Molecular and Functional Properties of a Calpain Activator Protein Specific for μ-Isoforms

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the mu-calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on mu-calpain independently from the source of the proteinase. The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation

Conformational Changes of Calpain from Human Erythrocytes in the Presence of Ca2

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca(2+) binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca(2+) promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca(2+) addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca(2+), but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution

Modulation of rat brain calpastatin efficiency by post-translational modifications

AbstractCalpains, the thiol proteinases of the calcium-dependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation.

Abstract Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation

Unexpected role of the L-domain of calpastatin during the autoproteolytic activation of human erythrocyte calpain.

Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca2+], through the conversion of the 80kDa catalytic subunit to a 75kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75kDa calpain-1 form also in vivo, in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of this study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 \ub5M) and low (5 \ub5M) [Ca2+]. Here we demonstrate that the L-DOM binds the 80kDa proenzyme in the absence of Ca2+. Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 \ub5M Ca2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca2+] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75kDa form. Taken together our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physio-pathological conditions having altered Ca2+ homeostasis

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

AbstractThe 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca2+-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations

The Calpain System Is Involved in the Constitutive Regulation of β-Catenin Signaling Functions

Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer

Selective Proinflammatory Activation of Astrocytes by High-Mobility Group Box 1 Protein Signaling

Abstract Extracellular high-mobility group box 1 protein (HMGB1) triggers inflammatory events in the brain. We demonstrate that astrocytes, the main glial cells in the brain, acquire a specific reactive phenotype when exposed to HMGB1. This cell activation, which involves the receptor for advanced glycation end-products and the MAPK/ERK1/2 cascade, results in the transcriptional/translational induction of a restricted number of inflammatory mediators, including cyclooxygenase-2, matrix metalloproteinase-9, and several chemokines of the CC and CXC families. The mixture of factors released by HMGB1-reactive astrocytes displays a potent chemotactic activity on human monocytic cells. This study is the first to suggest that HMGB1/astrocyte interaction plays a specific functional role in the progression of inflammatory processes in the CNS by facilitating local leukocyte infiltration

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR

AbstractWe are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca2+-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients