Type ATPase Research Articles

Dynein Light Intermediate Chain in Aspergillus nidulans Is Essential for the Interaction between Heavy and Intermediate Chains

Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus.

Metal Binding Affinities of Arabidopsis Zinc and Copper Transporters: Selectivities Match the Relative, but Not the Absolute, Affinities of their Amino-Terminal Domains,

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.

Alteration of RANKL-Induced Osteoclastogenesis in Primary Cultured Osteoclasts From SERCA2+/− Mice

RANKL is essential for the terminal differentiation of monocytes/macrophages into osteoclasts. RANKL induces long-lasting oscillations in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) only after 24 h of stimulation. These Ca(2+) oscillations play a switch-on role in NFATc1 expression and osteoclast differentiation. Which Ca(2+) transporting pathway is induced by RANKL to evoke the Ca(2+) oscillations and its specific role in RANKL-mediated osteoclast differentiation is not known. This study examined the effect of a partial loss of sarco/endoplasmic reticulum Ca(2+) ATPase type 2 (SERCA2) on osteoclast differentiation in SERCA2 heterozygote mice (SERCA2(+/-)). The BMD in the tibias of SERCA2(+/-) mice increased >1.5-fold compared with wildtype mice (WT). RANKL-induced [Ca(2+)](i) oscillations were generated 48 h after RANKL treatment in the WT mice but not in the SERCA2(+/-) bone marrow-derived macrophages (BMMs). Forty-eight hours after RANKL treatment, there was a lower level of NFATc1 protein expression and markedly reduced translocation of NFATc1 into the nucleus during osteoclastogenesis of the SERCA2(+/-) BMMs. In addition, RANKL treatment of SERCA2(+/-) BMMs incompletely induced formation of multinucleated cells, leading to reduced bone resorption activity. These results suggest that RANKL-mediated induction of SERCA2 plays a critical role in the RANKL-induced [Ca(2+)](i) oscillations that are essential for osteoclastogenesis.

Caenorhabditis elegansP5B‐type ATPase CATP‐5 operates in polyamine transport and is crucial for norspermidine‐mediated suppression of RNA interference

Physiological polyamines are required in various biological processes. In the current study, we used norspermidine, a structural analog of the natural polyamine spermidine, to investigate polyamine uptake in the model organism Caenorhabditis elegans. Norspermidine was found to have two remarkable effects: it is toxic for the nematode, without affecting its food, Escherichia coli; and it hampers RNA interference. By characterizing a norspermidine-resistant C. elegans mutant strain that has been isolated in a genetic screen, we demonstrate that both effects, as well as the uptake of a fluorescent polyamine-conjugate, depend on the transporter protein CATP-5, a novel P(5B)-type ATPase. To our knowledge, CATP-5 represents the first P(5)-type ATPase that is associated with the plasma membrane, being expressed in the apical membrane of intestinal cells and the excretory cell. Moreover, genetic interaction studies using C. elegans polyamine synthesis mutants indicate that CATP-5 has a function redundant to polyamine synthesis and link reduced polyamine levels to retarded postembryonic development, reduced brood size, shortened life span, and small body size. We suggest that CATP-5 represents a crucial component of the pharmacologically important polyamine transport system, the molecular nature of which has not been identified so far in metazoa.

Crystal structures of cisplatin bound to a human copper chaperone.

Copper trafficking proteins, including the chaperone Atox1 and the P(1B)-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

Histological characterization of Darier's disease in Tunisian families

Darier's disease (OMIM 124200) is an autosomal-dominant skin disorder characterized by warty papules and plaques in seborreheic areas, palmo-plantar pits and distinctive nail abnormalities. The disease has complete penetrance in adults and variable expressivity. It is caused by mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca(2+) ATPase type 2 isoform (SERCA2). We report histological investigations of six unrelated Tunisian families including 15 affected individuals with Darier's disease mutations. The typical histological features of Darier's disease have been observed in the 15 patients. Variable histological features have been observed among Tunisian patients ranging from mild to moderate lesions of Darier's disease. A significant correlation has been observed between the clinical presentation of the Darier's disease (mild or moderate) and the intensity of the histological features. Isolated acral form of Darier's disease was seen in one case. Two distinct original associations have been observed: Darier's disease/pemphigus vulgaris in one patient and Darier's disease/ichtyosis in the other patient. Our findings confirmed the clinical heterogeneity of Darier's disease on the basis of histological study. The intensity of the histological features could be closely correlated to the severity of Darier's disease clinical presentation.

CzcP is a novel efflux system contributing to transition metal resistance inCupriavidus metalliduransCH34

Cupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel P(IB4)-type ATPase CzcP is characterized, which belongs to the plasmid pMOL30-mediated cobalt-zinc-cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance in C. metallidurans was compared with that of three P(IB2)-type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal-sensitive Escherichia coli strains were functionally substituted in trans with CzcP and the three P(IB2)-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the K(m) values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as 'resistance enhancer': this P(IB4)-type ATPase exports transition metals Zn(2+), Cd(2+) and Co(2+) much more rapidly than the three P(IB2)-type proteins. However, a basic resistance level has to be provided by the P(IB2)-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.

Solution Structures of the Actuator Domain of ATP7A and ATP7B, the Menkes and Wilson Disease Proteins

ATP7A and ATP7B are two human P(1B)-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorylated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fold of the domain (such as Gly860Val in ATP7A) or affect the network of communication within the domain (such as Leu873Arg in ATP7A) or with the other domains of the enzyme (such as Gly853Arg in ATP7A).

Junctin – the quiet achiever

Recent advances in understanding skeletal and cardiac muscle function have evolved with recognition of the active role played by the intracellular sarcoplasmic reticulum (SR) Ca2+ store in contraction. The key proteins in this store are the Ca2+ binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) Ca2+ release channel and triadin and junctin (Beard et al. 2004). The CSQ–triadin–junctin–RyR complex (Fig. 1) in the SR lumen forms a ‘Ca2+ transduction machine’ that is central to EC coupling and to normal muscle development. Other proteins in the lumen of the SR, including the histidine rich calcium binding protein (HRC) (Suk et al. 1999), JP-45 (Anderson et al. 2003) and SRP-27 (Bleunven et al. 2008), must also contribute to control of SR intraluminal Ca2+ load, but the precise nature of their role remains undetermined. JP-45 in particular is ideally placed to communicate store load to the excitation–contraction (EC) coupling process as it binds to both CSQ and the dihydropyridine receptor (DHPR) in the surface/transverse tubule membrane. The importance of the luminal proteins has been underlined by the recent discovery that changes in Ca2+ signalling due to mutations in CSQ or to lack of its expression can result in sudden cardiac death (Viatchenko-Karpinski et al. 2004). Furthermore, studies in animal models show that changes in CSQ, junctin, triadin and HRC expression can lead to defective Ca2+ signalling. The review by Pritchard & Kranius (2009) examines the roles of junctin and HRC in Ca2+ cycling and their potential significance in heart failure. This commentary focuses on protein-protein interactions between CSQ, triadin, junctin and RyR proteins that may underlie their roles in Ca2+ cycling. Figure 1 Illustration of the possible interactions between triadin, junctin, CSQ1 and RyR1 in the lumen of the SR

Conformational Dynamics of Metal-Binding Domains in Wilson Disease Protein: Molecular Insights into Selective Copper Transfer

ATP7A/B are human P(1B)-type ATPases involved in cellular Cu homeostasis. The N-terminal parts of these multidomain proteins contain six metal-binding domains (MBDs) connected by linkers. The MBDs are similar in structure to each other and to the human copper chaperone Atox1, although their distinct roles in Cu transfer appear to vary. All domains have the ferredoxin-like fold and a solvent-exposed loop with a MXCXXC motif that can bind Cu(I). Here, we investigated the dynamic behavior of the individual MBDs (WD1-WD6) in ATP7B in apo forms using molecular dynamic simulations. We also performed simulations of three Cu-bound forms (WD2c, WD4c, and WD6c). Our results reveal molecular features that vary distinctly among the MBDs. Whereas WD1, WD2, and WD6 have well-defined Cu loop conformations stabilized by a network of interactions, WD4 and WD5 exhibit greater loop flexibility and, in WD4, helix alpha1 unwinds and rewinds. WD3, which has the lowest sequence identity, behaves differently and its Cu loop is rigid with respect to the rest of the domain. Cu coordination reduces structural dynamics in all domains but WD4c. In agreement with predictions on individual domains, simulations of the six possible Atox1-WD heterocomplexes show that Atox1 interactions with WD4 are the strongest. This study provides molecular explanations for reported Cu transfer and protein-protein interaction specificity.

Exercise capacity is related to calcium transients in ventricular cardiomyocytes

The aim of the present study was to evaluate the Ca2+ handling and contractility properties of cardiomyocytes isolated from rats with high intrinsic aerobic exercise capacity. Standard-performance (SP) and high-performance (HP) rats were categorized with a treadmill progressive exercise test according to the exercise time to fatigue (TTF). The SP group included rats with TTF between 16.63 and 46.57 min, and the HP group included rats with TTF>46.57 min. Isolated ventricular cardiomyocytes were dissociated from the hearts of SP and HP rats, and intracellular global Ca2+ ([Ca2+]i) transients were measured. The [Ca2+]i transient peak was increased in the HP group relative to the SP group (5.54+/-0.31 vs. 4.18+/-0.12 F/F0; P<or=0.05) and was positively correlated with the TTF attained during the progressive test (r=0.81). We also performed contractility measurements in isolated cardiomyocytes and found higher amplitude of contraction in the HP group compared with the SP group (6.7+/-0.2 vs. 6.0+/-0.3% resting cell length; P<or=0.05). To reinforce the intrinsic differences between SP and HP rats, we performed Western blot experiments and observed increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase type 2a (1.30+/-0.07 vs. 1.74+/-0.18 arbitrary units; P<or=0.05) and ryanodine receptor type 2 (1.86+/-0.13 vs. 3.57+/-0.12 arbitrary units; P<or=0.05) in HP rats. In summary, our data showed important intrinsic differences in cardiomyocyte properties that could explain some of the divergence observed in rats with high intrinsic aerobic exercise capacity.

Effect of tea catechins on erythrocyte Ca++-pump in type 2 diabetes mellitus

An altered intracellular calcium metabolism is known to play an essential role in the pathophysiology of diabetes and its complications. Tea [Camellia sinensis L. (Theaceae)] contains polyphenolic compounds collectively known as catechins belonging to the flavonoids family. Catechins have been reported to possess several biological properties including an antidiabetic effect. The present paper reports the effect of tea catechins on erythrocyte membrane Ca++-pump in normal and type 2 diabetic patients. The activity of erythrocyte Ca++-ATPase was significantly decreased in type 2 diabetes. In vitro incubation with tea catechins (10−3-10−8 M) caused an increase in the activity of Ca++-ATPase in both normal and type 2 diabetic subjects; the effect was more pronounced in type 2 diabetes and was observed at much lower concentrations (10−5 M) compared to normal (10−3 M). The order of effectiveness of individual catechins was in the order: EGCG > ECG > EC > EGC. The increase in Ca++-ATPase activity after incubation with tea catechins may be explained on the basis of alteration in fluidity due to a direct effect of catechins on the membrane.

Compartment‐specific regulation of plasma membrane calcium ATPase type 2 in the chick auditory brainstem

Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes after transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.

An NMR Study of the Interaction of the N-terminal Cytoplasmic Tail of the Wilson Disease Protein with Copper(I)-HAH1

ATP7B is a human P(1B)-type ATPase that has a crucial role in maintaining copper(I) homeostasis. Mutations in the corresponding gene are the cause of Wilson disease. Among its various distinguishing features is a long ( approximately 630 amino acids) N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We expressed the entire tail as a single construct in Escherichia coli and investigated its interaction with its copper chaperone (i.e. HAH1) by solution NMR spectroscopy. We observed that all six of the metal-binding domains were metallated by Cu(I)-HAH1, with the first, the second, and the fourth domains forming an adduct with it. This behavior is different from that of the highly similar human ATPase ATP7A, in which only two domains form such an adduct. The distinct behaviors of the different domains were analyzed in terms of the energetics of Cu(I) transfer, hinting at a specific role of the interaction with copper(I)-HAH1 in the overall functional process.

Role of α-Subunit VISIT-DG Sequence Residues Ser-347 and Gly-351 in the Catalytic Sites of Escherichia coli ATP Synthase

This paper describes the role of alpha-subunit VISIT-DG sequence residues alphaSer-347 and alphaGly-351 in catalytic sites of Escherichia coli F(1)F(o) ATP synthase. X-ray structures show the very highly conserved alpha-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues alphaArg-376, betaArg-182, betaLys-155, and betaArg-246 in the phosphate-binding subdomain. Mutations alphaS347Q and alphaG351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F(1)F(o) in membranes by 100- and 150-fold, respectively, whereas alphaS347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of alphaS347Q mutant was not inhibited, and the alphaS347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and alphaG351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and alphaG351Q mutant ATPase essentially completely, ATPase in alphaS347A or alphaS347Q mutant was inhibited maximally by approximately 80-90%, although reaction still occurred at residue betaTyr-297, proximal to the alpha-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts inbetaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and alphaG351Q mutant but not in alphaS347Q or alphaS347A mutant. The results demonstrate that alphaSer-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, alphaGly-351, although strongly conserved and clearly important for function, appears not to play a direct role.

Molecular Simulation Study of Kinesin: Coupling between ATPase Domain Conformational Change and Mechanical Stepping

Conventional two-headed kinesin is a motor protein that moves unidirectionally by stepping in hand-over-hand manner (akin to human walking) along microtubule (MT) driven by ATP hydrolysis free energy. In the absence of MT, X-ray crystallography revealed primarily two conformations of the head, ATPase domain; “T” structure preferred with a bound ATP and “D” structure preferred with a bound ADP.However, relations among the ATPase conformations, stepping motion, and type of bound nucleotide are still rather unclear. Here, we investigated the coupling mechanism between the enzyme structure and mechanical stepping. For this purpose, we performed molecular dynamics simulations with coarse-grained structure-based models. In particular, to investigate structural preference between T and D in front and rear heads bound on MT, we applied multiple-basin energy landscape model (Okazaki et al., 2006).Through simulations, we found the followings. (1) Enzyme structure can regulate its affinity to MT by the difference in the contact surface area: “T” structure has higher affinity to MT than “D”, which is consistent with experiments. (2) The internal-strain between two heads can regulate the ATPase structural preference: The rear head with forward-directed neck-linker prefer T structure, while the front head with backward-directed neck-linker prefer D structure.

Tributyltin sensitivity of vacuolar-type Na+-transporting ATPase from Enterococcus hirae

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of H(+)-transporting or Na(+)-transporting F-ATPase as well as H(+)-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na(+)-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na(+)-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 microM TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 microM. Active sodium transport by E. hirae cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 microM TBT. By contrast, IC50 of H(+)-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 microM. E. hirae V-ATPase is thus extremely less sensitive to TBT.

Novel mutations of the ATP2A2 gene in two families with Darier’s disease

Darier's disease (DD) is an autosomal dominant genodermatology. Mutations in the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium pumping ATPase type 2 (SERCA2) have been identified as the molecular basis of DD. The aim of this study was to report two Chinese pedigree of DD and to explore the genetic mutations. Polymerase chain reaction was carried out to amplify the exons and flanking intron boundaries of the ATP2A2 gene followed by direct sequencing. Two novel missense mutations were identified, a change of C203 to A (A68E) in exon 3 was found in one family and a change of C2759 to T (S920F) in exon 19 in the other, which were located within the transmembrane domain of SERCA2, highly conserved during evolution. The A68E and S920F mutations might be regarded as the causes of the disease in two Chinese families, but these were not tested functionally. Additional functional experiments are necessary to verify the relevance and suitability of these findings for future use in genetic counseling and prenatal diagnosis.

New insights into structure‐function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0)

Adenosine triphosphate, ATP, is the energy currency of living cells. While ATP synthases of archae and ATP synthases of pro- and eukaryotic organisms operate as energy producers by synthesizing ATP, the eukaryotic V-ATPase hydrolyzes ATP and thus functions as energy transducer. These enzymes share features like the hydrophilic catalytic- and the membrane-embedded ion-translocating sector, allowing them to operate as nano-motors and to transform the transmembrane electrochemical ion gradient into ATP or vice versa. Since archaea are rooted close to the origin of life, the A-ATP synthase is probably more similar in its composition and function to the "original" enzyme, invented by Nature billion years ago. On the contrary, the V-ATPases have acquired specific structural, functional and regulatory features during evolution. This review will summarize the current knowledge on the structure, mechanism and regulation of A-ATP synthases and V-ATPases. The importance of V-ATPase in pathophysiology of diseases will be discussed.

Dynamics of Human Myocardial Progenitor Cell Populations in the Neonatal Period

Pluripotent cardiac progenitor cells resident in myocardium offer a potentially promising role in promoting recovery from injury. In pediatric congenital heart disease (CHD) patients, manipulation of resident progenitor cells may provide important new approaches to improving outcomes. Our study goals were to identify and quantitate populations of progenitor cells in human neonatal myocardium during the early postnatal period and determine the proliferative capacity of differentiated cardiac myocytes. Immunologic markers of cell lineage (stage-specific embryonic antigen 4 [SSEA-4], islet cell antigen 1 [Isl1], c-kit, Nkx2.5, sarcoplasmic reticulum calcium-regulated ATPase type 2 [SERCA2]) and proliferation (Ki67) were localized in right ventricular biopsies from 32 CHD patients aged 2 to 93 days. Neonatal myocardium contains progenitor cells and transitional cells expressing progenitor and differentiated myocyte marker proteins. Some cells expressed the pluripotent cell marker c-kit and also coexpressed the myocyte marker SERCA2. Multipotent progenitor cells, identified by the expression of Isl1, were found. Ki67 was expressed in some myocytes and in nonmyocyte cells. A few cells expressing SSEA-4 and Isl1 were observed during the early postnatal period. Cells expressing c-kit, the premyocyte marker Nkx2.5, and Ki67 were found throughout the first postnatal month. A progressive decline in cell density during the first postnatal month was observed for c-kit+ cells (p = 0.0013) and Nkx2.5+ cells (p = 0.0001). The percentage of cells expressing Ki67 declined during the first 3 postnatal months (p = 0.0030). Cells in an incomplete state of cardiomyocyte differentiation continue to reside in the infant heart. However, the relative density of progenitor cells declines during the first postnatal month.