Cystic fibrosis improves COVID-19 survival and provides clues for treatment of SARS-CoV-2.

Systemic pools of ATP are elevated in individuals homozygous for cystic fibrosis (CF) as evidenced by elevated blood and plasma ATP levels. This elevated ATP level seems to provide benefit in the presence of advanced solid tumors (Abraham et al., Nature Medicine 2(5):593-596, 1996).

Interactions between the transmembrane domains of CD39: identification of interacting residues by yeast selection.

Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, is anchored to the membrane by two transmembrane domains at the two ends of the molecule. The transmembrane domains are important for enzymatic activity, as mutants lacking one or both of these domains have a fraction of the enzymatic activity of the wild-type CD39. We investigated the interactions between the transmembrane domains by using a strain of yeast that requires surface expression of CD39 for growth.

Glycans pattern the phase behaviour of lipid membranes.

Hydrated networks of glycans (polysaccharides)--in the form of cell walls, periplasms or gel-like matrices--are ubiquitously present adjacent to cellular plasma membranes. Yet, despite their abundance, the function of glycans in the extracellular milieu is largely unknown. Here we show that the spatial configuration of glycans controls the phase behaviour of multiphase model lipid membranes: inhomogeneous glycan networks stabilize large lipid domains at the characteristic length scale of the network, whereas homogeneous networks suppress macroscopic lipid phase separation.

Purification of membrane proteins.

Membrane proteins are pivotal players in biological processes. In order to understand how a membrane protein works, it is important to purify the protein to fully characterize it. Membrane proteins are difficult to purify because they are present in low levels and they require detergents to become soluble in an aqueous solution.

Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39.

CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.

CD39, NTPDase 1, is attached to the plasma membrane by two transmembrane domains. Why?

Since the identification of CD39 and other members of the e-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) family as the primary enzymes responsible for cell surface nucleotide hydrolysis, one of their most intriguing features has been their unusual topology. The active site lies in the large extracellular region, but instead of being anchored in the membrane by a single transmembrane domain or lipid link like other ectoenzymes, CD39 has two transmembrane domains, one at each end. In this review we discuss evidence that the structure and dynamics of the transmembrane helices are intricately connected to enzymatic function.

Bilayer mechanical properties regulate the transmembrane helix mobility and enzymatic state of CD39.

CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In this study, we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics.

A eukaryotic carboxyl-terminal signal sequence translocating large hydrophilic domains across membranes.

Yeast Golgi ecto-ATPase Ynd1p is an unusual type III membrane protein with the longest translocated N-terminus reported. Sequential deletion analysis reveals that translocation of this 500-residue-long hydrophilic domain across the membranes requires the C-terminal transmembrane domain of Ynd1p and its flanking regions. Additional studies indicate that the topogenic sequence of Ynd1p overrides the effect of a reverse signal-anchor sequence present at the N-terminus of Ynd1p, while it is not affected by a classic signal sequence at the N-terminus.

Distinctive roles of endoplasmic reticulum and golgi glycosylation in functional surface expression of mammalian E-NTPDase1, CD39.

CD39 is a membrane-bound ecto-nucleoside triphosphate diphosphohydrolase that is involved in the regulation of purinergic signaling. It has been previously reported that N-linked glycosylation is essential for the surface localization of CD39 and for its cellular activity. Here we have addressed the roles of different stages of N-linked glycosylation on CD39's activity and surface expression by using various glycosylation inhibitors, glycosylation deficient CHO cells, and oligosaccharide removal enzymes.

Amino acid signaling through the mammalian target of rapamycin (mTOR) pathway: Role of glutamine and of cell shrinkage.

Mammalian target of rapamycin (mTOR) mediates a signaling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth rate, participating to a versatile checkpoint that inspects the energy status of the cell. The pathway is activated by branched-chain amino acids (BCAA), leucine being the most effective, whereas amino acid dearth and ATP shortage lead to its deactivation. Glutamine- or amino acid-deprivation and hyperosmotic stress induce a fast cell shrinkage (with marked decrease of the intracellular water volume) associated to mTOR-dependent S6K1 dephosphorylation.

Dynamic motions of CD39 transmembrane domains regulate and are regulated by the enzymatic active site.

The two transmembrane domains flanking the active site of CD39 regulate its activity, but little is known about the structural and dynamic features underlying their importance. Here we use a disulfide crosslinking strategy to examine transmembrane helix interactions and dynamics and to correlate these features with activity and substrate binding. We find strong intrasubunit TM1-TM2 interactions, as well as TM1-TM1' and TM2-TM2' interactions between dimer subunits, near the extracellular side of the membrane but only weak interactions near the cytoplasmic end.

Proreceptor dimerization is required for insulin receptor post-translational processing.

The insulin receptor is a transmembrane protein dimer composed of two alphabeta monomers held together by inter-alpha-chain disulfide bonds. In a previous report we described a monomeric insulin receptor obtained by replacing Cys-524, -682, -683, and -685 with serine. The membrane-bound monomeric insulin receptors could be cross-linked to dimers in the presence of insulin, indicating that although covalent interactions had been abolished, noncovalent dimerization could still occur in the membrane.

ATP uptake in the Golgi and extracellular release require Mcd4 protein and the vacuolar H+-ATPase.

Extracellular nucleotides signal via a large group of purinergic receptors. Although much is known about these receptors, the mechanism of nucleotide transport out of the cytoplasm is unknown. We developed a functional screen for ATP release to the extracellular space and identified Mcd4p, a 919-amino acid membrane protein with 14 putative transmembrane domains, as a participant in glucose-dependent ATP release from Saccharomyces cerevisiae.

Construction and characterization of a monomeric insulin receptor.

A mutant insulin receptor was constructed by replacing cysteine residues Cys(524), Cys(682), Cys(683), and Cys(685) with serine. The mutant was expressed in COS7 and Chinese hamster ovary cells, did not form covalently linked dimers, and was present at the cell surface. There was half as much insulin binding activity at the cell surface in cells expressing the mutant compared with that in cells expressing the wild type receptor.

Transmembrane domains confer different substrate specificities and adenosine diphosphate hydrolysis mechanisms on CD39, CD39L1, and chimeras.

Members of the ecto-nucleoside triphosphate diphosphohydrolase (eNTPDase) family exhibit distinctive substrate specificities, but how such specificities are achieved by enzymes with identical putative catalytic domains is unknown. Previously we showed that H59G substitution changes CD39 from an apyrase to an adenosine diphosphatase (ADPase) in a manner that depends on intact associations of both transmembrane domains with the membrane. Here we show that the extracellular domain of CD39L1 ecto-adenosine triphosphatase (ecto-ATPase) has the same 3:1 ATP:ADP hydrolysis ratio as the extracellular domain of CD39, suggesting that the transmembrane domains are required to confer the native substrate specificities on each enzyme.

Glucose-dependent, cAMP-mediated ATP efflux from Saccharomyces cerevisiae.

Extracellular ATP plays an important role in the physiology of multicellular organisms; however, it is unknown whether unicellular organisms such as yeast also release ATP extracellularly. Experiments are described here which show that Saccharomyces cerevisiae releases ATP to the extracellular fluid. This efflux required glucose and the rate was increased dramatically by the proton ionophores nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; ATP efflux was also increased by the plasma membrane proton pump inhibitor diethylstilbestrol.