DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.

Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility.

Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor.

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway.

StUbEx PLUS-A Modified Stable Tagged Ubiquitin Exchange System for Peptide Level Purification and In-Depth Mapping of Ubiquitination Sites.

Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.

Integrative proteome and transcriptome analysis of extramedullary erythropoiesis and its reversal by transferrin treatment in a mouse model of beta-thalassemia.

Beta-thalassemia results from mutations of the ß-hemoglobin (Hbb) gene and reduced functional Hbb synthesis. Excess α-Hb causes globin chain aggregation, oxidation, cytoskeletal damage, and increased red blood cell clearance. These events result in anemia, altered iron homeostasis, and expansion of extramedullary erythropoiesis. Serum transferrin (Tf) is suggested to be an important regulator of erythropoiesis in murine models of thalassemia. The present study was conducted to establish a quantitative proteomic and transcriptomic analysis of transferrin-modulated extramedullary erythropoiesis in the spleen of wild type and thalassemic Hbb(th3/+) mice. Our LC-MS/MS protein analysis and mRNA sequencing data provide quantitative expression estimates of 1590 proteins and 24,581 transcripts of the murine spleen and characterize key processes of erythropoiesis and RBC homeostasis such as the whole heme synthesis pathway as well as critical components of the red blood cell antioxidant systems and the proliferative cell cycling pathway. The data confirm that Tf treatment of nontransfused Hbb(th3/+) mice induces a systematic correction of these processes at a molecular level. Tf treatment of Hbb(th3/+) mice for 60 days leads to a complete molecular restoration of the normal murine spleen phenotype. These findings support further investigation of plasma-derived Tf as a treatment for thalassemia.

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

GSNOR deficiency promotes tumor growth via FAK1 S-nitrosylation.

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.

FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Evaluation of SCO1 deletion on Saccharomyces cerevisiae metabolism through a proteomic approach.

The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.

Proteomic profile identifies dysregulated pathways in Cornelia de Lange syndrome cells with distinct mutations in SMC1A and SMC3 genes.

Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS, we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, SMC1A and SMC3. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the c-MYC gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS.

The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation.

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.

Proteomic analysis identifies differentially expressed proteins after HDAC vorinostat and EGFR inhibitor gefitinib treatments in Hep-2 cancer cells.

Several solid tumors are characterized by poor prognosis and few effective treatment options, other than palliative chemotherapy in the recurrent/metastatic setting. Epidermal growth factor receptor (EGFR) has been considered an important anticancer target because it is involved in the development and progression of several solid tumors; however, only a subset of patients show a clinically meaningful response to EGFR inhibition, particularly to EGFR tyrosine kinase inhibitors such as gefitinib. We have recently demonstrated synergistic antitumor effect of the histone deacetylase inhibitor vorinostat combined with gefitinib. To further characterize the interaction between these two agents, cellular extracts from Hep-2 cancer cells that were untreated or treated for 24 h with either vorinostat or gefitinib alone or with a vorinostat/gefitinib combination were analyzed using 2-D DIGE. Software analysis using DeCyder was performed, and numerous differentially expressed protein spots were visualized between the four examined settings. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified; some of these were validated by Western blotting. Finally, a pathway analysis of experimental data performed using MetaCore highlighted a relevant relationship between the identified proteins and additional potential effectors. In conclusion, we performed a comprehensive analysis of proteins regulated by vorinostat and gefitinib, alone and in combination, providing a useful insight into their mechanisms of action as well as their synergistic interaction.

Magnitude of Ubiquitination Determines the Fate of Epidermal Growth Factor Receptor Upon Ligand Stimulation.

Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modeling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor-α and epiregulin. Whereas only little differences in the order of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.

Liver-fibrosis-activated transcriptional networks govern hepatocyte reprogramming and intra-hepatic communication.

Liver fibrosis is a strong predictor of long-term mortality in individuals with metabolic-associated fatty liver disease; yet, the mechanisms underlying the progression from the comparatively benign fatty liver state to advanced non-alcoholic steatohepatitis (NASH) and liver fibrosis are incompletely understood. Using cell-type-resolved genomics, we show that comprehensive alterations in hepatocyte genomic and transcriptional settings during NASH progression, led to a loss of hepatocyte identity. The hepatocyte reprogramming was under tight cooperative control of a network of fibrosis-activated transcription factors, as exemplified by the transcription factor Elf-3 (ELF3) and zinc finger protein GLIS2 (GLIS2). Indeed, ELF3- and GLIS2-controlled fibrosis-dependent hepatokine genes targeting disease-associated hepatic stellate cell gene programs. Thus, interconnected transcription factor networks not only promoted hepatocyte dysfunction but also directed the intra-hepatic crosstalk necessary for NASH and fibrosis progression, implying that molecular "hub-centered" targeting strategies are superior to existing mono-target approaches as currently used in NASH therapy.

In vitro effects of Echis carinatus venom on the human plasma proteome.

Echis carinatus venom (EV) is a complex mixture of toxins that contribute to its lethality. EV proteolytic activity was analyzed by zymography, chromogenic assays, and SDS-PAGE. To understand the molecular mechanism of the envenomation, we investigated the in vitro effect of EV on human plasma proteins. We looked for EV protein substrates and their proteolytic fragments. We analyzed EV proteolytic activity on standard proteins such as prothrombin or fibrinogen. To set up the optimal EV:plasma protein ratio conditions, plasma was incubated with EV (treated plasma), depleted of abundant proteins, and subjected to SDS-PAGE. Samples from control and treated plasma were also analyzed by 2-DE/MALDI-TOF MS, leading to the identification of four classes of plasma proteins cleaved by EV: proteases, protease inhibitors, binding proteins, and transporters. EV mainly proteolyzes entire proteins but can also act on physiological fragments. In summary, the physiological effects of EV proteases involve other important processes in addition to blood coagulation; complement activation and hemoglobin metabolism are also affected. In particular, the cleavage of protease inhibitors appears to be the mechanism through which the venom neutralizes the body's defenses.

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study.

We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.

Kappa- opioid receptor regulates human sperm functions via SPANX-A/D protein family.

The kappa-opioid receptor (KOR) is involved in the regulation of the fertilizing capacity of human sperm. Recently, a testicular-specific protein family, SPANX-A/D, has also been found to be involved in regulating this process. In order to determine if KOR has a role in the regulation of sperm fertility through the SPANX-A/D protein family, we activated the kappa opioid receptor adding its selective agonist, U50488H to normozoospermic human spermatozoa. Then, we performed immunofluorescence assays and immunoprecipitation experiments followed by LC-MS/MS. According to our results, KOR activation may cause the translocation of SPANX-A/D into the nucleus of human spermatozoa. Phosphoproteomic studies show that KOR does not cause phosphorylation changes in SPANX-A/D residues. However, interactome assays demonstrate that KOR activation provokes changes in SPANX-A/D potential interactors involved in sperm motility, energy metabolism and nuclear processes. Taking these results into account, KOR may regulate human sperm fertility through SPANX-A/D protein family, modifying its subcellular location and interactions. Although further studies are needed, this finding could help us describing the molecular mechanisms underlying sperm fertility as well as developing new strategies for treating infertility.

SPANX-A/D protein subfamily plays a key role in nuclear organisation, metabolism and flagellar motility of human spermatozoa.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. Present only in hominids, SPANX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. Combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SPANX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SPANX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SPANX-A/D family members, therefore, might be promising targets for sperm fertility management.

Multi-omics characterization of a diet-induced obese model of non-alcoholic steatohepatitis.

To improve the understanding of the complex biological processes underlying the development of non-alcoholic steatohepatitis (NASH), a multi-omics approach combining bulk RNA-sequencing based transcriptomics, quantitative proteomics and single-cell RNA-sequencing was used to characterize tissue biopsies from histologically validated diet-induced obese (DIO) NASH mice compared to chow-fed controls. Bulk RNA-sequencing and proteomics showed a clear distinction between phenotypes and a good correspondence between mRNA and protein level regulations, apart from specific regulatory events discovered by each technology. Transcriptomics-based gene set enrichment analysis revealed changes associated with key clinical manifestations of NASH, including impaired lipid metabolism, increased extracellular matrix formation/remodeling and pro-inflammatory responses, whereas proteomics-based gene set enrichment analysis pinpointed metabolic pathway perturbations. Integration with single-cell RNA-sequencing data identified key regulated cell types involved in development of NASH demonstrating the cellular heterogeneity and complexity of NASH pathogenesis.

The proteome speciation of an immortalized cystic fibrosis cell line: New perspectives on the pathophysiology of the disease.

Cystic Fibrosis (CF) is a recessively inherited disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR has a pivotal role in the onset of CF, and several proteins are involved in its homeostasis. To study CFTR interactors at protein species level, we used a functional proteomics approach combining 2D-DIGE, mass spectrometry and enrichment analysis. A human bronchial epithelial cell line with cystic fibrosis (CFBE41o-) and the control (16HBE14o-) were used for the comparison. 73 differentially abundant spots were identified and some validated by western-blot. Enrichment analysis highlighted molecular pathways in which ezrin, HSP70, endoplasmin and lamin A/C, in addition to CFTR, were considered central hubs in CFTR homeostasis. These proteins acquire different functions through post-translational modifications, emphasizing the importance of studying the CF proteome at protein species level. Moreover, serpin H1, prelamin A/C, protein-SET and cystatin-B were associated to CF, demonstrating the importance of heat shock response, cross-talk between the cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating in the pathophysiology of the disease. These results open new perspectives for the understanding of the proteostasis network, characteristic of CF pathology, and could provide a springboard for new therapeutic strategies. BIOLOGICAL SIGNIFICANCE: Homeostasis of CFTR is a dynamic process managed by multiple proteostatic pathways. The used gel-based proteomic approach and enrichment analysis pointed out protein species variations among Human Bronchial (16HBE14o-) and Cystic Fibrosis Bronchial Epithelial cell lines (CFBE41o-) and specific molecular mechanisms involved in CF. In particular, we have highlighted HSP70 (HSP7C), HSP90 (endoplasmin), ERM proteins (ezrin), and lamin-A/C as central hubs of the functional analysis. Moreover, for the first time we consider serpin H1, lamin A/C, protein-SET and cystatin-B important player in CF, affecting acute exacerbation, cytoskeleton reorganization, CFTR gating and chronic inflammation in CF. Due to the presence of different spots corresponding to the same protein, we focalize our attention on the idea that a "protein species discourse" is mandatory to well-define functional roles of proteins. Our approach has permitted to pay attention to the molecular mechanisms which regulate pathways directly or indirectly involved with CFTR defects: heat shock response, cross-talk between cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating. Our data could open new perspectives into the understanding of CF, identifying potential targets for drug treatments in order to alleviate Δ508CFTR membrane instability and consequently increase life expectancy for CF patients.