Understanding and addressing the needs of people with cystic fibrosis in the era of CFTR modulator therapy.

Cystic fibrosis is a multiorgan disease caused by impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR). Since the introduction of the CFTR modulator combination elexacaftor-tezacaftor-ivacaftor (ETI), which acts directly on mutant CFTR to enhance its activity, most people with cystic fibrosis (pwCF) have seen pronounced reductions in symptoms, and studies project marked increases in life expectancy for pwCF who are eligible for ETI. However, modulator therapy has not cured cystic fibrosis and the success of CFTR modulators has resulted in immediate questions about the new state of cystic fibrosis disease and clinical challenges in the care of pwCF.

Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression.

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P.

Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD.

Commentary on Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder.

Significant progress has been made in understanding pseudoxanthoma elasticum (PXE), which results from mutations in ABCC6. The low prevalence of PXE and its heterotypic presentation confound genotype-phenotype correlations and the characterization of many identified variants. Kowal et al.

Serralysin family metalloproteases protects Serratia marcescens from predation by the predatory bacteria Micavibrio aeruginosavorus.

Micavibrio aeruginosavorus is an obligate Gram-negative predatory bacterial species that feeds on other Gram-negative bacteria by attaching to the surface of its prey and feeding on the prey's cellular contents. In this study, Serratia marcescens with defined mutations in genes for extracellular cell structural components and secreted factors were used in predation experiments to identify structures that influence predation. No change was measured in the ability of the predator to prey on S.

Structural analysis reveals pathomechanisms associated with pseudoxanthoma elasticum-causing mutations in the ABCC6 transporter.

Mutations in ABC subfamily C member 6 (ABCC6) transporter are associated with pseudoxanthoma elasticum (PXE), a disease resulting in ectopic mineralization and affecting multiple tissues. A growing number of mutations have been identified in individuals with PXE. For most of these variants, no mechanistic information is available regarding their role in normal and pathophysiologies.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR.

Factors Related to Self-rated Health in Older Adults: A Clinical Approach Using the International Classification of Functioning, Disability, and Health (ICF) Model.

Background And Purpose: A growing population of older adults will require health care professionals to become increasingly knowledgeable in geriatric care. Patient ratings, functional measures, and emphasis on health and wellness should be part of geriatric physical therapy practice. The purpose of the current study was to examine relationships between self-rated health (SRH) and movement-related variables in older adults using the International Classification of Functioning, Disability, and Health (ICF) as a research framework.

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS.

Stabilization of Nucleotide Binding Domain Dimers Rescues ABCC6 Mutants Associated with Pseudoxanthoma Elasticum.

ABC transporters are polytopic membrane proteins that utilize ATP binding and hydrolysis to facilitate transport across biological membranes. Forty-eight human ABC transporters have been identified in the genome, and the majority of these are linked to heritable disease. Mutations in the ABCC6 (ATP binding cassette transporter C6) ABC transporter are associated with pseudoxanthoma elasticum, a disease of altered elastic properties in multiple tissues.

Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR.

Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens.

The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined.

Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions.

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown.

Inducible polymerization and two-dimensional assembly of the repeats-in-toxin (RTX) domain from the Pseudomonas aeruginosa alkaline protease.

Self-assembling proteins represent potential scaffolds for the organization of enzymatic activities. The alkaline protease repeats-in-toxin (RTX) domain from Pseudomonas aeruginosa undergoes multiple structural transitions in the presence and absence of calcium, a native structural cofactor. In the absence of calcium, this domain is capable of spontaneous, ordered polymerization, producing amyloid-like fibrils and large two-dimensional protein sheets.

Modulation of the epithelial sodium channel (ENaC) by bacterial metalloproteases and protease inhibitors.

The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia.

Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence.

Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers.

Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D-CFTR.

Cystic fibrosis (CF) is caused by dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). One strategy to restore function to CF mutants is to suppress defects in CFTR processing and function using revertant mutations. Here, we investigate the effects of the revertant mutations G550E and 4RK (the simultaneous disruption of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K and R766K) on the CF mutant G551D, which impairs severely channel gating without altering protein processing and which affects a residue in the same α-helix as G550 and R555.

Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier-dependent pathway.

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation.

Activation of the epithelial sodium channel (ENaC) by the alkaline protease from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection.

Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease.

Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection.

Molecular modeling tools and approaches for CFTR and cystic fibrosis.

Cystic fibrosis is a multi-faceted disease resulting from the dysfunction of the CFTR channel. Understanding the structural basis of channel function and the structural origin of the defect is imperative in the development of therapeutic strategies. Here, we describe molecular modeling tools that, in conjunction with complementary experimental tools, lead to significant findings on CFTR channel function and on the effect of the pathogenic mutant F508del.

ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes.

The number of intermediate-conductance, Ca(2+)-activated K(+) channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.

The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations.

NMR evidence for differential phosphorylation-dependent interactions in WT and DeltaF508 CFTR.

The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DeltaF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and DeltaF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region.