Revisiting the Role of Leukocytes in Cystic Fibrosis

Cystic fibrosis in characterized by pulmonary bacterial colonization and hyperinflammation. Lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells of patients with CF express functional CFTR and are directly affected by altered CFTR expression/function, impairing their ability to resolve infections and inflammation. However, the mechanism behind and the contribution of leukocytes in the pathogenesis of CF are still poorly characterized. The recent clinical introduction of specific CFTR modulators added an important tool not only for the clinical management of the disease but also to the investigation of the pathophysiological mechanisms related to CFTR dysfunction and dysregulated immunity. These drugs treat the basic defect in cystic fibrosis (CF) by increasing CFTR function with improvement of lung function and quality of life, and may improve clinical outcomes also by correcting the dysregulated immune function that characterizes CF. Measure of CFTR function, protein expression profiling and several omics methods were used to identify molecular changes in freshly isolated leukocytes of CF patients, highlighting two roles of leukocytes in CF: one more generally related to the mechanism(s) causing immune dysregulation in CF and unresolved inflammation, and another more applicative role, which identifies in myeloid cells, an important tool predictive of the therapeutic response of CF patients. In this review we will summarize available data on CFTR expression and function in leukocyte populations and will discuss potential clinical applications based on available data

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

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

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

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

Principi di biochimica

Durante lo studio del metabolismo biomolecolare si pu\uf2 facilmente perdere di vista il significato generale di quanto si legge, ovvero come le diverse vie metaboliche si intreccino e si integrino per contribuire a creare un organismo funzionale. In tutto il libro, e in particolare nelle sezioni Il metabolismo nel contesto, si insiste sulle relazioni tra i prodotti metabolici, utilizzando un esempio ricorrente relativo all\u2019uso delle risorse bioenergetiche, mentre gli approfondimenti clinici e biologici rendono espliciti i diversi modi in cui la biochimica influenza, a volte in modo sorprendente, molti aspetti del mondo in cui viviamo. L\u2019apprendimento \ue8 inoltre reso pi\uf9 efficace e solido dai Quesiti (le cui risposte sono riportate a fine capitolo), dalle Strutture a margine che forniscono un breve promemoria sulla struttura di una molecola o di un gruppo funzionale incontrato in precedenza, dalle brevi digressioni costituite dalle Note a margine e dai numerosi Problemi proposti per ogni capitolo

Revisiting the role of leukocytes in cystic fibrosis

Cystic fibrosis in characterized by pulmonary bacterial colonization and hyperinflammation. Lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells of patients with CF express functional CFTR and are directly affected by altered CFTR expression/function, impairing their ability to resolve infections and inflammation. However, the mechanism behind and the contribution of leukocytes in the pathogenesis of CF are still poorly characterized. The recent clinical introduction of specific CFTR modulators added an important tool not only for the clinical management of the disease but also to the investigation of the pathophysiological mechanisms related to CFTR dysfunction and dysregulated immunity. These drugs treat the basic defect in cystic fibrosis (CF) by increasing CFTR function with improvement of lung function and quality of life, and may improve clinical outcomes also by correcting the dysregulated immune function that characterizes CF. Measure of CFTR function, protein expression profiling and several omics methods were used to identify molecular changes in freshly isolated leukocytes of CF patients, highlighting two roles of leukocytes in CF: one more generally related to the mechanism(s) causing immune dysregulation in CF and unresolved inflammation, and another more applicative role, which identifies in myeloid cells, an important tool predictive of the therapeutic response of CF patients. In this review we will summarize available data on CFTR expression and function in leukocyte populations and will discuss potential clinical applications based on available data

Biochimica

Questa settima edizione di Biochimica rende conto dei pi\uf9 recenti progressi attraverso un linguaggio chiaro e accessibile anche per quegli studenti che si avvicinano per la prima volta allo studio della disciplina. Le caratteristiche che, fin dalla prima edizione, rendono la Biochimica di Stryer uno dei libri di testo pi\uf9 diffusi sono: l\u2019organizzazione del testo semplice e logica: guida il lettore attraverso i processi e lo aiuta a districarsi nella complessit\ue0 dei meccanismi e delle vie metaboliche; le illustrazioni dedicate a singoli concetti: ogni immagine riesce a descrivere e spiegare un meccanismo oppure a raccontare tutto ci\uf2 che accade in una determinata via metabolica o in un dato processo biochimico senza distrarre l\u2019attenzione con un eccesso di dettagli; la rilevanza fisiologica: il testo aiuta lo studente a collegare la biochimica alla propria vita; le vie metaboliche e i processi sono presentati sempre nel loro contesto fisiologico, in modo che si possa apprezzare come la biochimica lavori nei diversi distretti dell\u2019organismo e in diverse condizioni, sia ambientali sia di stimolazione ormonale; gli approfondimenti clinici: ovunque fosse appropriato, le vie metaboliche e/o i meccanismi sono stati applicati alla fisiologia e alla patologia; le prospettive evoluzionistiche: l\u2019evoluzione \ue8 evidente sia nelle strutture sia nelle vie metaboliche biochimiche ed \ue8 puntualmente evidenziata

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\u2013calpastatin 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\u201360% 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 100 kD + 70 kD 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 35 kD and 20 kD, 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. \ua9 2011 Elsevier B.V. All rights reserve