ca2þ Channels

Considerable advances have been made in our understanding of the mechanisms by which cells regulate their selective permeability to Ca2þ. Control of calcium permeability through calciumselective channels in the plasma membrane is one of the main processes by which cells regulate [Ca2þ]i. Different types of calcium channels are characterized by differences in their gating properties, ion selectivity, and sensitivity to toxins (Chang et al. 1991; Chao et al. 1984; Choi et al. 1997). Most ion channels have four functional characteristics: ion selectivity, electrical conductance, iongating kinetics, and a sensor for chemical or electrical signals. Ion selectivity is accomplished by steric constraints (size) and more importantly by the relative binding strength of ions to sites within the channel. Conductance of a channel is a measure of the ease with which ions pass through the channel. Ion-gating characteristics include the three distinguishable processes of activation, deactivation, and inactivation of the gate function. Finally, Ca2þ channels are divided into four major classes based on the sensing mechanisms. Ca2þ channels opened by changes in membrane potential are voltage-dependent Ca2þ channels (voltage-operated Ca2þ channels (VOCCs)). Voltage-independent channels may be operated by depletion of intracellular Ca2þ stores (store-operated Ca2þ channels (SOCCs)) or by second messengers (second messenger-operated Ca2þ

channels (SMOCs)). Finally, Ca2þ channels that open in response to activation of an associated receptor are referred to as receptor-operated calcium channels (ROCs). Each of these major classes is extensively further subdivided based on function and molecular cloning.

2.30.3 Components of Intracellular Ca2þ Homeostasis – Ca2þ Channels 2.30.3.1

Voltage-Operated Ca2þ Channels

The major pathway for entry of Ca2þ into excitable cells such as muscle, neural, and secretory cells is through Ca2þ-permeable channels, which are activated by depolarization of the cell. These VOCCs trigger neurotransmitter and hormone release, muscle contraction, and many other cellular functions. At least six types of VOCCs have been identified by function: the low-voltage activated channel (T) and the high-voltage activated channels L, N, P, Q, and R. These channels are distinguished by several properties, including their different sensitivity to the 1,4-dihydropyridines (Varadi et al. 1995). The bestcharacterized Ca2þ channels are the L-type Ca2þ channels, or those that mediate long-lasting depolarization. The L-type Ca2þ channel is a heteropentameric complex of a1, a2, b, c, and n polypeptides, which are highly expressed in skeletal muscle. Cloning studies have identified six a1 gene transcripts and numerous a2, b, and c gene transcripts. L-type channels are blocked by dihydropyridines, phenylalkylamines, and benzothiazepines as discussed elsewhere in this series. Beyond manipulation of the Ca2þ channels by pharmacologic agents and toxins, VOCCs are sensitive to many toxic metals. The mechanisms by which metals modulate Ca2þ currents are both multiple and complex (Busselberg 1995) (Figure 3). First, the different valences, ionic forms, and metal–ligand complexes in physiological solutions determine, in part, which part of the channel a given metal may react with. Second, metals or their complexes may react specifically with the cell membrane, at the entrance to the Ca2þ channel (lead or zinc), or within the Ca2þ channel (mercury, methyl mercury, aluminum). Finally, metals and other toxicants may indirectly perturb VOCC function by their effects on Ca2þ and other signaling processes in the cytoplasm. Given the importance of Ca2þ channels in regulating and mediating cell functions, it is likely

Calcium and Proteases

Ca2+ 2+

Hg MeHg

Al3+ MeHg

Pb2+,Zn2+ MeHg

Extracellular

PKC

Cytoplasm

Pb2+ [Ca2+]

i

Figure 3 Schematic representation illustrating multiple interactions of toxic metals with voltage-operated calcium channels. Reproduced from Busselberg, D. Toxicol Lett. 1995, 82–83, 255–261.

that VOCCs are important targets for the toxicity of many metals (Tomsig and Suszkiw 1991).

2.30.3.2 Voltage-Independent Ca2þ Channels Agonist-induced activation of Ca2þ entry across the plasma membrane in nonexcitable cells is a less wellunderstood process. It has been suggested that agonistinduced activation of Ca2þ influx might be by direct interaction between an agonist receptor and a Ca2þ channel present in the plasma membrane (i.e., ROCs), by the action of second messengers such as cyclic adenosine monophosphate (cAMP), inositol phosphates, or G proteins (i.e., SMOCs), or simply by sensing the storage state of the intracellular Ca2þ pools (i.e., SOCCs). Thus, voltage-independent Ca2þ channels include the SMOCs, which are activated via signals and transducing processes directly triggered by receptor activation, and the SOCCs, which are activated as a consequence of depletion of the rapidly exchanging Ca2þ stores in the cytoplasm (Clementi and Meldolesi 1996). The SMOC family includes channels opened as a direct consequence of receptor activation and, although expressed primarily by excitable cells, SMOCs are also found in nonexcitable cells. SMOCs appear to comprise a heterogeneous family of channels regulated by a variety of second messengers including inositol 1,4,5 trisphosphate (IP3), inositol 1,3,4,5 trisphosphate (IP4), G proteins, cyclic guanosine monophosphate (cGMP), protein kinase C (PKC), and Ca2þ (Clementi and Meldolesi 1996).

593

These channels are activated by receptor agonists acting on G-protein receptors, ATP, and histamine. Evidence suggests that SMOCs are also activated by cytokines and growth factors. These channels appear to be permeable to Ba2þ and Mn2þ, but their permeability and sensitivity to many toxic metals have not been specifically characterized. SOCCs predominate in nonexcitable cells and are present in many, although probably not all, excitable cells. The precise mechanism of SOCC activation and the nature of the signal are still a matter of debate. Several drugs have been shown to inhibit SOCCs and SMOCs, including cytochrome P450inhibiting drugs of diverse structure such as -naphthoflavone, micronazole, and RS-73520. The mechanism of action of channel inhibition is not well characterized; however, most evidence suggests that inhibition of cytochrome P450 activity has little, if anything, to do with the actual mechanism of SOCC blockade. It should be noted, however, that because SOCCs appear heterogeneous and cytochrome P450s constitute a large family of enzymes, the existence of cytochrome P450-dependent or cytochrome P450-independent channels cannot be excluded. Finally, other drugs including cyclooxygenase and lipooxygenase inhibitors exert inhibitory effects on SMOCs and SOCCs. An ROC and a sustained Ca2þ influx have been observed in a wide variety of cell types (Tsunoda 1993). In most cases, the influx of external Ca2þ lags temporally behind the release of intracellular Ca2þ. An increase in [Ca2þ]i due to external Ca2þ influx is usually smaller than that released from intracellular stores. This sustained small increase in [Ca2þ]i is a necessary intermediate for long-term maintenance of cellular response. The mechanism by which agonists acting on receptors increase Ca2þ influx is not well defined. Examples of ROCs include smooth muscle channels coupled to ATP receptors and channels in neurons opened by activation of receptors for the neurotransmitter N-methyl-D-aspartate (NMDA). Studies to evaluate the direct effects of metals on ROCs show complex and selective actions. For example, several studies suggest that lead exposure disrupts NMDA receptor complex function both in vivo and in cultured cells. The inhibitory action of Pb2þ on this ROC seems to be specific for NMDA-activated Ca2þ currents, with only minor actions on kainate- or quisqualate-activated Ca2þ currents. Most evidence suggests that Pb2þ does not block the channel per se, but rather alters the binding of agonists. The actions of lead on the NMDA ROCs may have important

594 Alterations in Cell Signaling

implications for the adverse effects of lead on learning and memory (Vijverberg et al. 1994). 2.30.3.3

Ca2þ Pumps

Maintenance of the Ca2þ gradient across the plasma membrane requires the expenditure of energy in the form of ATP hydrolysis. ATP-driven removal of Ca2þ from the cytoplasm is achieved by membrane protein pumps that are located in both the plasma membrane and the SR/ER. These so-called Ca2þATPases pump Ca2þ out into the extracellular milieu or sequester Ca2þ in intracellular organelles. 2.30.3.4 The Plasma Membrane Ca2þ-ATPase The plasma membrane Ca2þ-ATPase or Ca2þ pump is a single polypeptide chain with a molecular mass of 134 kDa; it is the only high-affinity Ca2þ transporting system present in plasma membranes, and it is ubiquitous in all animal and plant cells (Carafoli 1994). The Ca2þ pump interacts with Ca2þ with high affinity but has a low total Ca2þ transporting capacity. For many cell types, the plasma membrane Ca2þ pump is the only Ca2þ-exporting system; however, in heart cells and neurons, the plasma membrane Ca2þ-ATPase is quantitatively second to a high-capacity, low-affinity Ca2þ exchanger, the Naþ/Ca2þ antiporter. The pump, originally discovered in erythrocyte membrane preparations, is present in the plasma membrane in very small amounts, representing less than 0.1% of the total membrane protein. Typical inhibitors of P-type pumps are orthovanadate and lanthanum ions (i.e., La3þ). The plasma membrane Ca2þ-ATPase is the product of a multigene family (i.e., plasma membrane calcium ATPases (PMCAs)) containing at least four genes in humans and rats (Hammes et al. 1994). Each of these isogenes produces additional isoforms through alternative splicing of primary transcripts. The distribution of the four isogene products and their splicing variants among human tissues shows that gene products 1 and 4 are expressed in all tissues, with isogene 1 generally being more abundant. The other gene products (2 and 3) are expressed in a relatively tissue-specific manner in muscle, brain, and kidney. Furthermore, developmental regulation of gene transcription also occurs. Tissue specificity of the various splice variants of the four isogenes has also been described especially in the central nervous system. The Ca2þ pump is modulated by calmodulin, acidic phospholipids (PLs), several protein kinases, and by dimerization via the calmodulin-

binding domain. Interaction with calmodulin stimulates the Vmax of the Ca2þ-ATPase and decreases its Km (Ca2þ) by about an order of magnitude (from 105 to 106 mol l1). The plasma membrane Ca2þ-ATPase is maintained in an inhibited state by its own carboxy-terminal calmodulin-binding domain, which upon binding calmodulin (or upon phosphorylation) induces a conformational change in the enzyme, exposing the active site. The affinity of the pump for calmodulin differs among the various isoforms, indicating that tissue-selective mechanisms of inhibition by toxic chemicals or drugs may exist. The plasma membrane Ca2þ pump belongs to the P class of ion-motive ATPases, in that it involves a phosphorylated aspartic acid residue during the reaction cycle. Several kinases including protein kinase A (PKA), PKC, and casein kinase-II have been shown to phosphorylate the pump in purified membrane preparations. Phosphorylation tends to increase the Vmax and/or the Km (Ca2þ) of the isolated pump; however, whether phosphorylation also occurs in vivo or whether the various isoforms of the pump exhibit kinase specificities remains to be determined. Although dimerization or oligomerization of the 134-kDa Ca2þ-ATPase has been demonstrated experimentally to enhance pump activity, its physiological relevance remains questionable given the low abundance of the Ca2þ-ATPase protein in the plasma membrane. 2.30.3.5 The Sarcoplasmic/Endoplasmic Reticulum Ca2þ-ATPase In addition to pumping Ca2þ out of the cell via PMCA, the cytoplasmic Ca2þ concentration is controlled by the regulated accumulation and release of Ca2þ from intracellular organelles. This compartmentalization in intracellular stores plays an important role in maintaining Ca2þ homeostasis and in regulating Ca2þ signaling functions. Compartmentalization intracellularly is necessary to enable pulsatile Ca2þ signals to be propagated and to maintain submicromolar and nontoxic Ca2þ concentrations in the cytosol. The energydependent loading of the intracellular stores with Ca2þ is accomplished by ATP-dependent Ca2þ pumps located on intracellular organelles, such as the SR in muscle cells and on the ER or a specialized organelle, the calciosome, in most other nonexcitable cells. Microsomal Ca2þ pumps belong to the sarco(endo)plasmic reticulum Ca2þ-ATPase (SERCA) family (Li et al. 1995; Lytton et al. 1992). A variety of tissue-specialized forms of the SERCA pumps have been characterized and/or cloned (Table 5) (Burk

Calcium and Proteases

595

Table 5 Properties of the sarco(endo)plasmic reticulum Ca2þ-ATPases Ca2þ affinity (mol l1)

Isoform

Tissue specificity

Inhibited by thapsigargin?

SERCA1a SERCA1b SERCA2a SERCA2b SERCA3

Skeletal muscle

Yes

K0.5 ¼ 0.4

Low

Heart Brain, heart, kidney, liver, lung Intestines, lymphoid tissue

Yes

K0.55 ¼ 0.4 K0.5 ¼ 0.27 K0.5 ¼ 1.1

Intermediate

Yes

et al. 1989; Wu et al. 1995). Molecular cloning has identified three distinct homologous Ca2þ-ATPase genes that encode five isoforms of the SERCA. Two alternatively spliced gene products, SERCA1a and SERCA1b, are expressed in a developmentally controlled manner in fast-twitch skeletal muscles. The SERCA2 gene also encodes two alternatively spliced isoforms: SERCA2a is expressed predominantly in slow-twitch muscles and cardiac muscles, whereas SERCA2b is ubiquitously expressed in all tissues examined to date; SERCA3 is another nonmuscle isoform whose expression is not as ubiquitous as SERCA2b. Like the PMCA family, the SERCA family of Ca2þ pumps is regarded as P-type ATPases forming an aspartyl phosphate intermediate during the reaction cycle, and they are sensitive to (a)

(b)

N

CaM-binding domain

CaM

N

CaM-binding C domain

Active

C

Active site

Phospholamban

C

N

N

P Active site Active site CaM-binding C domain

C Active site Active

Active 2þ

C

N

Proteolysis

N

Active site

Inhibited SR Ca2+ pump

CaMK PKA

PKC Calpain

High

inhibition by vanadate ions. SERCAs have high affinity for Ca2þ (Km 0.1–0.4 mmol l1), and these pumps having a higher Vmax than the PMCA pumps are crucial for maintaining low [Ca2þ]i. SERCA Ca2þ pumps are homologous to the PMCA family Ca2þ pumps with respect to domain structure, but lack the carboxyterminal calmodulin-binding regulatory domain. Instead, the SR pump is inhibited by interaction with the membrane protein phospholamban, which binds to the SR pump in a region near its active site, which is analogous to the calmodulin-binding site in the plasma membrane Ca2þ-ATPase. However, for the SR ATPase, the inhibition of pump activity is released by phosphorylation of phospholamban as opposed to calmodulin binding (Figure 4) (James et al. 1995).

Inhibited Plasma membrane Ca2+ pump

Active site C

Relative sensitivity to vanadate

C

P

P

Active

Figure 4 (a) The sarcoplasmic reticulum (SR) Ca pump is regulated by an external inhibitory domain in a manner analogous to the plasma membrane Ca2þ pump. The small membrane protein phospholamban interacts with a site close to the aspartyl phosphate-forming active site, inhibiting the pump. The inhibition is released by phosphorylation, causing dissociation of phospholamban from the pump, just as phosphorylation of the calmodulin (CaM)-binding domain in the plasma membrane pump causes a drop in the affinity of the domain for the active site. (b) CaM and calpain proteolysis also cause activation by removing the CaM-binding domain from the active site. CaMK, calmodulin kinase PKC, protein kinase C; PKA, protein kinase. A. James, P.; Vorherr, T.; Carafoli, E. Trends Biochem. Sci. 1995, 20 (1), 38–42.

596 Alterations in Cell Signaling

Agonist

GTP

? C

X( = CIF? SMG?)

PL

R

GDP

Gp

?

GTP

?

GDP

Ca2+

cADPr

RR

2.30.3.6

Ca2+-mobilizing messengers

Ca2+

IR

Use of inhibitors of the microsomal Ca2þ-ATPase has proven invaluable in studies of agonist-induced Ca2þ signaling in nonexcitable cells. The most popular inhibitory compound is the sesquiterpene lactone thapsigargin, which is a root extract from the plant Thapsia garganica. The utility of thapsigargin lies in its ability to produce a rise in [Ca2þ]i by depleting intracellular IP3-sensitive Ca2þ stores without the generation of inositol phosphates. Thus, thapsigargin evolved into a useful tool for studying the relationship between intracellular Ca2þ release and Ca2þ entry. In several cell types, including parotid acinar cells, platelets, lymphocytes, neutrophils, macrophages, hepatocytes, adrenal chromaffin cells, and fibroblasts, thapsigargin has been shown to cause the release of the agonist-stimulated IP3-sensitive Ca2þ store. The observation that thapsigargin reproduces the effects of agonists on Ca2þ entry provides strong evidence in support of the capacitative entry hypothesis first postulated by Putney and colleagues (Putney 1990; Putney and Bird 1993a,b, 1994; Takemura et al. 1989). The essential feature of the capacitative entry model is that the depletion of the intracellular IP3-sensitive Ca2þ store somehow stimulated the opening of plasma membrane Ca2þ channels to replenish the intracellular Ca2þ pool from the extracellular space. It is postulated that a diffusible second messenger, other than IP3 itself, or a guanosine triphosphate (GTP)-regulated protein links the filling state of the intracellular Ca2þ storage site to the activation of plasma membrane Ca2þ channels (Figure 5) (Fagan et al. 1996; Mullaney et al. 1988; Putney and Bird 1994; Randriamampita and Tsien 1993). By mechanisms similar but not identical to thapsigargin, the hydroquinone 2,5-di-(t-butyl)-1,4benzohydroquinone has been shown to stimulate capacitative Ca2þ entry in hepatocytes or lymphocytes (Llopis et al. 1991). The SERCA expressed in lymphoid cells may be an important target for a variety of toxicants, since polyaromatic hydrocarbons (Krieger et al. 1995) and the organotin compound tributyltin (Chow et al. 1992) have both been shown to inhibit Ca2þATPase activity in lymphoid cells. In fact, since tributyltin has multiple effects on Ca2þ homeostasis, it serves as an excellent conceptual model for demonstrating the capacity of toxicants to initiate Ca2þmediated cell injury (Figure 6) (Orrenius et al. 1992).

IP3

Ca2+

Figure 5 Calcium-mobilizing messengers: in electrically nonexcitable cells, signaling is generally initiated when an agonist activates a surface membrane receptor (R) that, usually through a G protein (Gp), activates a phospholipase C (PLC) that degrades phosphatidylinositol 4,5-bisphosphate, releasing the soluble messenger 1,4,5 trisphosphate (IP3). IP3 activates an IP3 receptor (IR) and thus releases calcium from an intracellular organelle to the cytoplasm. In excitable cells, calcium is released either by calcium-induced calcium release or by the action of cyclic ADP ribose (CADPR) on the ryanodine receptor calcium channel. Recent reports suggest that the release of calcium from the organelle causes a diffusible signal (X) to be generated or released, which activates a plasma membrane calcium entry pathway (represented as a channel, but the precise nature of this pathway is not known). This signal might be the activity termed calcium influx factor or a small G protein (SMG). Alternatively, SMG or some other cellular entity acting through hydrolysis of guanosine triphosphate (GTP) may be involved in either the formation or action of the diffusible messenger. Putney, J. W., Jr.; Bird, G. S. Trends Endocrinol. Metab. 1994, 5 (6), 256–260.

Tributyltin

Inhibition of plasma membrane Ca2+ -ATPase Emptying of intracellular Ca2+ store(s) Opening of Ca2+ channels

Diminished Ca2+ efflux Increased [Ca2+]i

Figure 6 Multiple mechanisms of [Ca2þ]i elevation by the immunotoxicant tributyltin (Dupont and Goldbeter 1992).

Disruption of Ca2þ Homeostasis

Large increases or sustained elevations in cytosolic free Ca2þ can result in numerous damaging effects on the cell. Increases in cytosolic free Ca2þ can activate

a number of degradative Ca2þ-dependent enzymes, such as phospholipases and proteinases. These enzymes can produce abnormalities in the structure

Calcium and Proteases

and function of cytoskeletal elements. Although the exact role of Ca2þ in toxicant-induced injury is unclear, the release of ER Ca2þ stores may be a key step in increasing cytosolic free Ca2þ and initiating the injury process (Costa 1990). For example, the release of ER Ca2þ activates calpains, which lead to disruption of ion homeostasis, cell swelling, cleavage of cytoskeleton proteins, and oncosis (Berridge 1993). In addition, prior depletion of ER Ca2þ stores protects renal proximal tubules from cell death produced by mitochondrial dysfunction and hypoxia (Hokin and Hokin 1953). In lethally injured cells, mitochondria are known to accumulate Ca2þ through a low-affinity, high-capacity Ca2þ transport system. This system plays a minor role in normal cellular Ca2þ regulation; however, under toxicant conditions, the uptake of Ca2þ may facilitate reactive oxygen species (ROS) formation and damage. 2.30.3.6.1

Renal cell Ca 2þ regulation

Cytosolic free Ca2þ levels in renal cells are maintained at approximately 100 nmol l1 by the action of 5

597

basolateral membrane Ca2þ-ATPase efflux pumps, the Naþ/Ca2þ exchanger, and the ER Ca2þATPase (Schrier et al. 1987). The ER and mitochondria also sequester Ca2þ as concentrations rise in the cytosol, which becomes important during cell injury (Figure 7). The release of ER Ca2þ is a key trigger in necrotic cell death in renal cells (Harriman et al. 2002). As stated above, thapsigargin inhibits the SERCA causing the release of ER Ca2þ and in the presence of ATP the cell can pump out the excess cytosolic Ca2þ, effectively depleting the ER Ca2þ store. Harriman et al. (2002) showed that thapsigargin exposure prior to ATP depletion, by mitochondrial inhibition, blocked calpain activation and necrotic cell death, establishing the importance of this intracellular pool of Ca2þ in the death of renal epithelial cells. Furthermore, they demonstrated that ATP depletion had to exceed 96% to block SERCA activity and release ER Ca2þ. Mitochondria have a large capacity for Ca2þ uptake, which is shuttled into the matrix by a ruthenium red (RR)-sensitive channel called the Ca2þ

Ca2+

Na+/Ca2+

Ca2+ ATPase

exchanger 6

Necrosis

Ca2+

Calpain

Ca2+

Voltagedependent channel

Apoptosis

4

1

Ca2+

2

ATP depletion

cyt c AIF smac/ diablo

SERCA MPT Ca2+

ER

3

Mitochondria

Figure 7 Schematic of the role of Ca2þ in renal cell death. (1) Cytosolic Ca2þ is regulated by numerous mechanisms and organelles inside renal cells. (2) Sarco(endo)plasmic reticulum Ca2þ-ATPase (SERCA) pumps on the endoplasmic reticulum (ER) pump Ca2þ into the lumen of the ER to buffer cytosolic levels and thereby participate in signaling. (3) During injury, cytosolic Ca2þ concentrations exceed 400–500 nmol l1 and mitochondria begin to actively sequester Ca2þ. This causes opening of the mitochondrial permeability transition (MPT) pore leading to mitochondrial swelling and inhibition of ATP synthesis. Mitochondrial swelling facilitates the release of cytochrome c (cyt c), apoptosis-inducing factor (AIF), and second mitochondria-derived activator of caspases (SMAC)/direct IAP binding protein with low PI (DIABLO) from the intermembrane space of mitochondria, which in the presence of ATP cause the cell to undergo apoptosis. If MPT has spread to a sufficient proportion of mitochondria and cellular ATP becomes depleted, the cell will undergo necrosis. (4) A rise in cytosolic Ca2þ also activates cytosolic calpain, a protease that mediates apoptosis and necrosis. (5) Membrane-bound Ca2þ efflux pumps use the Naþ gradient and ATP to remove Ca2þ from the cytosol. (6) Ca2þ enters from the extracellular space through a voltage-gated channel.

598 Alterations in Cell Signaling

uniporter (Bernardi 1999). In the renal cells, substantial mitochondrial Ca2þ uptake occurs after cytosolic concentrations reach 400 nmol l1 or greater, which is usually seen only during cell injury (Weinberg 1991). There is a negative correlation among the matrix Ca2þ concentration and oxidative phosphorylation and ATP production (Edelstein and Schrier 2001). Furthermore, mitochondrial Ca2þ accumulation favors the opening of the mitochondrial permeability transition (MPT) pore, which depolarizes the inner membrane and compounds the injury with futile ATP hydrolysis by F(1)F(0)-ATP synthase in reverse mode in an attempt to restore mitochondrial membrane potential (Schwerzmann and Pedersen 1986). In summary, Ca2þ is an important signal from the ER and to the mitochondria during renal cell death. The MPT pore is a channel that spans the mitochondrial inner and outer membranes allowing the equilibration of molecules up to 1.5 kDa (Lemasters et al. 2002). While the identity of all the proteins that make up the pore remains controversial (He and Lemasters 2002), opening of the classical MPT pore is inhibited by the immunosuppressant CsA, which binds to cyclophillin D, a proposed component of the pore. Bongkrekic acid (BKA) also inhibits MPT by binding to the adenine nucleotide translocase, suggesting a role for this protein in the makeup of the pore also. Other inhibitors of the MPT include high inner membrane potential and the Ca2þ uniporter inhibitor RR (Bernardi 1999). While in most cases Ca2þ accumulation in the mitochondrial matrix is a requirement for the MPT, factors such as free fatty acids (FFAs, especially unsaturated; Sultan and Sokolove 2001), inorganic phosphate, ROS, and low inner membrane potential reduce the threshold matrix concentration of Ca2þ required for pore opening (Bernardi 1999). MPT causes mitochondrial swelling due to oncotic forces generated by the high matrix protein concentration (Halestrap et al. 2002) and as Kþ enters the mitochondria down its concentration gradient (Kaasik et al. 2006). The swelling associated with MPT is a major mechanism by which cytochrome c and other apoptotic mediators are released into the cytosol, and depending on the rate at which MPT spreads through the mitochondria of a cell, and the ability of the cell to generate extramitochondrial ATP, the MPT can lead to apoptosis or necrosis (Lemasters et al. 2002). In summary, oxidative stress and Ca2þ dysregulation converge on mitochondria and induce dysfunction, which can lead to either type of cell death.

2.30.4 Calpains 2.30.4.1

The Calpain Family of Proteases

Calpains are defined as Ca2þ-activated, nonlysosomal cysteine proteases that are active at neutral pH (Goll et al. 2003; Guroff and Neutral 1964; Murachi et al. 1980). Calpain was first isolated from porcine skeletal muscle and described by Dayton et al. (1976a,b). After this initial isolation of calpain, which was later named m-calpain (calpain 2), two other calpain family members were identified, -calpain (calpain 1) and calpastatin, the endogenous inhibitor of calpain (Dayton et al. 1981; De Martino 1981; DeMartino and Croall 1982; Kishimoto et al. 1981; Mellgren 1980; Murakami et al. 1981; Nishiura et al. 2001; Sakon et al. 1981). Since these founding members of the calpain family were identified, numerous ubiquitous (Thompson and Goll 2000) or tissue-specific (Sorimachi et al. 1993a,b, 1994) as well as vertebrate and invertebrate calpains have been identified (Goll et al. 2003). To date, 15 calpain-like genes have been described for mammals, 4 in Drosophila, 12 in Caenorhabditis elegans, and 1 in plants (Sorimachi and Suzuki 2001). With the exception of the well-studied calpains, m-calpain, -calpain, and calpastatin, all other calpain isoforms have only been identified based on gene homology and therefore have not been purified or characterized in the protein form. Of the 15 mammalian members of the calpain family, there are 14 large subunit isoforms and 1 small subunit, plus the endogenous inhibitor calpastatin (Goll et al. 2003). Currently, the mammalian calpain family is divided into two groups, typical and atypical, based on domain structure homology (Table 6). The typical calpains, composed of calpains 1–3, 8, 9, 11, 12, and 14, are described as those that have domain structures similar to the prototypical calpains 1 and 2, containing the calcium-binding penta-EF-hand domain, domain IV. The atypical calpains (calpains 5–7, 10, 13, and 15) are distinguished from the typical calpains in that they lack the calcium-binding penta-EF-hand domain and instead contain a highly divergent fourth domain (Evans and Turner 2007; Goll et al. 2003; Sorimachi and Suzuki 2001) (Table 6).

2.30.4.2

Calpain Structure

The large subunit (80 kDa) of the typical calpains contains four distinct domains, distinguished as domains I–IV from N- to C-terminus (Figure 8).

Calcium and Proteases

599

Table 6 Mammalian calpain family: nomenclature, distribution, and splice variants

Gene

Protein product

Typical calpains CAPN1 Calpain 1 CAPN2 Calpain 2 CAPN3 Calpain 3

Alternative names

CAPN4

Calpain 4

CAPN8 CAPN9 CAPN11 CAPN12 CAPN14

Calpain 8 Calpain 9 Calpain 11 Calpain 12 Calpain 14

-Calpain m-calpain p94, nCL-1 Lp82, Lp85 Rt88 Calpain small subunit, 30K nCL-2 nCL-4 -

Atypical calpains CAPN5 Calpain 5 CAPN6 Calpain 6 CAPN7 Calpain 7 CAPN10 Calpain 10 CAPN13 Calpain 13 CAPN15 Calpain 15

htra3, nCL-3 CAPNX, calpamodulin palBH CAPN8a SOLH

EF-hand domain

þ þ þ

Expression

Alternative splicing

No No Yes (8)

þ

Ubiquitous Ubiquitous Skeletal muscle, lens, retina Ubiquitous

þ þ þ þ þ

Stomach mucosa Digestive tract Testis Hair follicle n.d.

Yes (2) Yes (2) No No Yes (2)

-

Testis, brain Placenta Ubiquitous Ubiquitous n.d. Ubiquitous

No No No Yes (8) No No

No

a Name no longer used. The currently accepted naming system for the calpain family of cysteine proteases is presented. Also, a clear distinction between the classification of typical and atypical calpains is outlined. Tissue-specific distribution is highlighted unless this characteristic has not been determined (n.d.). Lastly, as a focal point, knowledge of genetic alternative splicing is provided. Reproduced from Huang and Wang (2001), Saez et al. (2006), Sorimachi and Suzuki (2001), Evans and Turner (2007), and Goll et al. (2003).

Domain I is a short sequence of 87 (calpain 1) or 76 (calpain 2) amino acids, which are autolytically cleaved during activation of the protease (Goll et al. 2003; Suzuki et al. 1981, 1988). Sequence homology of domain I between species is about 72–86%; however, this domain has no homology to any other sequenced protein domains (Goll et al. 2003). Domain II is the proteolytic domain that contains the characteristic catalytic triad of cysteine proteases (cysteine, histidine, and asparagine), and is divided into two subdomains IIa and IIb. Domain IIa contains the active site cysteine (105/calpain 1, 115/calpain 2), while domain IIb contains the active site histidine (272/calpain1, 262/calpain 2) and asparagine (296/calpain 1, 286/calpain 2). Amino acid sequence homology of domain II between species ranges from 85 to 93%; interestingly, amino acid sequence homology of domain II between different isoforms of calpains is much more variable, ranging from 30 to 88% (Goll et al. 2003). It has recently been shown that Ca2þ can bind two nonEF-hand sites in domain II and this Ca2þ-binding event is proposed to be vital for the activity of the protease (Hosfield et al. 1999; Moldoveanu et al. 2002).

Domain III is thought to function as a PL-binding domain, potentially allowing the protease to interact with membranes; additionally, PL binding has been shown to increase the Ca2þ-binding capabilities of the enzyme (Tompa et al. 2001). Domain III is also thought to be capable of direct Ca2þ binding, as two Ca2þbinding sites have been identified within the domain (Goll et al. 2003). It has also been shown that domain III is involved in essential electrostatic interactions important for enzyme activity (Hosfield et al. 1999; Strobl et al. 2000). Domain IV is a calmodulin-like domain containing a penta-EF-hand motif that is responsible for Ca2þ binding and heterodimerization (Sorimachi and Suzuki 2001). Domain IV is highly similar to domain VI of the small subunit providing a basis for the interaction of the heterodimer, as the fifth EFhand of the large subunit and that of the small subunit interact with each other establishing the heterodimer (Sorimachi and Suzuki 2001). The remaining four EF-hands bind Ca2þ and are important for maximal activation of calpain; however, Ca2þ binding of these four sites is not currently thought to be the major Ca2þ-binding event leading to activation.

600 Alterations in Cell Signaling

Typical calpains

Calpain 4 small subunit Calpains 1,2,8,9,11,12,14

V Cys I

IIa

His Asn IIb

III

Cys Calpain 3

NS

I

VI

IV

His Asn

IIa

IS-1

5EF hand domain

IIb

III

IS-2

IV

Atypical calpains Cys (*Lys) His Asn Calpains 5,6*,10

I

IIa

IIb

III

T

Cys His Asn Calpain 7

IIa

IIb Cys

Calpain 13

IIa

PBH

III

His Asn IIb Cys His Asn

Calpain 15

Zn

IIa

IIb

SOH

Figure 8 Schematic representation of calpain isoforms. Domain structures of the currently known human isoforms of the calpain family are represented. The calpain family is subdivided into typical and atypical calpains based on the presence (typical) or absence (atypical) of the penta-EF-hand domain (domain IV is depicted as a blue domain with yellow ovals). Cys, His, and Asn (cysteine, histidine, and asparagine, respectively) represent the location of the three residues that comprise the catalytic triad (calpain 6 is noted for its replacement of the cysteine with a lysine). Reproduced from Sorimachi et al. (2001) and Evans et al. (2007).

Recent crystallographic structures of calpain 2 have brought new light on the boundaries of the domains, and a new domain classification system has been proposed (Goll et al. 2003; Hosfield et al. 1999). The new domain classification differs from the conventional amino acid sequence-derived system. In this system, domain I is not considered as a domain due to its trivial size, and instead of dividing the proteolytic domain into two subdomains, it is split into two distinct domains, domain I and domain II, while domains III and IV remain the same (Hosfield et al. 1999). The new classification system has been slowly accepted by the calpain field; however, the majority of the literature still uses the conventional classification system, and so will be used throughout this discussion. The small subunit (28 kDa) (calpain 4) of calpains 1 and 2 is identical and consists of two distinct

domains identified as domains V and VI from N- to C-terminus (Figure 8) (Goll et al. 2003). Domain V, a glycine-rich region in which 40 of the first 64 amino acids are glycine, is referred to as a hydrophobic domain and therefore has been suggested to interact with PLs (Imajoh et al. 1986); however, the exact function is still not known. The carboxy-terminal domain of the small subunit, domain VI, contains five EF-hand structures four of which are involved in binding Ca2þ, while the final is involved in dimerization with the large subunit (Maki et al. 1997; Xie et al. 2001). The fifth EF-hand of domain IV and the fifth EF-hand of domain VI interact with one another allowing heterodimerization of the small and large subunit of the typical calpains (Figure 9). The atypical calpains lack the characteristic calmodulin-like penta-EF-hand domain, domain IV, found in the typical calpains (Figure 8) (Goll et al. 2003). The

Calcium and Proteases

V

I

IIa

IIb

III

601

VI

IV

Figure 9 Schematic diagram of key factors in calpain activation: representation of a typical calpain heterodimer. The fifth EF-hand structure of domain IV (large subunit) and that of domain VI (small subunit) are involved in heterodimerization (double-headed arrow). Calcium binds multiple sites along the calpain molecule (orange stars indicate calcium-binding sites). Autolysis of an N-terminal peptide from domain I is associated with calpain activation (dashed line). Reproduced from Moldoveanu, T., et al. Cell 2002, 108 (5), 649–660.

atypical calpains do contain domains I–III, which are homologous to the typical domains I–III, and therefore retain the catalytic triad of the proteolytic domain. The absence of domain IV leads to speculation about the ability of these atypical calpains to interact with the small subunit, and it is currently thought that they do not heterodimerize with the small subunit. As a result, it is not clear whether the atypical calpains are as Ca2þ-dependent as the typical calpains, due to their lack of Ca2þ-binding sites. However, in view of the recent discovery of the importance of Ca2þ binding to non-EF-hand sites in domain II (discussed below), Ca2þ may still regulate the activity of these atypical calpains. 2.30.4.3

Calpain Activation

Understanding the mechanisms of calpain activation is an extremely important part of calpain biology, as calpain activity/inactivity has been shown to be involved in multiple disease processes. A major issue with in vivo calpain activation is that the requirements of Ca2þ concentrations for activation are outside of physiologically attainable concentrations. Normal physiological intracellular concentrations of Ca2þ are generally thought to be 50–300 nmol l1 (Maravall et al. 2000), while Ca2þ concentrations required for half-maximal activity are on the order of micromole and millimole levels for calpain-1 (-calpain) and calpain-2 (m-calpain) respectively (Goll et al. 2003). Many investigators have focused on understanding how calpains can become active at Ca2þ concentrations that are orders

of magnitude below their requirements. Some studies have focused on identifying mechanisms that lower the Ca2þ requirements for autolysis of the enzymes, while others have tried to identify molecules that would interact with the calpains to reduce their calcium requirements (Goll et al. 2003) (Figure 10). Other proposed mechanisms for calpain activation have included looking at microenvironments and the potential for Ca2þ concentrations being much higher in specific subcellular locations. Therefore, a current hypothesis is that calpain activation could be dependent on the subcellular localization of the enzymes and the local concentrations of Ca2þ in those locations. It has been proposed that calpain interaction with PLs especially those in the plasma membrane could position calpain near Ca2þ channels, which would allow calpain to be exposed to high Ca2þ concentrations thereby allowing for activation (Goll et al. 2003). Yet others in the field have questioned whether it is even necessary for calpain to achieve full activation in vivo, as less than half-maximal activation may be sufficient for its as yet unidentified physiological function. Additionally, the full activation of calpain may occur only during pathological conditions, in which intracellular Ca2þ concentrations can reach much higher levels. The role of posttranslational modifications in calpain activation has also been explored, as the regulation of many cellular processes is controlled by phosphorylation and other posttranslational modifications. Studies using phospho-specific antibodies and matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) proteomics have shown that calpains 1 and 2 are

602 Alterations in Cell Signaling

Ca++

EGF EGFR

Dissociation of subunits

ERK/ MAPK

FA K

+ PO4

Integrin

Calpastatin

Autolysis of domain I

Active calpain

rad eg

ati

on

– PO4

id binding Phospholip

location

Membrane trans

D

PKA

Isovalerylcarnitine Acyl CoA-binding protein UK114 Other protein activators

Figure 10 Mechanism of calpain activation. Depicted are the known mechanisms of typical calpain activation. These mechanisms include direct Ca2þ activation, autolysis, dissociation of the large and small subunits, degradation of the endogenous inhibitor calpastatin, membrane translocation, phospholipid binding, phosphorylation, and interaction with various ‘activator’ molecules. EGF, epidermal growth factor; EGFR, EGF receptors; ERK, extracellular signal-regulated kinases; MAPK, mitogen activated protein; PKA, protein kinase A.

phosphorylated at nine and eight sites, respectively (Goll et al. 2003). These phosphorylation sites are located in clusters at the N- and C-terminal sides of domain II, and analysis of the amino acid sequences surrounding these phosphorylation sites has predicted these sites to be PKA (calpain 1) and PKC (calpain 2) sites (Goll et al. 2003). Not all sites are thought to be phosphorylated at one time, as only 2–4 phosphates per molecule are generally found. The phosphorylation of the calpains is still not clearly understood as more questions about the role of phosphorylation have arisen from these initial detection studies. 2.30.4.4 Physiological and Pathological Roles of Calpain The calpain system has been implicated in many physiological and pathological conditions. In some cases specific isoforms of calpain have been

identified, while in others they have not. The prototypical calpains (calpains 1 and 2) have been studied much more extensively and therefore will be the subject of a general discussion on the multiple physiological roles of calpain. Despite the great efforts that have been put forth over the last 30 years, the exact role of calpains in physiological and pathological conditions is still controversial and remains quite elusive (Goll et al. 2003). The lack of specific tools, such as specific inhibitors and diagnostic substrates, has delayed the advancement of the knowledge of calpain biology and pathobiology. Calpain is known to cleave a diverse array of substrates without having an apparent specific consensus sequence. It has been proposed that the conformation of the three-dimensional environment surrounding the cleavage site, and not just the primary amino acid sequence, is the key factor in subsite specificity of calpain cleavage, further complicating the ascertainment of genuine

Calcium and Proteases

calpain targets (Goll et al. 2003). In contrast to other protease families, calpain is notorious for being able to cleave substrates at limited sites, leaving apparently functional shortened versions of the original substrate (Goll 1999; Goll et al. 1992). This pattern of specific limited proteolysis has led to hypotheses that implicate calpains not as digestive proteases (lysosomal or proteasomal) but as regulators of tightly controlled processes (Goll et al. 2003) such as cell cycle progression (Choi et al. 1997), regulation of gene expression (Hirai et al. 1991), apoptosis (Kidd et al. 2000), signal transduction (Kishimoto et al. 1989), and cell motility (Glading et al. 2002). 2.30.4.5 Physiological Roles of Typical Calpains Since their isolation in 1978, calpains 1 and 2 have been extensively studied; however, a definitive role in a physiological process has not been identified thus far (Goll et al. 2003). An overwhelming number of roles for these typical calpains have been proposed and it is not clear if all or any of them will be significant in vivo. As noted above, calpains 1 and 2 have been extensively studied and therefore this discussion will refer to these members, except when noted otherwise. 2.30.4.6

Calpain in Cell Cycle

Calpain has been shown to be vital for the proper progression of the cell through the cell cycle, specifically the G1- to S-phase transition (Choi et al. 1997; Goll et al. 2003). This important cell cycle transition is known to be controlled by cyclin-dependent kinases, such as cdk-2 and cdk-4, in addition to other important molecules such as cyclin D or cyclin E (Nigg 1995). Many of these regulators of cell cycle including cyclin D, cyclin E, and p27kip1 have been shown to be degraded by calpain (Carragher 2006). Calpain-dependent degradation of cyclin D1 has been demonstrated in NIH-3T3 cells during serum starvation, and inhibition of calpain reverses cell cycle arrest, indicating a negative regulatory role for calpain (Carragher et al. 2002; Choi et al. 1997). In contrast, another study indicated that inhibition of calpain in WI-38 fibroblasts blocks cell cycle progression, indicating a positive regulatory role for calpain (Mellgren 1997). However, some of these studies were conducted using nonspecific calpain inhibitors that are known to inhibit the proteasome (which has also been shown to degrade the cyclins), complicating the interpretation of these results. Yet

603

other studies found calpain-dependent degradation of p53 to be involved in the progression of the cell cycle, again implicating calpain as a positive regulator of cell cycle progression (Zhang et al. 1997). Additionally, a recent study using fibroblasts from a calpain 4 (small subunit) knockout mouse demonstrated that these cells progress through the cell cycle and proliferate at similar rates as their wildtype counterparts (Arthur et al. 2000). This would indicate that neither calpain 1 nor calpain 2 is vital for progression of the cell cycle, and questions the validity of previous studies using nonspecific inhibitors of the calpains. In summary, the role of calpains in cell cycle progression and mitosis remains a controversial topic that is yet to be fully understood. 2.30.4.7 Calpain in Regulation of Gene Expression Calpain has been implicated in the cleavage of proteins that regulate gene expression, such as the transcription factors c-Fos, c-Jun, and p53, suggesting a role for calpain in the control of gene expression (Hirai et al. 1991; Kubbutat and Vousden 1997; Pariat et al. 1997, 2000). However, it is also known that the proteasome is responsible for degrading these transcription factors (Goll et al. 2003). Therefore, it is unclear at this point if calpain does in fact degrade these transcription factors in vivo, or if these are just nonspecific effects resulting from inadequate tools (Gonen et al. 1997; Salvat et al. 1999). The currently accepted hypothesis is that these three transcription factors are mostly degraded by the proteasome pathway; however, it is thought that under certain conditions calpains may be involved in their degradation (Salvat et al. 1999), and whether this small amount of seemingly specific degradation is physiologically or pathologically important remains unclear. 2.30.4.8

Calpain in Apoptosis

The role of calpain in apoptosis or programmed cell death is complicated and controversial; some reports suggest that calpain promotes apoptosis, whereas others claim that it is antiapoptotic (Goll et al. 2003). As a result of the varied and contrasting roles that have been proposed, it is currently thought that the involvement of calpain in apoptosis is probably highly cell- and stimuli-specific (Kidd et al. 2000). The caspases, another family of cysteine proteases, are the principal regulators of apoptosis (Danial and Korsmeyer 2004; Thornberry and Lazebnik 1998).

604 Alterations in Cell Signaling

Multiple members of the caspase family have been shown to be cleaved directly by calpain leading to their inactivation (Chua et al. 2000), suggesting an important antiapoptotic role for calpain in the modulation and regulation of known apoptotic molecules. Conversely, calpain 1 has been shown to cleave caspase-12 producing an activated proapoptotic form of the caspase. Additionally, calpain 1 has been shown to mediate a cleavage event that transforms the antiapoptotic Bcl-xl into a proapoptotic form (Nakagawa and Yuan 2000). Furthermore, calpain 1 has recently been implicated as a molecule that can cleave apoptosis-inducing factor (AIF) from the inner mitochondrial membrane to induce apoptosis (Polster et al. 2005). Given these selected examples, it is clear that the role of calpain in apoptosis is not completely understood and will require much more investigation to elucidate a specific role. Additional details on apoptotic cell death can be found in Chapter 2.28 of this volume. 2.30.4.9

Calpain in Signal Transduction

An early report in 1989 provided evidence for calpain-mediated cleavage of PKC resulting in a constitutively active form of the kinase (Kishimoto et al. 1989). This report led to a field of calpain research centered on signal transduction. Many other signaling molecules (including kinases, phosphatases, and cytoskeletal proteins) were identified as in vitro substrates of calpain, further supporting calpain as a regulator of signal transduction; however, none of the molecules could be validated as signal transduction-related calpain substrates in vivo (Goll et al. 2003). Despite this apparent inability to validate these substrates in vivo, the role of calpain in signal transduction is still being evaluated. Most recently, calpain has been implicated in integrin-mediated signal transduction pathways – specifically, the -integrins are cleaved by calpain (Pfaff et al. 1999). 2.30.4.10

Calpain in Cell Motility

Perhaps the most definitive evidence of physiological importance for calpain is seen in the work on cytoskeletal/membrane attachments and cell motility. Multiple cytoskeletal and focal adhesion complex components, such as II-spectrin, talin, ezrin, focal adhesion kinase (FAK), paxillin, vimentin, and desmin (Carragher 2006), have been established as in vitro calpain substrates. Additionally, calpain-dependent degradation of focal adhesion attachments at

both the leading and trailing edge of motile cells is required for mobility (Glading et al. 2002). Furthermore, fibroblasts from calpain 4 (small subunit) knockout mice, as mentioned above, are able to proliferate; however, their ability to migrate is markedly reduced (Arthur et al. 2000; Dourdin et al. 2001). Fibroblasts from these mice, which have no detectable calpain activity, do not possess the ability to degrade talin, whereas their wild-type counterparts retain this ability. The use of fibroblasts from the calpain 4 knockout mice has provided strong molecular support for the role of calpain in cell motility, escaping the lack of specificity afforded by the use of inhibitors. As alluded to previously, the generation of a genetic knockout of the calpain small subunit has provided very useful information about the physiological role of calpain. The fact that two separately developed CAPN4 knockout mice were embryonic lethal tells us that calpain activity is absolutely required for development (Arthur et al. 2000; Zimmerman et al. 2000). It was determined that lack of all calpain activity resulted in devastating defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitor cells (Arthur et al. 2000). Since the activity of both calpain 1 and calpain 2 was abolished, it is difficult to determine if one or both of the calpains are responsible. To elucidate this question, two subsequent studies were performed: (1) a calpain 1 knockout mouse was developed, which displayed no embryonic lethality but displayed a phenotype of deficient platelet aggregation (Azam et al. 2001); and (2) a calpain 2 knockout mouse was developed, which was embryonic lethal, revealing that calpain 2 is the calpain isoform that is vital for embryogenesis (Dutt et al. 2006). Clearly, the calpain system has vital physiological implications, which remain to be elucidated; however, the advancement of molecular approaches will undoubtedly provide further advances in our understanding of the many physiological roles of calpain. 2.30.4.11 Calpains

Pathological Roles of Typical

It is generally thought that the role of calpains in pathological conditions can originate from either of two conditions: (1) insufficient calpain activity or (2) excessive activation of calpain. Therefore, calpain activity can be described as a double-edged sword, and some as yet undefined intermediate amount of highly regulated calpain activity is required for normal physiological function, while alterations of

Calcium and Proteases

605

Table 7 Summary of calpain isoforms associated with disease pathologies Calpain isoform

Associated disease pathology

Proposed mechanism

Calpain 1/calpain 2

Muscular dystrophy Stroke Traumatic brain injury Spinal cord injury Alzheimer’s disease Neurodegenerative disorders Cataracts Cancer Atherosclerosis Limb-girdle muscular dystrophy type 2A Cataracts Gastric cancer Type 2 diabetes mellitus

Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis Loss of Ca2þ homeostasis and/or genetic mutations Loss of Ca2þ homeostasis Genetic mutations Loss of Ca2þ homeostasis Genetic mutations Genetic mutations

Calpain 3 Calpain 9 Calpain 10

Reproduced from Carragher, N. O. Curr. Pharm. Des. 2006, 12(5), 615–638; Goll, D. E., et al. Physiol. Rev. 2003, 83 (3), 731–801.

activity in either direction can have negative consequences. For example, calpains have been implicated in multiple pathological conditions including type 2 diabetes, cataracts, Duchene’s muscular dystrophy, Parkinson’s disease, Alzheimer’s disease, rheumatoid arthritis, ischemia, stroke, platelet disorders, gastric cancer, ovarian cancer, and limb-girdle muscular dystrophy, all with proposed mechanisms that involve altered calpain function (Table 7) (Carragher 2006; Goll et al. 2003; Zatz and Starling 2005). However, of these proposed calpainopathies, only limb-girdle muscular dystrophy has been definitively linked to a dysfunction of calpain (calpain 3), while the others still maintain only correlative associations (Zatz and Starling 2005). The proposed underlying mechanisms of these disorders generally fall into two categories: (1) genetic, resulting from gain-of-function or loss-of-function mutations of calpain genes or (2) disorders involving disruption of Ca2þ homeostasis (Goll et al. 2003).

in which calpain inhibitors are used alone, caution must be used when interpreting the results. Calpain has been implicated as a mediator of injury in other ischemic models such as renal proximal tubular injury used in our laboratory. Renal cell oncosis produced by a variety model toxicants can be blocked with multiple calpain inhibitors (Harriman et al. 2000, 2002; Liu et al. 2001, 2002; Schnellmann and Williams 1998; Waters et al. 1997). Calpain was shown to be involved in promoting the permeability of the plasma membrane during toxicant-induced oncosis (a model for ischemia-induced cell injury) (Waters et al. 1997). Ultimately, a specific role for calpain in progressive plasma membrane permeability was identified, involving calpain-dependent degradation of the cytoskeletal components paxillin, talin, and vinculin (Liu and Schnellmann 2003), which are important for the structure of the cytoskeleton and integrity of the plasma membrane.

2.30.4.13 2.30.4.12

Calpains in Ischemia

Increases in intracellular Ca2þ levels after ischemic insults provide an ideal situation for calpain activation. Much of the research on calpain and ischemic insults has been performed in model systems of neuronal ischemia, providing theories that overactivation of calpain, resulting from loss of Ca2þ homeostasis, facilitates neuronal injury (Bartus et al. 1995; Neumar et al. 2001). Indeed, calpain inhibition has been shown to protect neuronal cells in multiple ischemic in vivo models of stroke (Bartus et al. 1994; Rami et al. 2007). However, as with any experiments

Calpains in Cancer

The role of calpain in cancer began with reports that calpain activity was increased in breast cancer tissues, especially those that were positive for estrogen receptor (Shiba et al. 1996). More recently, multiple studies have shown a correlation between increased calpain expression/activity and cancer development/progression in renal cell carcinoma (Braun et al. 1999), squamous cell/basal cell carcinoma (Reichrath et al. 2003), and chronic lymphocytic leukemia (Witkowski et al. 2002). These studies indicated a tumor promoter role for calpains, while other studies have discovered a tumor suppressor

606 Alterations in Cell Signaling

role for calpain. Calpain 9, a digestive tract-specific calpain, is downregulated in gastric cancer tissues and cell lines (Yoshikawa et al. 2000). Furthermore, specific knockdown of the calpain 9 isoform results in cell transformation and tumorigenesis, providing strong support for this calpain isoform in tumor development (Liu et al. 2000). Perhaps the strongest evidence was seen with specific knockdown of calpain 2 resulting in reduced invasion of the human prostate carcinoma cell line DU 145 both in vitro and in vivo (Mamoune et al. 2003). Additionally, calpain 10 has been linked to polycystic ovarian cancer through genetic screening approaches; however, molecular mechanisms of this association have not yet been explored (Urbanek 2007).

2.30.4.14

Mitochondrial Calpains

While calpains had generally been considered as ubiquitously expressed cytosolic enzymes, there were reports of calpain-like activity localized to the mitochondria of rat liver in 1982 (Beer et al. 1982) and, 10 years later, in 1991 (Tavares and DuqueMagalhaes 1991), which went largely unnoticed. However, in 1998, Gores and colleagues demonstrated that this mitochondrial calpain-like protease was involved in the induction of the MPT pore (Aguilar et al. 1996; Gores et al. 1998), drawing much more attention to the identification of the specific isoform of calpain responsible. As evidenced from the initial reports of mitochondrial calpain-like activity, it had been assumed that there were actually multiple sources of calpain-like activity localized to multiple submitochondrial compartments. The first identification of a specific calpain isoform being localized to the mitochondria took place in 2005 by Garcia et al., purporting calpain 1 (-calpain) as the resident mitochondrial calpain in rat brain cortex. Prior to this report, investigators had shown that purified calpain 1 was capable of cleaving AIF, a resident mitochondrial protein involved in caspaseindependent apoptosis (Polster et al. 2005). Indeed, subsequent studies using small interfering RNA (siRNA) technology confirmed that calpain 1 is capable of cleaving AIF allowing for its release from the mitochondria in neuronal cell cultures (Cao et al. 2007). Additionally, both calpastatin, the endogenous inhibitor of calpain, and calpain 1 have been identified in the mitochondria of pulmonary smooth muscle (Kar et al. 2007, 2008). Clearly, there is rapidly accumulating evidence that calpain 1 is not

exclusively a cytosolic protease and is likely targeted to the mitochondria. As predicted by the early mitochondrial calpainlike protease studies, another calpain isoform was identified. Almost simultaneously with the identification of calpain 1, our laboratory identified a separate calpain isoform, calpain 10, in the mitochondria from the cortex of the kidney of rabbits (Arrington et al. 2006). This particular mitochondrial calpain has Ca2þ-inducible activity (not Ca2þ dependent) that is inhibited by calpain inhibitors, calpeptin and E-64, at submicromolar concentrations in isolated mitochondria. Mitochondrial calpain 10 was positively identified in all subfractions of mitochondria using three separate antibodies targeted to different portions of the protein. After being transfected into NIH-3T3 cells, a calpain 10-green fluorescent protein (GFP) fusion protein was targeted to the mitochondria, and the N-terminal 15 amino acids of calpain 10 were found to be sufficient for mitochondrial targeting (Arrington et al. 2006). Functionally, mitochondrial calpain 10 was implicated in Ca2þ-induced mitochondrial dysfunction as measured by mitochondrial swelling and decreased mitochondrial respiration, the latter of which was inhibited by calpeptin at submicromolar levels. Two proteolytic targets for mitochondrial calpain 10 were identified as NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and NADH dehydrogenase 6 (ND6), two proteins that are part of complex I of the mitochondrial electron transport chain. Additional studies are needed to completely elucidate the role of calpain 10 in cellular injury. 2.30.4.15

Phospholipase A2

PLA2 enzymes catalyze the hydrolysis of the sn-2 ester bond of PLs. The action of PLA2 enzymes generates FFAs and lysophospholipids (PLs without an sn-2 fatty acid), both of which have biological and/or pathological functions (Anliker and Chun 2004; Das 2006; Montrucchio et al. 2000; Penzo et al. 2004). Lysophospholipids can differ in the polar head group attached at the third position of the glycerol backbone, in the nature of the sn-1 fatty acid, and in the type of linkage at the sn-1 position (i.e., ester vs vinyl ether) (Bruni et al. 1988; Meyer and McHowat 2007). The fatty acids released by PLA2 hydrolysis can be saturated (no double bonds), unsaturated (one double bond), or polyunsaturated (two or more double bonds). The family of PLA2 enzymes includes over 20 isoforms, which differ in their Ca2þ

Calcium and Proteases

requirement, biological function, and localization (Schaloske and Dennis 2006). Two different naming and classification systems for PLA2 have emerged in the literature. Historically, PLA2 were classified as secretory PLA2 (sPLA2), cytosolic/Ca2þ-dependent PLA2 (cPLA2), calcium-independent phospholipases A2 (iPLA2), platelet-activating factor acetylhydrolases (PAF-AHs), or lysosomal PLA2 (LPLA2) based on their Ca2þ requirement, localization, and/or function. Recently, Dennis and colleagues have developed a grouping system, based on nucleotide sequence homology and other conserved features, which consists of 15 groups, some consisting of multiple subgroups or individual isoforms (Schaloske and Dennis 2006; Six and Dennis 2000).

2.30.5 Conclusion Calcium is an important messenger in a variety of cell signaling pathways and it functions in the regulation of diverse cellular processes. Disruption in calcium concentration results in cellular dysfunction and activation of proteases. As insufficient or increased protease activation can cause cellular dysfunction, calcium must be tightly regulated within the intra- and extracellular compartments.

Acknowledgments We need to acknowledge the calpain grant NIH ESO12239, the mito grant NIH GMO84147, and the PLA2 grant NIH DKO62028.

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