Temperature-Dependent Olive Pomace Extraction for Obtaining Bioactive Compounds Preventing the Death of Murine Cortical Neurons.

High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50 v/v) at 180 °C for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca2+-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca2+-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.

Modulation of Plasmatic Matrix Metalloprotease 9: A Promising New Tool for Understanding the Variable Clinical Responses of Patients with Cystic Fibrosis to Cystic Fibrosis Transmembrane Conductance Regulator Modulators.

BACKGROUND: The most recent modulator combination, elexacaftor/tezacaftor/ivacaftor (Trikafta®), has been shown to improve clinical outcomes in most patients with cystic fibrosis (PwCF). Unfortunately, the clinical benefits are sometimes variable; thus, improving our knowledge of the possible causes of this variability can help reduce it. METHODS: Circulating mononuclear cells (CMCs) and plasma were collected from 16 PwCF (including those on Trikafta® therapy) and 4 non-CF subjects. Cystic fibrosis transmembrane conductance regulator (CFTR) activity and matrix metalloprotease 9 (MMP9) expression were monitored before and after therapy, together with some clinical parameters. The relationship between MMP9 expression and the modulation of the extracellular-regulated 1/2 (ERK1/2) and nuclear factor-kB (NF-kB) pathways was also analyzed. RESULTS: MMP9, markedly expressed in the CMCs and plasma of all the patients included in the study, was downregulated in the clinically responsive PwCF. In the non-responder, the MMP9 levels remained high. The modulation of MMP9 following treatment with Trikafta® may be controlled by the NF-kB pathway. CONCLUSIONS: These data strongly suggest that MMP9 downregulation is a potential biomarker of therapy efficacy and that it could be useful in understanding the molecular events underlying the variable clinical responses of patients to Trikafta®. This knowledge could be helpful for future studies of personalized medicine and thereby ensure improvements in individual responses to therapies.

Possible etiological association of ovine papillomaviruses with bladder tumors in cattle.

INTRODUCTION: Bladder tumors of cattle are very uncommon accounting from 0.1% to 0.01% of all bovine malignancies. Bladder tumors are common in cattle grazing on bracken fern-infested pasturelands. Bovine papillomaviruses have a crucial role in tumors of bovine urinary bladder. AIM OF THE STUDY: To investigate the potential association of ovine papillomavirus (OaPV) infection with bladder carcinogenesis of cattle. METHODS: Droplet digital PCR was used to detect and quantify the nucleic acids of OaPVs in bladder tumors of cattle that were collected at public and private slaughterhouses. RESULTS: OaPV DNA and RNA were detected and quantified in 10 bladder tumors of cattle that were tested negative for bovine papillomaviruses. The most prevalent genotypes were OaPV1 and OaPV2. OaPV4 was rarely observed. Furthermore, we detected a significant overexpression and hyperphosphorylation of pRb and a significant overexpression and activation of the calpain-1 as well as a significant overexpression of E2F3 and of phosphorylated (activated) PDGFßR in neoplastic bladders in comparison with healthy bladders, which suggests that E2F3 and PDGFßR may play an important role in OaPV-mediated molecular pathways that lead to bladder carcinogenesis. CONCLUSION: In all tumors, OaPV RNA could explain the causality of the disease of the urinary bladder. Therefore, persistent infections by OaPVs could be involved in bladder carcinogenesis. Our data showed that there is a possible etiologic association of OaPVs with bladder tumors of cattle.

Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis.

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC-MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl- transport in CF.

A Bioactive Olive Pomace Extract Prevents the Death of Murine Cortical Neurons Triggered by NMDAR Over-Activation.

We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.

A new human calpastatin skipped of the inhibitory region protects calpain-1 from inactivation and degradation.

Several human acute and chronic diseases involve calpain over-activation. However, the mechanistic linkages between the etiology and the progression of cell damages are not yet completely understood. Here we show that different human cells and tissues, including brain tumor specimens, cell lines of nerve origin, breast tumor samples and peripheral blood mononuclear cells from healthy donors, express a calpastatin form that lacks all the exons coding for the domains responsible of calpain inhibition. The open reading frame of this new form of calpastatin, named hcast 3-25, starts inside the L-domain (exons 2 and 3) and continues with the exons from 25 to 29 that code for the conserved C-terminal tail shared by all the full-length calpastatins. We have here observed that unlike the other calpastatins forms, that are predominantly Δ3 splice variants, hcast 3-25 is endowed with exon 3. At a functional level, recombinant hcast 3-25 operates as a positive modulator of calpain-1 in vitro by preventing 1) calpain-1-mediated proteolytic degradation of the activated enzyme and 2) binding to calpain-1 of inhibitory calpastatins that contain the L-domain. Thus hcast 3-25 can be considered as a novel member and possible modulator of the calpain/calpastatin system acting by a mechanism alternative to inhibition.

Production and Purification of Recombinant Calpastatin.

The production of recombinant calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific calpastatin species that can be present concomitantly with other calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various calpastatin species for several reasons among which: calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other calpastatin species deriving from different tissues or organisms and calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/calpastatin system in vitro.

Isolation of Endogenous Calpastatin.

We here describe the purification of calpastatin from human erythrocytes. When calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca2+ to specifically identify the protein. Thus, the purification steps necessary to obtain the inhibitor protein were originally designed to obtain calpain from the same tissue. For this reason, in addition to calpastatin purification, we also include a method for purifying human erythrocyte calpain and globin. We routinely use these two components for assaying calpastatin inhibition.

Immunoblotting for Calpastatin Expression.

Immunoblotting is a procedure routinely used to analyze calpastatin expression. However, immunoblotting alone may not be adequate for this task, since calpastatin isoforms can vary by tissue, can be modified by partial digestion, and can undergo posttranslational modifications. Here we describe a method for more precise evaluation of calpastatin expression by combining immunoblot analysis with an assay for the inhibitory activity of a single calpastatin species isolated by SDS-PAGE and protein elution from the gel.

Cell protection from Ca2+-overloading by bioactive molecules extracted from olive pomace.

We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca2+-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca2+-induced cell damages by reducing the activation of the Ca2+-dependent protease calpain, acting possibly through the modification of the permeability to Ca2+ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca2+ homeostasis.

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 80-kDa catalytic subunit into a 75-kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75 kDa 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 the present study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 µM) and low (5 µM) [Ca2+]. Here we demonstrate that the L-DOM binds the 80 kDa proenzyme in the absence of Ca2+ Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM 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 75 kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75 kDa 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 physiopathological conditions having altered Ca2+ homeostasis.

Interference with Ca2+-Dependent Proteolysis Does Not Alter the Course of Muscle Wasting in Experimental Cancer Cachexia.

Protein hypercatabolism significantly contributes to the onset and progression of muscle wasting in cancer cachexia. In this regard, a major role is played by the ATP-ubiquitin-proteasome-dependent pathway and by autophagy. However, little is known about the relevance of the Ca2+-dependent proteolytic system. Since previous results suggested that this pathway is activated in the skeletal muscle of tumor hosts, the present study was aimed to investigate whether inhibition of Ca2+-dependent proteases (calpains) may improve cancer-induced muscle wasting. Two experimental models of cancer cachexia were used, namely the AH-130 Yoshida hepatoma and the C26 colon carcinoma. The Ca2+-dependent proteolytic system was inhibited by treating the animals with dantrolene or by overexpressing in the muscle calpastatin, the physiologic inhibitor of Ca2+-dependent proteases. The results confirm that calpain-1 is overexpressed and calpastatin is reduced in the muscle of rats implanted with the AH-130 hepatoma, and show for the first time that the Ca2+-dependent proteolytic system is overactivated also in the C26-bearing mice. Yet, administration of dantrolene, an inhibitor of the Ca2+-dependent proteases, did not modify tumor-induced body weight loss and muscle wasting in the AH-130 hosts. Dantrolene was also unable to reduce the enhancement of protein degradation rates occurring in rats bearing the AH-130 hepatoma. Similarly, overexpression of calpastatin in the tibialis muscle of the C26 hosts did not improve muscle wasting at all. These observations suggest that inhibiting a single proteolytic system is not a good strategy to contrast cancer-induced muscle wasting. In this regard, a more general and integrated approach aimed at targeting the catabolic stimuli rather than the proteolytic activity of a single pathway would likely be the most appropriate therapeutic intervention.

Abnormal activation of calpain and protein kinase Cα promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients.

Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.

Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex.

We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.

Interaction between calpain-1 and HSP90: new insights into the regulation of localization and activity of the protease.

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca2+. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca2+, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca2+]i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca2+-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.

Differential regulation of the calpain-calpastatin complex by the L-domain of calpastatin.

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain-calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca(2+)] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.

Calpain inhibition promotes the rescue of F(508)del-CFTR in PBMC from cystic fibrosis patients.

A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F(508)del-CFTR from the cell surface. In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F(508)del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F(508)del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.

Evidence for alteration of calpain/calpastatin system in PBMC of cystic fibrosis patients.

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.

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

We 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 Ca(2+)-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.

Role of calpain in the regulation of CFTR (cystic fibrosis transmembrane conductance regulator) turnover.

The level of the mature native 170 kDa form of CFTR (cystic fibrosis transmembrane conductance regulator) at the plasma membrane is under the control of a selective proteolysis catalysed by calpain. The product of this limited digestion, consisting of discrete fragments still associated by strong interactions, is removed from the plasma membrane and internalized in vesicles and subject to an additional degradation. This process can be monitored by visualizing the accumulation of a 100 kDa fragment in a proliferating human leukaemic T-cell line and in human circulating lymphocytes. In reconstructed systems, and in intact cells, the conversion of native CFTR into the 100 kDa fragment linearly correlated with calpain activation and was prevented by addition of synthetic calpain inhibitors. A reduction in Ca2+ influx, by blocking the NMDA (N-methyl-D-aspartate) receptor Ca2+ channel, inhibited the conversion of the native 170 kDa fragment into the 100 kDa fragment, whereas an endosome acidification blocker promoted accumulation of the digested 100 kDa CFTR form. An important role in calpain-mediated turnover of CFTR is exerted by HSP90 (heat-shock protein 90), which, via association with the protein channel, modulates the degradative effect of calpain through a selective protection. Taken together these results indicate that CFTR turnover is initiated by calpain activation, which is induced by an increased Ca2+ influx and, following internalization of the cleaved channel protein, and completed by the lysosomal proteases. These findings provide new insights into the molecular mechanisms responsible for the defective functions of ion channels in human pathologies.