Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein.

CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.

Simple binding of protein kinase A prior to phosphorylation allows CFTR anion channels to be opened by nucleotides.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.

Selective profiling of N- and C-terminal nucleotide-binding sites in a TRPM2 channel.

Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.

A novel approach for local abdominal aortic aneurysm growth quantification.

Although aneurysm size still remains the most accepted predictor of rupture risk, abdominal aortic aneurysms (AAAs) with maximum diameter smaller than 5 cm may also rupture. Growth rate is an additional marker for rupture risk as it potentially reflects an undesirable wall remodeling that leads to fast regional growth. Currently, an indication for surgery is an expansion rate >10 mm/year, measured as change in maximum diameter over time. However, as AAA expansion is non-uniform, it is questionable whether measurement of maximum diameter change over time can capture increased localized remodeling activity. A method for estimating AAA surface area growth is introduced, providing a better measure of local wall deformation. The proposed approach is based on the non-rigid iterative closest point algorithm. Optimization and validation is performed using 12 patient-specific AAA geometries artificially deformed to produce a target surface with known nodal displacements. Mesh density sensitivity, range of uncertainty, and method limitations are discussed. Application to ten AAA patient-specific follow-ups suggested that maximum diameter growth does not correlate strongly with the maximum surface growth (R 2 = 0.614), which is not always colocated with maximum diameter, or uniformly distributed. Surface growth quantification could reinforce the quality of aneurysm surveillance programs.

The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 µM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies.

Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage.

Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.

Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2'-phosphate.

Transient receptor potential melastatin 2 (TRPM2), a Ca(2+)-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid-adenine-dinucleotide (NAAD), and NAAD-2'-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified-specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2'-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest.

The transmembrane protein KpOmpA anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load.

In Klebsiella pneumoniae the transmembrane ß-barrel forming outer membrane protein KpOmpA mediates adhesion to a wide range of immune effector cells, thereby promoting respiratory tract and urinary infections. As major transmembrane protein OmpA stabilizes Gram-negative bacteria by anchoring their outer membrane to the peptidoglycan layer. Adhesion, osmotic pressure, hydrodynamic flow, and structural deformation apply mechanical stress to the bacterium. This stress can generate tensile load to the peptidoglycan-binding domain (PGBD) of KpOmpA. To investigate how KpOmpA reacts to mechanical stress, we applied a tensile load to the PGBD and observed a detailed unfolding pathway of the transmembrane ß-barrel. Each step of the unfolding pathway extended the polypeptide connecting the bacterial outer membrane to the peptidoglycan layer and absorbed mechanical energy. After relieving the tensile load, KpOmpA reversibly refolded back into the membrane. These results suggest that bacteria may reversibly unfold transmembrane proteins in response to mechanical stress.

Electrodelivery of drugs into cancer cells in the presence of poloxamer 188.

In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.

Cytotoxic activity of platinum(II) and palladium(II) complexes of N-3-pyridinylmethanesulfonamide: the influence of electroporation.

The series of complexes: cis-[Pd(PMSA)2X2], cis-[Pt(PMSA)2X2], trans-[Pt(PMSA)2I2] and [Pt(PMSA)4]Cl2 (PMSA = N-3-pyridinylmethanesulfonamide; X = Cl, Br, I), previously synthesized and characterized by us, as well as the free ligand PMSA, were tested for their cytotoxic activity without electroporation -- against murine leukemia F4N and human SKW-3 and MDA-MB-231 tumour cell lines -- and with electroporation -- against the latter two cell lines. The majority of the complexes exhibited cytotoxic effects (IC50 < 100 micromol/l) under the conditions of electroporation. Both cis- and trans-[Pt(PMSA)2I2] had pronounced cytotoxic effects (29-61 micromol/l against MDA-MB-231 cells).