Sungjo Park

Interaction of Asymmetric ABCC9-Encoded Nucleotide Binding Domains Determines KATP Channel SUR2A Catalytic Activity

Nucleotide binding domains (NBDs) secure ATP-binding cassette (ABC) transporter function. Distinct from traditional ABC transporters, ABCC9-encoded sulfonylurea receptors (SUR2A) form, with Kir6.2 potassium channels, ATP-sensitive K+ (K ATP) channel complexes. SUR2A contains ATPase activity harbored within NBD2 and, to a lesser degree, NBD1, with catalytically driven conformations exerting determinate linkage on the Kir6.2 channel pore. While homodomain interactions typify NBDs of conventional ABC transporters, heterodomain NBD interactions and their functional consequence have not been resolved for the atypical SUR2A protein. Here, nanoscale protein topography mapped assembly of monodisperse purified recombinant SUR2A NBD1/NBD2 domains, precharacterized by dynamic light scattering. Heterodomain interaction produced conformational rearrangements inferred by secondary structural change in circular dichroism, and validated by atomic force and transmission electron microscopy. Physical engagement of NBD1 with NBD2 translated into enhanced intrinsic ATPase activity. Molecular modeling delineated a complemental asymmetry of NBD1/NBD2 ATP-binding sites. Mutation in the predicted catalytic base residue, D834E of NBD1, altered NBD1 ATPase activity disrupting potentiation of catalytic behavior in the NBD1/NBD2 interactome. Thus, NBD1/NBD2 assembly, resolved by a panel of proteomic approaches, provides a molecular substrate that determines the optimal catalytic activity in SUR2A, establishing a paradigm for the structure-function relationship within the K ATP channel complex.

Functional studies of frataxin

Mitochondria generate adenosine triphosphate (ATP) but also dangerous reactive oxygen species (ROS). One‐electron reduction of dioxygen in the early stages of the electron transport chain yields a superoxide radical that is detoxified by mitochondrial superoxide dismutase to give hydrogen peroxide. The hydroxyl radical is derived from decomposition of hydrogen peroxide via the Fenton reaction, catalyzed by Fe2+ ions. Mitochondria require a constant supply of Fe2+ for heme and iron‐sulfur cluster biosyntheses and therefore are particularly susceptible to ROS attack. Two main antioxidant defenses are known in mitochondria: enzymes that catalytically remove ROS, e.g. superoxide dismutase and glutathione peroxidase, and low molecular weight agents that scavenge ROS, including coenzyme Q, glutathione, and vitamins E and C. An effective defensive system, however, should also involve means to control the availability of pro‐oxidants such as Fe2+ ions. There is increasing evidence that this function may be carried out by the mitochondrial protein frataxin. Frataxin deficiency is the primary cause of Friedreichs ataxia (FRDA), an autosomal recessive degenerative disease. Frataxin is a highly conserved mitochondrial protein that plays a critical role in iron homeostasis. Respiratory deficits, abnormal cellular iron distribution and increased oxidative damage are associated with frataxin defects in yeast and mouse models of FRDA. The mechanism by which frataxin regulates iron metabolism is unknown. The yeast frataxin homologue (mYfhlp) is activated by Fe(II) in the presence of oxygen and assembles stepwise into a 48‐subunit multimer (α48) that sequesters <2000 atoms of iron in a ferrihydrite mineral core. Assembly of mYfhlp is driven by two sequential iron oxidation reactions: a fast ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (αα3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding α48. Depending on the ionic environment, stepwise assembly is associated with the sequestration of 50–75 Fe(II)/subunit. This Fe(II) is initially loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. However, as iron oxidation and mineralization proceed, Fe(III) becomes progressively inaccessible and a stable iron‐protein complex is produced. In conclusion, by coupling iron oxidation with stepwise assembly, frataxin can successively function as an iron chaperon or an iron store. Reduced iron availability and solubility and increased oxidative damage may therefore explain the pathogenesis of FRDA.

KATP channel Kir6.2 E23K variant overrepresented in human heart failure is associated with impaired exercise stress response

ATP-sensitive K+ (KATP) channels maintain cardiac homeostasis under stress, as revealed by murine gene knockout models of the KCNJ11-encoded Kir6.2 pore. However, the translational significance of KATP channels in human cardiac physiology remains largely unknown. Here, the frequency of the minor K23 allele of the common functional Kir6.2 E23K polymorphism was found overrepresented in 115 subjects with congestive heart failure compared to 2,031 community-based controls (69 vs. 56%, Pxa0<xa00.001). Moreover, the KK genotype, present in 18% of heart failure patients, was associated with abnormal cardiopulmonary exercise stress testing. In spite of similar baseline heart rates at rest among genotypic subgroups (EE: 72.2xa0±xa02.3, EK: 75.0xa0±xa01.8 and KK: 77.1xa0±xa03.0xa0bpm), subjects with the KK genotype had a significantly reduced heart rate increase at matched workload (EE: 32.8xa0±xa02.7%, EK: 28.8xa0±xa02.1%, KK: 21.7xa0±xa02.6%, Pxa0<xa00.05), at 75% of maximum oxygen consumption (EE: 53.9xa0±xa03.9%, EK: 49.9xa0±xa03.1%, KK: 36.8xa0±xa05.3%, Pxa0<xa00.05), and at peak VO2 (EE: 82.8xa0±xa06.0%, EK: 80.5xa0±xa04.7%, KK: 59.7xa0±xa08.1%, Pxa0<xa00.05). Molecular modeling of the tetrameric Kir6.2 pore structure revealed the E23 residue within the functionally relevant intracellular slide helix region. Substitution of the wild-type E residue with an oppositely charged, bulkier K residue would potentially result in a significant structural rearrangement and disrupted interactions with neighboring Kir6.2 subunits, providing a basis for altered high-fidelity KATP channel gating, particularly in the homozygous state. Blunted heart rate response during exercise is a risk factor for mortality in patients with heart failure, establishing the clinical relevance of Kir6.2 E23K as a biomarker for impaired stress performance and underscoring the essential role of KATP channels in human cardiac physiology.

Lipid metabolism greases the stem cell engine.

Metabolic plasticity is increasingly postulated to be vital in the transition between stemness maintenance andxa0lineage specification. Knobloch etxa0al. (2012) now demonstrate that regulation of lipogenesis by fatty acid synthase and Spot14-dependent malonyl-CoA supply determines the proliferative activity of resident neural stem cells, contributing to adult neurogenesis.

Role for SUR2A ED Domain in Allosteric Coupling within the KATP Channel Complex

Allosteric regulation of heteromultimeric ATP-sensitive potassium (KATP) channels is unique among protein systems as it implies transmission of ligand-induced structural adaptation at the regulatory SUR subunit, a member of ATP-binding cassette ABCC family, to the distinct pore-forming K+ (Kir6.x) channel module. Cooperative interaction between nucleotide binding domains (NBDs) of SUR is a prerequisite for KATP channel gating, yet pathways of allosteric intersubunit communication remain uncertain. Here, we analyzed the role of the ED domain, a stretch of 15 negatively charged aspartate/glutamate amino acid residues (948–962) of the SUR2A isoform, in the regulation of cardiac KATP channels. Disruption of the ED domain impeded cooperative NBDs interaction and interrupted the regulation of KATP channel complexes by MgADP, potassium channel openers, and sulfonylurea drugs. Thus, the ED domain is a structural component of the allosteric pathway within the KATP channel complex integrating transduction of diverse nucleotide-dependent states in the regulatory SUR subunit to the open/closed states of the K+-conducting channel pore.

Quaternary structure of KATP channel SUR2A nucleotide binding domains resolved by synchrotron radiation X-ray scattering.

Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K(+) (K(ATP)) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168x80x37A(3) and 175x81x37A(3) based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein-protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of K(ATP) channel SUR2A regulatory domains.

Functionalized Carbon Nanotube and Graphene Oxide Embedded Electrically Conductive Hydrogel Synergistically Stimulates Nerve Cell Differentiation

Nerve regeneration after injury is a critical medical issue. In previous work, we have developed an oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel incorporated with positive charges as a promising nerve conduit. In this study, we introduced cross-linkable bonds to graphene oxide and carbon nanotube to obtain the functionalized graphene oxide acrylate (GOa) and carbon nanotube poly(ethylene glycol) acrylate (CNTpega). An electrically conductive hydrogel was then fabricated by covalently embedding GOa and CNTpega within OPF hydrogel through chemical cross-linking followed by in situ reduction of GOa in l-ascorbic acid solution. Positive charges were incorporated by 2-(methacryloyloxy)ethyltrimethylammonium chloride (MTAC) to obtain rGOaCNTpega-OPF-MTAC composite hydrogel with both surface charge and electrical conductivity. The distribution of CNTpega and GOa in the hydrogels was substantiated by transmission electron microscopy (TEM), and strengthened electrical conductivities were determined. Excellent biocompatibility was demonstrated for the carbon embedded composite hydrogels. Biological evaluation showed enhanced proliferation and spreading of PC12 cells on the conductive hydrogels. After induced differentiation using nerve growth factor (NGF), cells on the conductive hydrogels were effectively stimulated to have robust neurite development as observed by confocal microscope. A synergistic effect of electrical conductivity and positive charges on nerve cells was also observed in this study. Using a glass mold method, the composite hydrogel was successfully fabricated into conductive nerve conduits with surficial positive charges. These results suggest that rGOa-CNTpega-OPF-MTAC composite hydrogel holds great potential as conduits for neural tissue engineering.

SDF-1-enhanced cardiogenesis requires CXCR4 induction in pluripotent stem cells.

Transformation of pluripotent stem cells into cardiac tissue is the hallmark of cardiogenesis, yet pro-cardiogenic signals remain partially understood. Preceding cardiogenic induction, a surge in CXCR4 chemokine receptor expression in the early stages of stem cell lineage specification coincides with the acquisition of pre-cardiac profiles. Accordingly, CXCR4 selection, in conjunction with mesoderm-specific VEGF type II receptor FLK-1 co-expression, segregates cardiogenic populations. To assess the functionality of the CXCR4 biomarker, targeted activation and disruption were here exploited in the context of embryonic stem cell-derived cardiogenesis. Implicated as a cardiogenic hub through unbiased bioinformatics analysis, induction of the CXCR4/SDF-1 receptor–ligand axis triggered enhanced beating activity in stem cell progeny. Gene expression knockdown of CXCR4 disrupted spontaneous embryoid body differentiation and blunted the expression of cardiogenic markers MEF2C, Nkx2.5, MLC2a, MLC2v, and cardiac-MHC. Exogenous SDF-1 treatment failed to rescue cardiogenic-deficient phenotype, demonstrating a requirement for CXCR4 expression in mediating SDF-1 effects. Thus, a pro-cardiogenic signaling role for the CXCR4/SDF1 axis is herein revealed within pluripotent stem cell progenitors, exposing a functional target to promote lineage-specific differentiation.

Covalent crosslinking of graphene oxide and carbon nanotube into hydrogels enhances nerve cell responses

Healing of nerve injuries is a critical medical issue. Biodegradable polymeric conduits are a promising therapeutic solution to provide guidance for axon growth in a given space, thus helping nerve heal. Extensive studies in the past decade reported that conductive materials could effectively increase neurite and axon extension in vitro and nerve regeneration in vivo. In this study, graphene oxide and carbon nanotubes were covalently functionalized with double bonds to obtain crosslinkable graphene oxide acrylate (GOa) sheets and carbon nanotube poly(ethylene glycol) acrylate (CNTpega). An electrically conductive reduced GOa-CNTpega-oligo(polyethylene glycol fumarate) (OPF) hydrogel (rGOa-CNTpega-OPF) was successfully fabricated by chemically crosslinking GOa sheets and CNTpega with OPF chains followed by in situ chemical reduction in l-ascorbic acid solution. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging showed homogenous distribution of GOa/CNTpega carbon content in the rGOa-CNTpega-OPF composite hydrogel, resulting in a significant increase of electrical conductivity compared with neutral OPF without carbon content. Cell studies showed excellent biocompatibility and distinguished PC12 cell proliferation and spreading on the rGOa-CNTpega-OPF composite hydrogel. Fluorescent microscopy imaging demonstrated robustly stimulated neurite development in these cells on a conductive rGOa-CNTpega-OPF composite hydrogel compared with that on neutral OPF hydrogels. These results illustrated a promising potential for the rGOa-CNTpega-OPF composite hydrogel to serve as conduits for neural tissue engineering.

KATP channels process nucleotide signals in muscle thermogenic response

Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K+ (KATP) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, KATP channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how KATP channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the KATP channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with KATP channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal KATP channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of KATP channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of KATP channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.