The HERCulean task of recognizing, ubiquitinating, and shielding misfolded integral membrane proteins.

During ER-associated decay, unfolded membrane-resident proteins are targeted for removal and degradation by ubiquitin ligases whose identities and precise operations remain unclear. In this issue, Guerriero and Brodsky discuss new results from Kamada et al. (https://doi.org/10.1083/jcb.202308003) showing the clearance of misfolded CFTR by the E3 ligase HERC3.

Heavy metals in cigarette smoke strongly inhibit pancreatic ductal function and promote development of chronic pancreatitis.

BACKGROUND AND AIMS: Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP. METHODS: We measured sweat chloride (Cl-) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3 - and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis. RESULTS: Sweat samples from smokers, both with and without CP, exhibited elevated Cl- concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3 - secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation. CONCLUSION: Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking. KEY POINTS: Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3 - secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers.

In vivo editing of lung stem cells for durable gene correction in mice.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Gene editing flows to the lungs.

Intravenous targeting of airway basal cells for cystic fibrosis gene therapy overcomes lung barriers.

Trivalent anions as probes of the CFTR channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel uses positively charged amino-acid side-chains to form binding sites for permeating anions. These binding sites have been investigated experimentally using a number of anionic probes. Mutations that alter the distribution of positive and negative charges within the pore have differential effects on the binding of monovalent versus divalent anions. This study uses patch clamp recording from wild-type and pore-mutant forms of CFTR to investigate small trivalent anions (Co(NO2)63-, Co(CN)3- and IrCl63-) as potential probes of anion binding sites. These anions caused weak block of Cl- permeation in wild-type CFTR (Kd ≥ 700 µM) when applied to the intracellular side of the membrane. Mutations that increase the density of positive charge within the pore (E92Q, I344K, S1141K) increased the binding affinity of these anions 80-280-fold, and also greatly increased the voltage-dependence of block, consistent with fixed charges in the pore affecting monovalent : multivalent anion selectivity. However, high-affinity pore block by Co(NO2)63-apparently did not alter channel gating, a hallmark of high-affinity binding of divalent Pt(NO2)42- ions within the pore. This work increases the arsenal of probes available to investigate anion binding sites within Cl- channel pores.

Impact of lumacaftor/ivacaftor on the bacterial and fungal respiratory pathogens in cystic fibrosis: a prospective multicenter cohort study in Sweden.

BACKGROUND: A significant decline in pulmonary exacerbation rates has been reported in CF patients homozygous for F508del treated with lumacaftor/ivacaftor. However, it is still unclear whether this reduction reflects a diminished microbiological burden. OBJECTIVES: The aim of this study was to determine the impact of lumacaftor/ivacaftor on the bacterial and fungal burden. DESIGN: The study is a prospective multicenter cohort study including 132 CF patients homozygous for F508del treated with lumacaftor/ivacaftor. METHODS: Clinical parameters as well as bacterial and fungal outcomes 1 year after initiation of lumacaftor/ivacaftor were compared to data from 2 years prior to initiation of the treatment. Changes in the slope of the outcomes before and after the onset of treatment were assessed. RESULTS: Lung function measured as ppFEV1 (p < 0.001), body mass index (BMI) in adults (p < 0.001), and BMI z-score in children (p = 0.007) were improved after initiation of lumacaftor/ivacaftor. In addition, the slope of the prevalence of Streptococcus pneumoniae (p = 0.007) and Stenotrophomonas maltophilia (p < 0.001) shifted from positive to negative, that is, became less prevalent, 1 year after treatment, while the slope for Candida albicans (p = 0.009), Penicillium spp (p = 0.026), and Scedosporium apiospermum (p < 0.001) shifted from negative to positive. CONCLUSION: The current study showed a significant improvement in clinical parameters and a reduction of some of CF respiratory microorganisms 1 year after starting with lumacaftor/ivacaftor. However, no significant changes were observed for Pseudomonas aeruginosa, Staphylococcus aureus, or Aspergillus fumigatus, key pathogens in the CF context.

Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids").

Unmet challenges in cystic fibrosis treatment with modulators.

INTRODUCTION: 'Highly effective' modulator therapies (HEMTs) have radically changed the Cystic Fibrosis (CF) therapeutic landscape. AREAS COVERED: A comprehensive search strategy was undertaken to assess impact of HEMT in life of pwCF, treatment challenges in specific populations such as very young children, and current knowledge gaps. EXPERT OPINION: HEMTs are prescribed for pwCF with definite genotypes. The heterogeneity of variants complicates treatment possibilities and around 10% of pwCF worldwide remains ineligible. Genotype-specific treatments are prompting theratyping and personalized medicine strategies. Improvement in lung function and quality of life increase survival rates, shifting CF from a pediatric to an adult disease. This implies new studies addressing long-term efficacy, side effects, emergence of adult co-morbidities and possible drug-drug interactions. More sensitive and predictive biomarkers for both efficacy and toxicity are warranted. As HEMTs cross the placenta and are found in breast milk, studies addressing the potential consequences of treatment during pregnancy and breastfeeding are urgently needed. Finally, although the treatment and expected outcomes of CF have improved dramatically in high- and middle-income countries, lack of access in low-income countries to these life-changing medicines highlights inequity of care worldwide.

Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

[A systemic gene therapy for the treatment of cystic fibrosis]. / Une thérapie génique systémique dans le traitement de la mucoviscidose.

Title: Une thérapie génique systémique dans le traitement de la mucoviscidose. Abstract: Le master I2VB se propose de donner les bases conceptuelles et pratiques des différents aspects de l'infectiologie. Il s'appuie sur une coopération exemplaire entre les équipes de recherche en infectiologie et en immunologie de l'Université de Tours, et celles, entre autres, de l'Unité Infectiologie et Santé Publique (ISP) du Centre INRAE de Tours-Nouzilly, concrétisée par une profonde interaction entre chercheurs et enseignants-chercheurs. Cette formation aborde aussi bien les aspects fondamentaux et appliqués de l'infectiologie et de l'immunologie allant de l'étude moléculaire des interactions entre le pathogène et son hôte, jusqu'à la conception et la mise sur le marché des produits de la vaccinologie, des biothérapies anti-infectieuses et des anticorps immuno-thérapeutiques. Le master I2VB (niveau M1) donne lieu aux parcours ICM, I&B et AcT (niveau M2). L'option Infectiologie Cellulaire et Moléculaire (ICM) (responsables : Françoise Debierre-Grockiego et Martine Braibant) a pour objectifs de : former des scientifiques dotés d'une culture générale et technique spécialisée dans les biotechnologies, l'infectiologie, les interactions hôte-pathogène et les mécanismes de la réponse immunitaire anti-infectieuse, contribuant à l'avancée des connaissances scientifiques et à ses applications industrielles, demandes sociétales en forte progression. former des pharmaciens, médecins, vétérinaires, ingénieurs agronomes aux enjeux actuels de l'infectiologie à la fois dans les domaines fondamentaux et appliqués. L'option Immunité et biomédicaments (I&B) (responsables : Anne di Tommaso et Isabelle Dimier-Poisson) a pour objectifs de : former des scientifiques dotés d'une culture générale et technique spécialisée dans les biotechnologies, l'infectiologie, la vaccinologie, les biomédicaments et les biothérapies anti-infectieuses contribuant à l'avancée des connaissances scientifiques et à ses applications industrielles, demandes sociétales en forte progression. former de jeunes scientifiques, pharmaciens, médecins, vétérinaires, ingénieurs agronomes aux enjeux actuels de l'infectiologie et des biomédicaments à la fois dans les domaines fondamentaux et appliqués. L'option Anticorps thérapeutiques (AcT) (responsables : Laurie Lajoie et Isabelle Dimier-Poisson) a pour objectifs de : former des scientifiques dotés d'une culture générale et technique spécialisée dans les biotechnologies, l'immunologie, la cancérologie et les biomédicaments dont les anticorps thérapeutiques, contribuant à l'avancée des connaissances scientifiques et à ses application industrielles et juridiques, demandes sociétales en forte progression. former de jeunes scientifiques, pharmaciens, médecins, vétérinaires, ingénieurs agronomes aux enjeux actuels de l'infectiologie et des biomédicaments à la fois dans les domaines fondamentaux et appliqués.

Anti-inflammatory effects of elexacaftor/tezacaftor/ivacaftor in adults with cystic fibrosis heterozygous for F508del.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1ß (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1ß and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes.

[Cystic fibrosis in childhood and adulthood]. / Mukoviszidose im Kindes- und im Erwachsenenalter.

BACKGROUND: Cystic fibrosis (CF, or mucoviscidosis) is one of the rare diseases with a fatal course and with the highest prevalence. Formerly known as a purely childhood disease, this multisystemic disease follows an autosomal recessive inheritance pattern and results in a malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to the production of viscous secretions. The prognosis and outcome of CF are determined by the severity of the involvement of the lungs. Other typically affected organs include the pancreas, liver and intestines. OBJECTIVE: This article reviews the clinical presentation and evolution of CF with a focus on the new era of the highly effective CFTR modulator treatment. MATERIAL AND METHODS: An overview of the current state of knowledge on the care for CF patients is presented. RESULTS AND DISCUSSION: The introduction of the CF newborn screening, the increased understanding of the disease and the development of novel treatment options have substantially increased the quality of life and life expectancy of people with CF. As a result, more than half of CF patients in Germany are now older than 18 years of age and the complications of a chronic disease as well as organ damage due to the intensive treatment are gaining in importance. The highly effective CFTR modulator treatment results in a significant improvement in CFTR function, lung function, body mass index and quality of life and is available to approximately 90% of patients in Germany, based on the genotype. Nevertheless, further research including the development of causal treatment, e.g., gene therapy, targeting the underlying defect in the remaining 10% of CF patients, is urgently needed. Even in adult patients, CF with a mild course or a CFTR-related disease should be considered, e.g., in cases of bronchiectasis and/or recurrent abdominal complaints.

Considerations for the use of inhaled antibiotics for Pseudomonas aeruginosa in people with cystic fibrosis receiving CFTR modulator therapy.

The major cause of mortality in people with cystic fibrosis (pwCF) is progressive lung disease characterised by acute and chronic infections, the accumulation of mucus, airway inflammation, structural damage and pulmonary exacerbations. The prevalence of Pseudomonas aeruginosa rises rapidly in the teenage years, and this organism is the most common cause of chronic lung infection in adults with cystic fibrosis (CF). It is associated with an accelerated decline in lung function and premature death. New P. aeruginosa infections are treated with antibiotics to eradicate the organism, while chronic infections require long-term inhaled antibiotic therapy. The prevalence of P. aeruginosa infections has decreased in CF registries since the introduction of CF transmembrane conductance regulator modulators (CFTRm), but clinical observations suggest that chronic P. aeruginosa infections usually persist in patients receiving CFTRm. This indicates that pwCF may still need inhaled antibiotics in the CFTRm era to maintain long-term control of P. aeruginosa infections. Here, we provide an overview of the changing perceptions of P. aeruginosa infection management, including considerations on detection and treatment, the therapy burden associated with inhaled antibiotics and the potential effects of CFTRm on the lung microbiome. We conclude that updated guidance is required on the diagnosis and management of P. aeruginosa infection. In particular, we highlight a need for prospective studies to evaluate the consequences of stopping inhaled antibiotic therapy in pwCF who have chronic P. aeruginosa infection and are receiving CFTRm. This will help inform new guidelines on the use of antibiotics alongside CFTRm.

Long-term therapy with CFTR modulators consistently improves glucose metabolism in adolescents and adults with cystic fibrosis.

BACKGROUND: Impaired glycemic control and the subsequent development of Cystic fibrosis Related Diabetes (CFRD) are prevalent complications, affecting up to 50 % of adults with cystic fibrosis (CF). CFTR modulator (CFTRm) therapies improve pulmonary functions, reduce exacerbation rates, increase survival in people with CF (pwCF) and appear to have a positive effect on extrapulmonary manifestations, such as nutritional state, improvements in upper respiratory symptoms, and quality of life. Initial findings indicate that CFTRm may have a positive impact on short-term glycemic control; however, long-term effects remain uncertain at present. METHODS: In this retrospective study, data were collected and analyzed on 15 pwCF, ages 13-37 years, started on CFTRm therapy. Oral Glucose Tolerance Test (OGTT) results were compared pre- and post-CFTRm therapy. RESULTS: The 120-min OGTT value decreased from 159.7 mg/dL to 130.4 mg/dL post-CFTRm (p = 0.047). The average time elapsed between the two OGTTs was 49.87 months (ranging 9-157 months, median 38 months). Glycemic status improved in six pwCF (two CFRD to normal (NGT)/indeterminate (INDET) glucose tolerance; two impaired glucose tolerance (IGT) to INDET; two INDET to NGT) and worsened in one (IGT to CFRD). Six pwCF and NGT remained stable with no changes in glycemic status throughout the follow-up period. CONCLUSIONS: CFTRm therapy may decelerate the glycemic control deterioration in pwCF over an extended period. These findings indicate the need for periodic OGTTs following the initiation of CFTRm therapy to appropriately adjust insulin requirements and prevent hypoglycemia. Further larger cohorts are required to authenticate and substantiate these findings.

[Cystic fibrosis primarily presenting with pseudo-Bartter syndrome: a report of three cases and literature review]. / 以假性Bartter综合征为主要表现的囊性纤维化患儿3ä¾‹å¹¶æ–‡çŒ®å¤ä¹ .

OBJECTIVES: To summarize the clinical characteristics and genetic variations in children with cystic fibrosis (CF) primarily presenting with pseudo-Bartter syndrome (CF-PBS), with the aim to enhance understanding of this disorder. METHODS: A retrospective analysis was performed on the clinical data of three children who were diagnosed with CF-PBS in Hunan Children's Hospital from January 2018 to August 2023, and a literature review was performed. RESULTS: All three children had the onset of the disease in infancy. Tests after admission showed hyponatremia, hypokalemia, hypochloremia, and metabolic alkalosis, and genetic testing showed the presence of compound heterozygous mutation in the CFTR gene. All three children were diagnosed with CF. Literature review obtained 33 Chinese children with CF-PBS, with an age of onset of 1-36 months and an age of diagnosis of 3-144 months. Among these children, there were 29 children with recurrent respiratory infection or persistent pneumonia (88%), 26 with malnutrition (79%), 23 with developmental retardation (70%), and 18 with pancreatitis or extrapancreatic insufficiency (55%). Genetic testing showed that c.2909G>A was the most common mutation site of the CFTR gene, with a frequency of allelic variation of 23% (15/66). CONCLUSIONS: CF may have no typical respiratory symptoms in the early stage. The possibility of CF-PBS should be considered for infants with recurrent hyponatremia, hypokalemia, hypochloremia, and metabolic alkalosis, especially those with malnutrition and developmental retardation. CFTR genetic testing should be performed as soon as possible to help with the diagnosis of CF.

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.