The role of phosphorylation in calmodulin-mediated gating of human AQP0.

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes.

Structural and functional analysis of aquaporin-2 mutants involved in nephrogenic diabetes insipidus.

Aquaporins are water channels found in the cell membrane, where they allow the passage of water molecules in and out of the cells. In the kidney collecting duct, arginine vasopressin-dependent trafficking of aquaporin-2 (AQP2) fine-tunes reabsorption of water from pre-urine, allowing precise regulation of the final urine volume. Point mutations in the gene for AQP2 may disturb this process and lead to nephrogenic diabetes insipidus (NDI), whereby patients void large volumes of highly hypo-osmotic urine.

Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup.

Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux.

Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST).

Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST).

High-yield overproduction and purification of human aquaporins from .

Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast and subsequent AQP purification. The protocol typically yields 1-5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into , although expression levels may vary.

Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells.

Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner.

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers.

Moonlighting of Haemophilus influenzae heme acquisition systems contributes to the host airway-pathogen interplay in a coordinated manner.

Nutrient iron sequestration is the most significant form of nutritional immunity and causes bacterial pathogens to evolve strategies of host iron scavenging. Cigarette smoking contains iron particulates altering lung and systemic iron homeostasis, which may enhance colonization in the lungs of patients suffering chronic obstructive pulmonary disease (COPD) by opportunistic pathogens such as nontypeable. NTHi is a heme auxotroph, and the NTHi genome contains multiple heme acquisition systems whose role in pulmonary infection requires a global understanding.

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f.

Haemophilus influenzae Type f Hijacks Vitronectin Using Protein H To Resist Host Innate Immunity and Adhere to Pulmonary Epithelial Cells.

The incidence of invasive Haemophilus influenzae type b (Hib) disease has significantly decreased since the introduction of an efficient vaccine against Hib. However, in contrast to Hib, infections caused by H. influenzae serotype f (Hif) are emerging.

Moraxella catarrhalis Binds Plasminogen To Evade Host Innate Immunity.

Several bacterial species recruit the complement regulators C4b-binding protein, factor H, and vitronectin, resulting in resistance against the bactericidal activity of human serum. It was recently demonstrated that bacteria also bind plasminogen, which is converted to plasmin that degrades C3b and C5. In this study, we found that a series of clinical isolates (n = 58) of the respiratory pathogen Moraxella catarrhalis, which is commonly isolated from preschool children and adults with chronic obstructive pulmonary disease (COPD), significantly binds human plasminogen.

Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

Background: Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P.

Haemophilus influenzae surface fibril (Hsf) is a unique twisted hairpin-like trimeric autotransporter.

The Haemophilus surface fibril (Hsf) is an extraordinary large (2413 amino acids) trimeric autotransporter, present in all encapsulated Haemophilus influenzae. It contributes to virulence by directly functioning as an adhesin. Furthermore, Hsf recruits the host factor vitronectin thereby inhibiting the host innate immune response resulting in enhanced survival in serum.

Haemophilus influenzae stores and distributes hemin by using protein E.

The human pathogen Haemophilus influenzae causes mainly respiratory tract infections such as acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease. We recently revealed the crystal structure of H. influenzeae protein E (PE), a multifunctional adhesin that is involved in direct interactions with lung epithelial cells and host proteins.

A fine-tuned interaction between trimeric autotransporter haemophilus surface fibrils and vitronectin leads to serum resistance and adherence to respiratory epithelial cells.

Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin, which inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We reported previously that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in the inhibition of MAC formation and the invasion of lung epithelial cells.

The unique structure of Haemophilus influenzae protein E reveals multiple binding sites for host factors.

Haemophilus influenzae protein E (PE) is a multifunctional adhesin involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data at 1.8 Å.

Crystallization and X-ray diffraction analysis of a novel surface-adhesin protein: protein E from Haemophilus influenzae.

Protein E (PE) is a ubiquitous multifunctional surface protein of Haemophilus spp. and other bacterial pathogens of the Pasteurellaceae family. H.