Biochemical Characteristics Of Calpain

Endopeptidase

Heterodimer: 80-kD catalytic subunit, 30-kD regulatory subunit —Calpain and |Ji-calpain are ubiquitously distributed cytosolic isozymes —Calpain and |Ji-calpain have identical regulatory subunits but distinctive catalytic subunits

—Calpain requires a higher concentration of Ca2+ for activation than |Ji-calpain

Phospholipids reduce the Ca2+ requirement

Substrates: cytoskeletal and membrane proteins and enzymes

FIGURE 15-27

Biochemical characteristics of calpain.

FIGURE 15-28

Calpain translocation. Proposed pathways of calpain activation and translocation. Both calpain subunits may undergo calcium (Ca2+)-mediated autolysis within the cytosol and hydrolyze cytoso-lic substrates. Calpains may also undergo Ca2+-mediated translocation to the membrane, Ca2+-mediated, phospholipid-facilitated autolysis and hydrolyze membrane-associated substrates. The autolyzed calpains may be released from the membrane and hydrolyze cytosolic substrates. (From Suzuki and Ohno [10], and Suzuki et al. [11]; with permission.)

2015105 0

CON TFEC +C12 BHQ +C12 TBHP +C12

CON TFEC +C12 BHQ +C12 TBHP +C12

FIGURE 15-29

A, B, Dissimilar types of calpain inhibitors block renal proximal tubular toxicity of many agents. Renal proximal tubular suspensions were pretreated with the calpain inhibitor 2 (CI2) or PD150606 (PD). CI2 is an irreversible inhibitor of calpains that binds to the active site of the enzyme. PD150606 is a reversible inhibitor of calpains that binds to the calcium (Ca2+)-binding

domain on the enzyme. The toxicants used were the haloalkane cysteine conjugate tetrafluoroethyl-L-cysteine (TFEC), the alkylating quinone bromohydroquinone (BHQ), and the model oxidant t-butylhydroperoxide (TBHP). The release of lactate dehydrogenase (LDH) was used as a marker of cell death. CON—control. (From Waters et al. [12]; with permission.)

FIGURE 15-30

One potential pathway in which calcium (Ca2+) and calpains play a role in renal proximal tubule cell death. These events are subsequent to mitochondrial inhibition and ATP depletion. 1) |x-Calpain releases endoplasmic reticulum (er) Ca2+ stores. 2) Release of er Ca2+ stores increases cytosolic free Ca2+ concentrations. 3) The increase in cytosolic free Ca2+ concentration mediates extracellular Ca2+ entry. (This may also occur as a direct result of er Ca2+ depletion.) 4) The influx of extracellular Ca2+ further increases cytosolic free Ca2+ concentrations. 5) This initiates the translocation of nonactivated m-calpain to the plasma membrane (6). 7) At the plasma membrane nonactivated m-calpain is autolyzed and hydrolyzes a membrane-associated substrate. 8) Either directly or indirectly, hydrolysis of the membrane-associated substrate results in influx of extracellular chloride ions (Cl-). The influx of extracellular Cl- triggers terminal cell swelling. Steps a-d represent an alternate pathway that results in extracellular Ca2+ entry. (Data from Waters et al. [12,13,14].)

PROPERTIES OF PHOSPHOLIPASE A2 GROUP

FIGURE 15-31

Biochemical characteristics of several identified phospholipase A2s.

Characteristics

Secretory

Cytosolic

Ca2+-Independent

Localization

Secreted

Cytosolic

Cytosolic

Membrane

Molecular mass

~14 kDa

~85 kDa

~40 kDa

Arachidonate preference

+

+

+

Ca2+ required

mM

(M

None

None

Ca2+ role

Catalysis

Memb. Assoc.

None

None

FIGURE 15-32

FIGURE 15-32

The importance of the cytosolic phospholipase A2 in oxidant injury. A, Time-dependent release of arachidonic acid (AA) from LLC-PK] cells exposed to hydrogen peroxide (0.5 mM). B and C, The concentration-dependent effects of hydrogen peroxide on LLC-PKj cell death (using lactate dehydrogenase [LDH] release as marker) after 3 hours' exposure. Cells were transfected with 1) the cytosolic PLA2 (LLC-cPLA2), 2) the secretory PLA2 (LLC-sPLA2), 3) vector (LLC-vector), or 4) were not transfected (LLC-PKi). Cells transfected with cytosolic PLA2 exhibited greater AA release and cell death in response to oxidant exposure than cells transfected with the vector or secretory PLA2 or not transfected. These results suggest that activation of cytosolic PLA2 during oxidant injury contributes to cell injury and death. (From Sapirstein et al. [15]; with permission.)

Inhibitor 1 is IL-1ß converting enzyme inhibitor 1 (YVAD-CHO) and inhibitor II is CPP32/apopain inhibitor (DEVD-CHO). These results suggest that caspases are activated after mitochondrial inhibition and that caspases may contribute to antimycin A-induced DNA damage and cell death. (From Kaushal et al. [16]; with permission.)

Potential role of caspases in cell death in LLC-PKi cells exposed to antimycin A. A, Time-dependent effects of antimycin A treatment on caspase activity in LLC-PKi cells. B, C, The effect of two capase inhibitors on antimycin A-induced DNA damage and cell death, respectively. Antimycin A is an inhibitor of mitochondrial electron transport.

Inhibitor 1 is IL-1ß converting enzyme inhibitor 1 (YVAD-CHO) and inhibitor II is CPP32/apopain inhibitor (DEVD-CHO). These results suggest that caspases are activated after mitochondrial inhibition and that caspases may contribute to antimycin A-induced DNA damage and cell death. (From Kaushal et al. [16]; with permission.)

Was this article helpful?

0 0

Post a comment