Un-Jin P. Zimmerman

The Calpain Small Subunit Gene Is Essential: Its Inactivation Results in Embryonic Lethality

Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild‐type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild‐type littermates. Genotyping of fetuses at different stages of development also revealed only wild‐type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.

Calcium-mediated breakdown of glial filaments and neurofilaments in rat optic nerve and spinal cord.

Disruptive effects of calcium upon neurofilaments and glial filaments were studied in white matter of rat optic nerve and spinal cord and in rat peripheral nerve. Filament ultrastructure and tissue protein composition were compared following a calcium influx into excised tissues. A calcium influx was induced by freeze-thawing tissues in media containing calcium (5 mM) while control tissues were freeze-thawed in the presence of EGTA (5 mM). Experimental and control tissues were either fixed by immersion in glutaraldehyde and processed for electron microscopic examination or homogenized in a solubilizing buffer and analyzed for protein content by SDS-polyacrylamide gel electrophoresis. Morphological studies showed that calcium influxes led to the loss of neurofilaments and glial filaments and to their replacement by an amorphous granular material. These morphological changes were accompanied by the loss of neurofilament triplet proteins and glial fibrillary acidic (GFA) protein from whole-tissue homogenates. In addition, a calcium-sensitive 58,000-mol-wt protein was identified in rat optic and peripheral nerve. The findings indicate the widespread occurrence of neurofilament proteolysis following calcium influxes into CNS and PNS tissues. The parallel breakdown of glial filaments and loss of GFA protein subunits suggest the presence of additional calcium-activated proteases(s) in astroglial cells.

Two-stage autolysis of the catalytic subunit initiates activation of calpain I

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.

Calcium‐activated Proteolysis of Intermediate Filamentsa

Intermediate filaments (IF) are a major component of the cytoskeleton in different cell types. While the functions of IF remain unclear, it is speculated that some IF, especially neurofilaments (NF), provide structural stability to the cytoskeleton, enabling markedly asymmetrical cell forms to be established and maintained. Large neurons, for example, contain axonal processes whose dimensions are several thousand fold greater than those of the parent neuronal cell body. It seems reasonable to suspect that the synthesis, assembly, and transport of N F down the axon play an important role in determining dimensions and configuration of the neurons, especially its axonal extension. Most studies on NF metabolism have focused on their synthesis and axonal transport. Much less information is available on the fate of these proteins although it is clear that the enormous quantities of NF proteins transported down the axon must be balanced by a comparable process of degradation and turnover. There is, in fact, considerable evidence suggesting that calcium-activated protease is a major factor regulating NF metabolism. This paper will review data on calcium-mediated breakdown of N F in situ, the isolation and characterization of calcium-activated proteases that degrade N F proteins, and the nature of the reaction whereby the enzyme degrades isolated N F proteins and N F in situ.

Mechanisms Underlying the Neuronal Response to Ischemic Injury. Ccalcium-Activated Proteolysis of Neurofilaments

Publisher Summary This chapter discusses the calcium-activated proteolysis of neurofilament (NF) by describing the substrate and its relationship to the neuronal cytoskeleton, by describing the reaction whereby NF are degraded by calcium, and by characterizing the enzyme that mediates the reaction. NF represents a major component of the neuronal cytoskeleton, thereby serving to establish and maintain the asymmetrical shape of the neuron. The synthesis and transport of NF tend to stabilize the axon, because NF comprises the structural foundation of the axon. Much of the turnover of NF proteins appears to occur at the distal end of the axon, mediated by a calciumactivated neutral protease (CANP) that can be isolated from neural and non-neural tissues. Activation of CANP at nerve endings may well be coupled to calcium influxes associated with neurotransmission. The enzyme, however, is not limited to synaptic endings but is also present throughout the neuronal cytoplasm.

Proteolysis of Synaptobrevin, Syntaxin, and Snap‐25 in Alveolar Epithelial Type II Cells

Synaptobrevin‐2, syntaxin‐1, and SNAP‐25 were identified in rat alveolar epithelial type II cells by Western blot analysis. Synaptobrevin‐2 was localized in the lamellar bodies, and syntaxin‐1 and SNAP‐25 were found in 0.4% Nonidet P40‐soluble and ‐insoluble fractions, respectively, of the type II cells. When the isolated type II cells were stimulated for secretion with calcium ionophore A23187 or with phorbol 12‐myristate 13‐acetate, these proteins were found to have been proteolyzed. Preincubation of cells with calpain inhibitor II (N‐acetylleucylleucylmethionine), however, prevented the proteolysis. Treatment of the cell lysate with exogenous calpain resulted in a time‐dependent decrease of these proteins. The data suggest that synaptobrevin, syntaxin, and SNAP‐25 are subject to proteolytic modification by activated calpain in intact type II cells stimulated for secretion.

Annexin II Binds to the Membrane of A549 Cells in a Calcium-Dependent and Calcium-Independent Manner

We investigated the nature of annexin II binding to the biological membranes using a lung epithelium-derived cell line A549. The cytosolic and membrane fractions of A549 cells were separated in the presence of 5 mM EGTA. Both fractions contain annexin II monomer and tetramer as evaluated by western blots using specific monoclonal antibodies against p36 and p11 subunits of annexin II. A substantial amount of annexin II was associated with the membrane fraction even after extensive washing with EGTA buffer, indicating the presence of two pools of annexin II. The EGTA-resistant membrane-bound annexin II could be partially extracted by 1% Triton X-100 or 60 mM n-octyl-beta-D-glucopyranoside, and completely by 30 mM CHAPS or 0.1% deoxycholate. This fraction of annexin II was also extracted by 0.1 M Na2CO3, pH 11 and partitioned into the aqueous phase after being treated with Triton X-114, demonstrating that the EGTA-resistant annexin II is a peripheral membrane protein. When the cells were lysed in varying concentrations of Ca2+, annexin II translocated from cytosolic fraction to membrane fraction at 4-25 microM Ca2+. To identify proteins closely associated with annexin II the membrane fraction was treated with the bifunctional chemical cross-linker disulfosuccinimidyl tartarate, followed by western blot analysis using anti-p36 or anti-p11 antibodies. We find that both p36 and p11 were cross-linked to a 51 kDa protein. In addition, p11 also binds to several proteins with molecular mass of 91, 65, 40 and 36 kDa. Our results suggest that annexin II may bind to the A549 cell membranes via specific membrane-associated proteins.

Calcium-Activated Protease and the Regulation of the Axonal Cytoskeleton

The synthesis, assembly, and intracellular dissemination of a prominent and elaborate cytoskeleton are basic properties of neurons that enable these cells to establish and maintain a highly asymmetric cell form. Variations in size and shape among different neurons are determined by the quantity and quality of their cytoskeletal components. A complex interplay among cytoskeletal elements undoubtedly underlies the formation of growth cones and their transformation into neurites as well as the subsequent maintenance of established neuritic processes. Similar interactions of cytoskeletal components are probably also rcponsible for the regeneration of cell processes that occurs following injury to the axonal extension of the cell.

Neurofilament proteolysis in rat peripheral nerve. Homologies with calcium-activated proteolysis of other tissues

Abstract Alterations of nerve proteins were studied in desheathed segments of rat sciatic nerves which were freeze-thawed and incubated in solutions containing calcium or EGTA. Calcium caused a selective loss of 69,000, 150,000, and 200,000 MW neurofilament triplet proteins as well as some loss of 55,000–57,000 MW proteins when nerve proteins were examined by SDS gel electrophoresis. The loss of nerve proteins was accompanied by a variable appearance of 25,000, 36,000, and 72,000 MW proteins. The calcium-induced changes did not occur in nerve segments which had been heated to 60°C for 30 min and were prevented by preincubation of tissues with 1 mM p-chloromercuribenzoate (PCMB), N-ethyl-malemide (NEM), or iodoacetamide. The calcium-induced alterations of nerve proteins were attributed to a calcium-activated thiol protease which preferentially degrades neurofilament proteins. Neurofilament protease of rat peripheral nerve was characterized by studying the alterations of nerve proteins under different incubational conditions. Calcium activated proteolysis was demonstrated between pH 6.0 and 8.8 and at 4, 20, and 37°C. Protein breakdown occurred with 50 μM calcium, and could be simulated by strontium and, to a lesser extent, by barium and lanthanum. Other metals had strong (Hg, Zn, Cd, Cu, Co), weak (Pb and Ag), or no (Al, Mg, and Mn) inhibitory effects on enzymatic activity. Proteolysis was also strongly inhibited by TLCK (1 mM) and by TPCK (1 mM) but not by PMSF (1 mM). The properties of neurofilament protease closely resemble those of calcium-activated proteases described in several tissues.

SECRETAGOGUE-INDUCED PROTEOLYSIS OF CAMP-DEPENDENT PROTEIN KINASE IN INTACT RAT ALVEOLAR EPITHELIAL TYPE II CELLS

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.