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Yuichi Takakuwa

Publications and source records attributed to Yuichi Takakuwa.

11 recordsLinked to original sources
PubMed2006

Regulation of protein 4.1R interactions with membrane proteins by Ca2+ and calmodulin.

Red blood cell protein 4.1 (4.1R) is essential for maintaining erythrocyte shape and controlling membrane mechanical properties, such as deformability and stability. The importance of 4.1R has been demonstrated by the dramatic erythrocyte alterations observed in patients lacking this protein. Indeed, 4.1R null red blood cells adopt an elliptical shape and are characterized by unstable membranes. The key role of 4.1R likely results from multiple protein-protein interactions: lateral interactions with the spectrin/actin network and vertical interactions with the cytoplasmic domain of transmembrane proteins glycophorin C (GPC), Band 3 (anion exchanger 1, AE1), and CD44. 4.1R promotes the formation of a ternary complex with GPC and p55 through its 30 kDa membrane-binding domain. Based on the primary structure of the prototypical 80 kDa isoform of 4.1R, functional domains and sites for binding partners have been identified. The others and we have been focusing on the structure and function of the 30 kDa NH2-terminal domain of 4.1R, which is responsible for 4.1R interaction with the transmembrane proteins described above. A major finding is that Ca2+, in association with calmodulin (CaM), plays a critical role in regulation of the interaction of the 30 kDa domain with its various binding partners. This review is a detailed report of our current knowledge regarding 4.1R, and more specifically, 4.1R 30 kDa domain: its primary structure, functions and modulation by Ca2+ and CaM. Emphasis is given on the relationships between structure and function that we have been able to establish through X-ray crystal structure analysis of the 30 kDa membrane-binding domain in 4.1R. Finally, we give insights into the potential roles of 4.1R in the dynamic organization of the membrane skeleton viewed as a complex system.

Alternative Splicing↗
PubMed2005

Transgenic over-expression of GATA-1 mutant lacking N-finger domain causes hemolytic syndrome in mouse erythroid cells.

Transcription factor GATA-1 is essential for erythroid cell differentiation. GATA-binding motifs have been found in the regulatory regions of various erythroid-specific genes, suggesting that GATA-1 contributes to gene regulation during the entire process of erythropoiesis. A GATA-1 germ-line mutation results in embryonic lethality due to defective primitive erythropoiesis and GATA-1-null embryonic stem cells fails to differentiate beyond the proerythroblast stage. Therefore, the precise roles of GATA-1 in the later stages of erythropoiesis could not be clarified. Under the control of a GATA-1 gene hematopoietic regulatory domain, a GATA-1 mutant lacking the N-finger domain (DeltaNF mutant) was over-expressed in mice. These mice exhibited abnormal morphology in peripheral red blood cells (RBCs), reticulocytosis, splenomegaly, and erythroid hyperplasia, indicating compensated hemolysis. These mice were extremely sensitive to phenylhydrazine (PHZ), an agent that induces hemolysis, and their RBCs were osmotically fragile. Importantly, the hemolytic response to PHZ was partially restored by the simultaneous expression of wild-type GATA-1 with the DeltaNF mutant, supporting our contention that DeltaNF protein competitively inhibits the function of endogenous GATA-1. These data provide the first in vivo evidence that the NF domain contributes to the gene regulation that is critical for differentiation and survival of mature RBCs in postnatal erythropoiesis.

Animals↗
PubMed2004

Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation.

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.

Actins↗
PubMed2003

In situ observation of mobility and anchoring of PKCbetaI in plasma membrane.

We employed fluorescence correlation spectroscopy (FCS) to analyze the characteristics of biomolecules in living cells. Protein kinase C (PKC) changes its subcellular localization from cytosol to the plasma membrane by its ligand. Using FCS, we found PKCbetaI labeled with enhanced green fluorescent protein freely diffusing in cytosol. Upon 12-O-tetradecanoylphorbol-13-acetate activation, a large part of PKCbetaI is anchored in the plasma membrane but some PKCbetaI still moves freely near the plasma membrane. These results indicate that a diffusion-driven transport mechanism is appropriate for the molecular mechanism of the PKCbetaI localization change.

Cell Line↗
PubMed2002

Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development.

Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.

Animals↗
PubMed2002

Flow cytometric analysis of reticulated platelets in patients with ischemic stroke.

Reticulated platelets are newly formed platelets containing a residual amount of RNA, and percentage of reticulated platelets (%RP) has been shown to reflect platelet turnover. Recently, a new flow cytometric approach for analyzing %RP in patients with thrombocytopenic disorders has been reported. We measured %RP by flow cytometry using the fluorescent dye thiazole orange (TO) to evaluate platelet kinetics in patients with different clinical categories of ischemic stroke. Patients with ischemic stroke were categorized into lacunar (n=25), atherothrombotic (n=26) and cardioembolic stroke (n=17). %RP was significantly higher in patients with cardioembolic stroke than in controls (n=140). Stepwise multiple regression analysis also showed cardioembolic stroke (R(2)=0.14) to be significant independent predictors of %RP among stroke patients even after adjustment for other factors. We concluded that %RP is increased in patients with cardioembolic stroke, which may reflect increased platelet turnover as a consequence of platelet consumption during thrombogenesis.

Aged↗
PubMed2002

Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability.

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin-actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.

Adenosine Triphosphate↗
PubMed2002

Useful markers for detecting decreased serum antioxidant activity in hemodialysis patients.

This study evaluates a novel application of a method for measuring serum antioxidant activity, based on the detection of erythrocyte membrane lipid peroxidation in cases of uremia. A human erythrocyte ghost membrane in Tris-HCl was mixed with adenosine 5'-diphosphate and iron chloride (FeCl3; ADP/Fe3+) solution (at a molar ratio of 17:1), and the mixture was incubated under aerobic conditions at 37 degrees C for 2 hours. The concentration of erythrocyte membrane thiobarbituric acid-reactive substances increased proportionally with respect to ADP/Fe3+ concentration, and this increase was inhibited by serum albumin in a dose-dependent manner. In patients undergoing chronic hemodialysis therapy, predialytic sera contained in this reaction mixture were weaker than postdialytic sera in terms of inhibitory effect against erythrocyte membrane lipid peroxidation, whereas serum albumin contents remained at levels equivalent to those of the normal control. A gradual increase in human mercaptalbumin nonmercaptalbumin ratio during hemodialysis treatment might be one of the major factors that leads to the recovery of decreased serum antioxidant activity. We clearly showed that the serum scavenging activity against erythrocyte membrane lipid peroxidation in hemodialysis patients decreases markedly, and this pathological condition is improved by hemodialysis.

Adenosine Monophosphate↗
PubMed2002

Hyperzincemia with systemic inflammation: a heritable disorder of calprotectin metabolism with rheumatic manifestations?

A boy had infantile-onset systemic inflammation, growth failure, hepatosplenomegaly, anemia, leukocytopenia, progressive muscular dystrophy, and hypercalprotectinemia, resulting in marked hyperzincemia. His mother had a history of chronic arthritis since childhood and also showed hypercalprotectinemia/hyperzincemia. We postulate an inherent defect in calprotectin metabolism.

Adolescent↗