Molecular features of copper binding proteins involved in copper homeostasis.

Copper has a wide and important role in biological systems, determining conformation and activity of many metalloproteins and enzymes, such as cytochrome oxidase and superoxide dismutase . Furthermore, due to its possible reactivity with nonspecific proteins and toxic effects, elaborate systems of absorption, concentration buffering, delivery to specific protein sites and elimination, require a complex system including small carriers, chaperones and active transporters. The P-type copper ATPases ATP7A and ATP7B provide an important system for acquisition, active transport, distribution and elimination of copper.

Biochemical characterization of P-type copper ATPases.

Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.

Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B.

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down-regulation of CTR1 and up-regulation of the Cu-ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance.

Ca(2+)/H (+) exchange, lumenal Ca(2+) release and Ca (2+)/ATP coupling ratios in the sarcoplasmic reticulum ATPase.

The Ca(2+) transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca(2+) in intracellular membrane bound compartments, thereby lowering cytosolic Ca(2+) to induce relaxation. The stored Ca(2+) is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca(2+), whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca(2+).

Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state.

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca(2+)-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca(2+) pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca(2+)-binding sites ready to accept new cytosolic Ca(2+).

Distinctive features of catalytic and transport mechanisms in mammalian sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases.

Ca(2+) (sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA)) and Cu(+) (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca(2+), demonstrated by the addition of ATP and Ca(2+) to SERCA deprived of Ca(2+) (E2) as compared with ATP to Ca(2+)-activated enzyme (E1·2Ca(2+)). Activation by Ca(2+) is slower at low pH (2H(+)·E2 to E1·2Ca(2+)) and little sensitive to temperature-dependent activation energy.

Analysis of calcium transients in cardiac myocytes and assessment of the sarcoplasmic reticulum Ca2+-ATPase contribution.

Ca(2+) signaling plays an essential role in several functions of cardiac myocytes. Transient rises and reductions of cytosolic Ca(2+), permitted by the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and other proteins, control each cycle of contraction and relaxation. Here we provide a practical method for isolation of neonatal rat cardiac myocytes and measurement of Ca(2+) transients in cultured cardiac myocytes, yielding information on kinetic resolution of the transients, variations of cytosolic Ca(2+) concentrations, and adequacy of intracellular Ca(2+) stores.

Regulation and rate limiting mechanisms of Ca2+ ATPase (SERCA2) expression in cardiac myocytes.

Involvement of the calcineurin/NFAT pathway in transcription of cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) was demonstrated (Prasad and Inesi, Am J Physiol Heart Circ Physiol 300(1):H173-H180, 2011) by upregulation of SERCA2 following calcineurin (CN) activation by cytosolic Ca(2+), and downregulation of SERCA2 following CN inhibition with cyclosporine (CsA) or CN subunits gene silencing. We show here that in cultured cardiac myocytes, competitive engagement of the CN/NFAT pathway is accompanied by downregulation of SERCA2 and Ca(2+) signaling alterations. In fact, SERCA2 downregulation occurs following infection of myocytes with adenovirus vectors carrying luciferase or SERCA1 cDNA under control of NFAT-dependent promoters, but not under control of CMV promoters that do not depend on NFAT.

The Ca2+-ATPase (SERCA1) is inhibited by 4-aminoquinoline derivatives through interference with catalytic activation by Ca2+, whereas the ATPase E2 state remains functional.

Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to μm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the μm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms.

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms.

Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase).

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673.

Silencing calcineurin A subunit reduces SERCA2 expression in cardiac myocytes.

Resting intracellular Ca(2+) can be raised, in neonatal rat cardiac myocytes, by exposure to very low concentration of thapsigargin (TG). Such a Ca(2+) rise yields calcineurin (CN) activation demonstrated by increased expression of transfected luciferase cDNA under control of nuclear factor of activated T-cells (NFAT) promoter and increased translocation of NFAT to nuclei. We found that exposure of cardiac myocytes to TG is followed by increase of sarcroplasmic reticulum Ca(2+) transport ATPase (SERCA2) expression, which is further increased when CN inactivation by CAMKII (calmodulin-dependent kinase) is prevented with KN93 (CAMKII inhibitor).

ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements.

ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate.

Comparative features of copper ATPases ATP7A and ATP7B heterologously expressed in COS-1 cells.

ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, prior or subsequent to treatment with endoglycosidase, and evidenced by protein or glycoprotein staining as well as Western blotting, indicates that the ATP7A protein is glycosylated while ATP7B is not.

High-yield heterologous expression of wild type and mutant Ca(2+) ATPase: Characterization of Ca(2+) binding sites by charge transfer.

High-yield heterologous SERCA1 (Ca(2+) ATPase) expression was obtained in COS-1 cells infected with recombinant adenovirus vector (rAdSERCA). Higher transcription and expression were obtained in the presence of a His(6) tag at the amino terminus, as compared with a His(6) tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes.

High yield heterologous expression of wild-type and mutant Cu+-ATPase (ATP7B, Wilson disease protein) for functional characterization of catalytic activity and serine residues undergoing copper-dependent phosphorylation.

ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector.

Reaction cycle of Thermotoga maritima copper ATPase and conformational characterization of catalytically deficient mutants.

Copper transport ATPases sustain important roles in homeostasis of heavy metals and delivery of copper to metalloenzymes. The copper transport ATPase from Thermotoga maritima (CopA) provides a useful system for mechanistic studies, due to its heterologous expression and stability. Its sequence comprises 726 amino acids, including the N-terminal metal binding domain (NMBD), three catalytic domains (A, N, and P), and a copper transport domain formed by eight helices, including the transmembrane metal binding site (TMBS).

Effects of thapsigargin and phenylephrine on calcineurin and protein kinase C signaling functions in cardiac myocytes.

Neonatal rat cardiac myocytes were exposed to 10 nM thapsigargin (TG) or 20 muM phenylephrine (PE) to compare resulting alterations of Ca(2+) homeostasis. Either treatment results in resting cytosolic [Ca(2+)] rise and reduction of Ca(2+) signals in myocytes following electrical stimuli. In fact, ATP-dependent Ca(2+) transport is reduced due to catalytic inhibition of sarcoplasmic reticulum ATPase (SERCA2) by TG or reduction of SERCA2 protein expression by PE.

Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima.

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 micromol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.

Effects of high-affinity inhibitors on partial reactions, charge movements, and conformational States of the Ca2+ transport ATPase (sarco-endoplasmic reticulum Ca2+ ATPase).

The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca(2+) ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca(2+) binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca(2+) in the absence of ATP.

The Ca2+ ATPase of cardiac sarcoplasmic reticulum: Physiological role and relevance to diseases.

The Ca(2+) ATPase of sarcoplasmic reticulum has a prominent role in excitation/contraction coupling of cardiac muscle, as it induces relaxation by sequestering Ca(2+) from the cytoplasm. The stored Ca(2+) is in turn released to trigger contraction. We review here experiments demonstrating that in cardiac myocytes Ca(2+) signaling and contractile activation are strongly altered by pharmacological inhibition or transcriptional down-regulation of SERCA.

Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase.

Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu).

Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin.

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues.