Takashi Morita

p38α Mitogen-Activated Protein Kinase Plays a Critical Role in Cardiomyocyte Survival but Not in Cardiac Hypertrophic Growth in Response to Pressure Overload

ABSTRACT The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38α is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38α in hearts. First, we generated mice with floxed p38α alleles and crossbred them with mice expressing the Cre recombinase under the control of the α-myosin heavy-chain promoter to obtain cardiac-specific p38α knockout mice. These cardiac-specific p38α knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38α plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.

Mitochondria-related male infertility

Approximately 15% of human couples are affected by infertility, and about half of these cases of infertility can be attributed to men, through low sperm motility (asthenozoospermia) or/and numbers (oligospermia). Because mitochondrial genome (mtDNA) mutations are identified in patients with fertility problems, there is a possibility that mitochondrial respiration defects contribute to male infertility. To address this possibility, we used a transmitochondrial mouse model (mito-mice) carrying wild-type mtDNA and mutant mtDNA with a pathogenic 4,696-bp deletion (ΔmtDNA). Here we show that mitochondrial respiration defects caused by the accumulation of ΔmtDNA induced oligospermia and asthenozoospermia in the mito-mice. Most sperm from the infertile mito-mice had abnormalities in the middle piece and nucleus. Testes of the infertile mito-mice showed meiotic arrest at the zygotene stage as well as enhanced apoptosis. Thus, our in vivo study using mito-mice directly demonstrates that normal mitochondrial respiration is required for mammalian spermatogenesis, and its defects resulting from accumulated mutant mtDNAs cause male infertility.

Crystal structure of an anticoagulant protein in complex with the Gla domain of factor X.

The γ-carboxyglutamic acid (Gla) domain of blood coagulation factors is responsible for Ca2+-dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla domain of factor X. The crystal structure of X-bp in complex with the Gla domain peptide of factor X at 2.3-Å resolution showed that the anticoagulation is based on the fact that two patches of the Gla domain essential for membrane binding are buried in the complex formation. The Gla domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.

Snake Venom Vascular Endothelial Growth Factors (VEGFs) Exhibit Potent Activity through Their Specific Recognition of KDR (VEGF Receptor 2)

Vascular endothelial growth factor (VEGF165) exhibits multiple effects via the activation of two distinct endothelial receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase-1) and KDR (kinase insert domain-containing receptor). KDR shows strong ligand-dependent tyrosine phosphorylation in comparison with Flt-1 and mainly mediates the mitogenic, angiogenic, and permeability-enhancing effects of VEGF165. Here we show the isolation of two VEGFs from viper venoms and the characterization of their unique biological properties. Snake venom VEGFs strongly stimulated proliferation of vascular endothelial cells in vitro. Interestingly, the maximum activities were almost twice that of VEGF165. They also induced strong hypotension on rat arterial blood pressure compared with VEGF165 in vivo. A receptor binding assay revealed that snake venom VEGFs bound to KDR-IgG with high affinity (Kd = ∼0.1 nm) as well as to VEGF165 but did not interact with Flt-1, Flt-4, or neuropilin-1 at all. Our data clearly indicate that snake venom VEGFs act through the specific activation of KDR and show potent effects. Snake venom VEGFs are a highly specific ligand to KDR and form a new group of the VEGF family.

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD.

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.

Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin-like heterodimers.

Snake venom contains a number of the hemostatically active C-type lectin-like proteins, which affect the interaction between von Willebrand factor (vWF) and the platelet glycoprotein (GP) Ib or platelet receptor to inhibit/induce platelet activation. Flavocetin-A (FL-A) is a high-molecular mass C-type lectin-like protein (149 kDa) isolated from the habu snake venom. FL-A binds with high affinity to the platelet GP Ibalpha-subunit and functions as a strong inhibitor of vWF-dependent platelet aggregation. We have determined the X-ray crystal structure of FL-A and refined to 2.5 A resolution. This is a first elucidation of a three-dimensional structure of the platelet GP Ib-binding protein. The overall structure reveals that the molecule is a novel cyclic tetramer (alphabeta)(4) made up of four alphabeta-heterodimers related by a crystallographic 4-fold symmetry. The tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the alpha-subunit and at the N-terminus of the beta-subunit in the neighboring alphabeta-heterodimer. The high affinity of FL-A for the platelet GP Ib alpha-subunit could be explained by a cooperative-binding action through the multiple binding sites of the tetramer.

Molecular Cloning of Glycoprotein Ib-Binding Protein, Flavocetin-A, which Inhibits Platelet Aggregation

Flavocetin-A is a strong platelet aggregation inhibitor isolated from the venom of Trimeresurus flavoviridis. It binds specifically to platelet glycoprotein Ib with high affinity and inhibits von Willebrand factor-dependent platelet aggregation. The apparent molecular weight of flavocetin-A is 149 kDa. It consists of two subunits, alpha (17 kDa) and beta (14 kDa). The amino acid sequences of the alpha and beta subunits were determined from cloned cDNAs. Deduced amino acid sequences showed signal peptide-sequences of 23 amino acids for both alpha and beta subunits, mature peptide sequences of 135 amino acids for the alpha subunit, and 125 amino acids for the beta subunit. The amino acid sequences of alpha and beta subunits show high degrees of homology to those of C-type lectin-like venom proteins such as habu coagulation factors IX/X-binding protein (IX/X-bp), botrocetin, and alboaggregin-B. The cysteinyl residues of flavocetin-A, IX/X-bp, and botrocetin are conserved, except that flavocetin-A contains Cys 135 in the alpha subunit and Cys 3 in the beta subunit. We assumed that the arrangements of disulfide bridges in flavocetin-A are similar to those of IX/X-bp and botrocetin, and the additional Cys 135 of the alpha subunit and Cys 3 of the beta subunit are involved in novel disulfide bridges. These findings suggested that the additional disulfide bridges formed with Cys 135 of the alpha subunit and Cys 3 of the beta subunit cause polymerization of C-type lectin-like heterodimers.

Damage-Induced Neuronal Endopeptidase Is Critical for Presynaptic Formation of Neuromuscular Junctions

Damage-induced neuronal endopeptidase (DINE) is a metalloprotease belonging to the neprilysin family. Expression of DINE mRNA is observed predominantly in subsets of neurons in the CNS and peripheral nervous system during embryonic development, as well as after axonal injury. However, the physiological function of DINE and its substrate remain unknown. We generated DINE-deficient mice to examine the physiological role of DINE. Shortly after birth, these mice died of respiratory failure resulting from a dysfunction of the diaphragm, which showed severe atrophy. As DINE was abundantly expressed in motor neurons and there was atrophy of the diaphragm, we analyzed the interaction between motor nerves and skeletal muscles in the DINE-deficient mice. Although there were no obvious deficiencies in numbers of motor neurons in the spinal cord or in the nerve trajectories from the spinal cord to the skeletal muscle in DINE-deficient mice, detailed histochemical analysis demonstrated a significant decrease of nerve terminal arborization in the diaphragm from embryonic day 12.5. In accordance with the decrease of final branching, the diaphragms from DINE-deficient mice exhibited only a few neuromuscular junctions. Similar changes in nerve terminal morphology were also apparent in other skeletal muscles, including the latissimus dorsi and the intercostal muscles. These data suggest that DINE is a crucial molecule in distal axonal arborization into muscle to establish neuromuscular junctions.

Calcium-binding analysis and molecular modeling reveal echis coagulation factor IX/factor X-binding protein has the Ca-binding properties and Ca ion-independent folding of other C-type lectin-like proteins

Many biologically active heterodimeric proteins of snake venom consist of two C‐type lectin‐like subunits. One of these proteins, habu IX/X‐bp, is a Gla domain‐binding protein whose subunits both bind to a Ca2+ ion, with a total of two Ca2+‐binding sites. The molecular modeling and Ca2+‐binding analysis of echis IX/X‐bp revealed that it lacks one of two Ca2+‐binding sites, though the folding of this subunit is conserved. It is concluded that heterodimeric C‐type lectin‐like proteins function independent of Ca2+ and have essentially a similar folding to habu IX/X‐bp.

Use of Snake Venom Inhibitors in Studies of the Function and Tertiary Structure of Coagulation Factors

C-type lectin-like proteins (CLPs) of snake venom have a variety of biological properties, acting for example as anticoagulants, procoagulants, and agonists/antagonists of platelet activation. The structural and functional studies of the first identified venom CLP, factors IX/X-binding protein (IX/X-bp), have contributed to our understanding of the roles of magnesium ions in the blood coagulation cascade reaction. The crystal structures of γ-carboxyglutamic acid (Gla) domains of coagulation factors X and IX have recently been clarified in structural studies of complexes between the Gla domain of factor X and X-bp (a venom CLP) and between the Gla domain of factor IX and IX-bp (a venom CLP).