A small molecule chaperone rescues keratin-8 mediated trafficking of misfolded podocin to correct genetic Nephrotic Syndrome.

Podocin is a key membrane scaffolding protein of the kidney podocyte essential for intact glomerular filtration. Mutations in NPHS2, the podocin-encoding gene, represent the commonest form of inherited nephrotic syndrome (NS), with early, intractable kidney failure. The most frequent podocin gene mutation in European children is R138Q, causing retention of the misfolded protein in the endoplasmic reticulum. Here, we provide evidence that podocin R138Q (but not wild-type podocin) complexes with the intermediate filament protein keratin 8 (K8) thereby preventing its correct trafficking to the plasma membrane. We have also identified a small molecule (c407), a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator protein defect, that interrupts this complex and rescues mutant protein mistrafficking. This results in both the correct localization of podocin at the plasma membrane and functional rescue in both human patient R138Q mutant podocyte cell lines, and in a mouse inducible knock-in model of the R138Q mutation. Importantly, complete rescue of proteinuria and histological changes was seen when c407 was administered both via osmotic minipumps or delivered orally prior to induction of disease or crucially via osmotic minipump two weeks after disease induction. Thus, our data constitute a therapeutic option for patients with NS bearing a podocin mutation, with implications for other misfolding protein disorders. Further studies are necessary to confirm our findings.

Theratyping cystic fibrosis patients to guide elexacaftor/tezacaftor/ivacaftor out-of-label prescription.

BACKGROUND: Around 20% of people with cystic fibrosis (pwCF) do not have access to the triple combination elexacaftor/tezacaftor/ivacaftor (ETI) in Europe because they do not carry the F508del allele on the CF transmembrane conductance regulator (CFTR) gene. Considering that pwCF carrying rare variants may benefit from ETI, including variants already validated by the US Food and Drug Administration (FDA), a compassionate use programme was launched in France. PwCF were invited to undergo a nasal brushing to investigate whether the pharmacological rescue of CFTR activity by ETI in human nasal epithelial cell (HNEC) cultures was predictive of the clinical response. METHODS: CFTR activity correction was studied by short-circuit current in HNEC cultures at basal state (dimethyl sulfoxide (DMSO)) and after ETI incubation and expressed as percentage of normal (wild-type (WT)) CFTR activity after sequential addition of forskolin and Inh-172 (ΔI ETI/DMSO%WT). RESULTS: 11 pwCF carried variants eligible for ETI according to the FDA label and 28 carried variants not listed by the FDA. ETI significantly increased CFTR activity of FDA-approved CFTR variants (I601F, G85E, S492F, M1101K, R347P, R74W;V201M;D1270N and H1085R). We point out ETI correction of non-FDA-approved variants, including N1303K, R334W, R1066C, Q552P and terminal splicing variants (4374+1G>A and 4096-3C>G). ΔI ETI/DMSO%WT was significantly correlated to change in percentage predicted forced expiratory volume in 1 s and sweat chloride concentration (p<0.0001 for both). G85E, R74W;V201M;D1270N, Q552P and M1101K were rescued more efficiently by other CFTR modulator combinations than ETI. CONCLUSIONS: Primary nasal epithelial cells hold promise for expanding the prescription of CFTR modulators in pwCF carrying rare mutants. Additional variants should be discussed for ETI indication.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

Correlating genotype with phenotype using CFTR-mediated whole-cell Cl- currents in human nasal epithelial cells.

Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR-related diseases (CFTR-RDs). Here, we investigate genotype-phenotype-CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non-CF subjects and individuals with CF and CFTR-RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole-cell patch-clamp technique or grown as primary cultures at an air-liquid interface after conditional reprogramming. The properties of cAMP-activated whole-cell Cl- currents in freshly isolated hNE cells identified them as CFTR-mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non-CF > CFTR residual function > CFTR minimal function. CFTR-mediated whole-cell Cl- currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR-mediated whole-cell Cl- currents in hNE cells correlated well with CFTR-mediated transepithelial Cl- currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl- concentration of study subjects. Thus, this study highlights the complexity of genotype-phenotype-CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. KEY POINTS: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body. This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity. CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient-derived cells. Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype-phenotype-CFTR function relationships.

Pharmacological chaperones improve intra-domain stability and inter-domain assembly via distinct binding sites to rescue misfolded CFTR.

Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein-lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules.

Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters.

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters.

Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.

Urinary Exosomes of Patients with Cystic Fibrosis Unravel CFTR-Related Renal Disease.

Background: The prevalence of chronic kidney disease is increased in patients with cystic fibrosis (CF). The study of urinary exosomal proteins might provide insight into the pathophysiology of CF kidney disease. Methods: Urine samples were collected from 19 CF patients (among those 7 were treated by cystic fibrosis transmembrane conductance regulator (CFTR) modulators), and 8 healthy subjects. Urine exosomal protein content was determined by high resolution mass spectrometry. Results: A heatmap of the differentially expressed proteins in urinary exosomes showed a clear separation between control and CF patients. Seventeen proteins were upregulated in CF patients (including epidermal growth factor receptor (EGFR); proteasome subunit beta type-6, transglutaminases, caspase 14) and 118 were downregulated (including glutathione S-transferases, superoxide dismutase, klotho, endosomal sorting complex required for transport, and matrisome proteins). Gene set enrichment analysis revealed 20 gene sets upregulated and 74 downregulated. Treatment with CFTR modulators yielded no significant modification of the proteomic content. These results highlight that CF kidney cells adapt to the CFTR defect by upregulating proteasome activity and that autophagy and endosomal targeting are impaired. Increased expression of EGFR and decreased expression of klotho and matrisome might play a central role in this CF kidney signature by inducing oxidation, inflammation, accelerated senescence, and abnormal tissue repair. Conclusions: Our study unravels novel insights into consequences of CFTR dysfunction in the urinary tract, some of which may have clinical and therapeutic implications.

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

Cis variants identified in F508del complex alleles modulate CFTR channel rescue by small molecules.

Molecules correcting the trafficking (correctors) and gating defects (potentiators) of the cystic fibrosis causing mutation c.1521_1523delCTT (p.Phe508del) begin to be a useful treatment for CF patients bearing p.Phe508del. This mutation has been identified in different genetic contexts, alone or in combination with variants in cis. Until now, 21 exonic variants in cis of p.Phe508del have been identified, albeit at a low frequency. The aim of this study was to evaluate their impact on the efficacy of CFTR-directed corrector/potentiator therapy (Orkambi). The analysis by minigene showed that two out of 15 cis variants tested increased exon skipping (c.609C > T and c.2770G > A). Four cis variants were studied functionally in the absence of p.Phe508del, one of which was found to be deleterious for protein maturation c.1399C > T (p.Leu467Phe). In the presence of p.Phe508del, this variant was the only to prevent the response to Orkambi treatment. This study showed that some patients carrying p.Phe508del complex alleles are predicted to poorly respond to corrector/potentiator treatments. Our results underline the importance to validate treatment efficacy in the context of complex alleles.

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-ß-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-ß-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-ß-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis.

Pseudomonas aeruginosa LasB protease impairs innate immunity in mice and humans by targeting a lung epithelial cystic fibrosis transmembrane regulator-IL-6-antimicrobial-repair pathway.

BACKGROUND: Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity. OBJECTIVE: We evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo. METHODS: Wild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models. RESULTS: We showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways. CONCLUSIONS: Our data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals.

rAAV-CFTRΔR Rescues the Cystic Fibrosis Phenotype in Human Intestinal Organoids and Cystic Fibrosis Mice.

RATIONALE: Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients with cystic fibrosis (CF). The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit. Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)-based gene therapy encouraged us to reexplore an rAAV approach for CF. OBJECTIVES: We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy. METHODS: We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRΔR) combined with a short promoter (CMV173) to ensure optimal gene expression. A rescue in chloride and fluid secretion after rAAV-CFTRΔR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in ΔF508 mice. MEASUREMENTS AND MAIN RESULTS: rAAV-CFTRΔR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function. Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRΔR-treated CF mice. CONCLUSIONS: Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.

Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP).

Efficient protein solubilization using detergents is required for in-depth proteome analysis, but successful LC-MS/MS analysis greatly depends on proper detergents removal. A commonly used sample processing method is the filter-aided sample preparation (FASP), which allows protein digestion and detergent removal on the same filtration device. Many optimizations of the FASP protocol have been published, but there is no information on the influence of the filtration unit typology on the detergents removal. The aim of this study was to compare the performance of conic and flat bottom filtration units in terms of number of proteins identified by LC-MS/MS. We have analyzed 1, 10 and 100 µg of total cell lysate prepared using lysis buffer with different SDS concentrations. We compared the FASP protocol using conic and flat bottom filtration units to ethanol precipitation method. Subsequently, we applied our most performant protocol to single murine pancreatic islet, and identified up to 2463 protein using FASP versus 1169 proteins using ethanol precipitation. We conclude that FASP performance depends strongly on the filter shape: flat bottom devices are better suited for low-protein samples, as they allow better SDS removal leading to the identification of greater number of proteins.

A new workflow for proteomic analysis of urinary exosomes and assessment in cystinuria patients.

Cystinuria is a purely renal, rare genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases, patients undergo nephrectomy, but given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow-up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study conducted on eight cystinuria patients versus 10 controls highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins, and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking the potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinary exosomes in different renal diseases and to search for diagnostic/prognostic markers. Data are available via ProteomeXchange with identifier PXD001430.

Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen-deuterium exchange.

Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.

Regulation of interleukin-6 in head and neck squamous cell carcinoma is related to papillomavirus infection.

The prevalence of head and neck squamous cell carcinoma (HNSCC) related to human papillomavirus (HPV) is increasing, unlike tobacco- and alcohol-associated cancers. To gain a clearer understanding of the molecular mechanisms implicated in HNSCC, depending on the presence or not of a viral sequence, we investigated the expression of proteins detected in the tumor regions of HNSCC patients. Twenty-two untreated HNSCC patients were selected according to the presence of HPV-16. For six patients, tumor and controlateral healthy tissues were tested for viral detection before quantitative proteomic analysis. After confirmation by Western blot, proteins were connected into a network, leading to investigate interleukin-6 (IL-6) by immunocytochemistry and ELISA. 41 ± 5% of proteins quantified by proteomics were differentially expressed in tumor compared with healthy regions. Among them, 36 proteins were retained as modulated in HPV-16 positive or negative tumors, including cytokeratins, tubulins, annexin A1, and serpin B1. Network analysis suggested a central role of IL-6, confirmed by overexpression of IL-6 in tumor tissues as in sera of HPV-negative HNSCC compared with HPV-16-positive tumors. This modulation may contribute to the survival and proliferation of cancer cells, although it was not related to tumor stage or to the level of HPV-16 DNA.