Biomedical subjects

Hirokazu Satoh

Publications and source records attributed to Hirokazu Satoh.

6 recordsLinked to original sources

PubMed2006

Establishment of a high sensitivity plasma assay for human pentraxin3 as a marker for unstable angina pectoris.

OBJECTIVE: Plasma pentraxin 3 (PTX3) levels are increased in patients with acute myocardial infarction, yet its involvement in unstable angina pectoris (UAP) remains unclear. To critically evaluate the role of PTX3 in UAP, a sensitive and precise measurement of PTX3 concentration is needed. METHODS AND RESULTS: We established a high sensitive plasma ELISA assay system for the detection of PTX3 using monoclonal antibodies. The lower limit of detection of our ELISA was 0.1 ng/mL, sensitivity far greater than the current commercially available kit. Plasma samples were obtained from 162 consecutive patients treated for hypertension, hyperlipidemia, diabetes mellitus, or cardiovascular disease at a physician's office. PTX3 was not associated with any known coronary risk factors. Additionally, we collected plasma samples from 252 consecutive subjects admitted to a university hospital for coronary artery assessment by coronary angiography. PTX3 was significantly increased in patients in whom coronary intervention was performed. We further analyzed the plasma level of PTX3 in 52 patients with effort angina (EAP) and 16 patients with UAP. Compared with the control group, PTX3 were significantly higher in the UAP group. CONCLUSIONS: The levels of plasma PTX3 were increased in patients with arterial inflammation, especially UAP. This PTX3 detection system will be useful for the prediction of UAP.

Adult↗

PubMed2004

The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1.

ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca(2+)-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca(2+) with FLAG-tagged Alix or FLAG-tagged Alix Delta EBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged Alix Delta ABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca(2+)-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca(2+)-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca(2+). Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCT Delta ABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca(2+)-dependent interaction with Alix.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Structures, functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins.

Penta-EF-hand (PEF) proteins comprise a family of Ca(2+)-binding proteins that have five repetitive EF-hand motifs. Among the eight alpha-helices (alpha1-alpha8), alpha4 and alpha7 link EF2-EF3 and EF4-EF5, respectively. In addition to the structural similarities in the EF-hand regions, the PEF protein family members have common features: (i) dimerization through unpaired C-terminal EF5s, (ii) possession of hydrophobic Gly/Pro-rich N-terminal domains, and (iii) Ca(2+)-dependent translocation to membranes. Based on comparison of amino acid sequences, mammalian PEF proteins are classified into two groups: Group I PEF proteins (ALG-2 and peflin) and Group II PEF proteins (Ca(2+)-dependent protease calpain subfamily members, sorcin and grancalcin). The Group I genes have also been found in lower animals, plants, fungi and protists. Recent findings of specific interacting proteins have started to gradually unveil the functions of the noncatalytic mammalian PEF proteins.

Amino Acid Sequence↗

PubMed2002

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner.

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.

Annexin A7↗

PubMed2002

ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner.

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.

Amino Acid Sequence↗

PubMed2002

Both ALG-2 and peflin, penta-EF-hand (PEF) proteins, are stabilized by dimerization through their fifth EF-hand regions.

ALG-2 (apoptosis-linked gene-2 protein) and peflin are Ca(2+)-binding proteins and belong to the penta-EF-hand (PEF) protein family, which includes calpain, sorcin, and grancalcin. ALG-2 forms either a homodimer or a heterodimer with peflin like other PEF proteins. In this study, we found that the fifth-EF-hand (EF-5) regions of both ALG-2 and peflin are essential for dimerization and their stabilities. Exogenously expressed EF-5-deletion (DeltaEF-5) mutants of ALG-2 and peflin were unstable and were not detected in HEK293 cells by Western blotting. In a pulse--chase experiment, the DeltaEF-5 mutants were rapidly degraded, but they were stabilized by treatment with a proteasome inhibitor, MG132. In MG132-treated cells, DeltaEF-5 mutants were recovered in the insoluble fractions. Transient coexpression of ALG-2 increased the peflin level. These results indicate that the absence of a fifth EF-hand results in rapid degradation by the proteasome. On the other hand, stable expression of exogenous peflin decreased the amount of endogenous peflin. The amount of peflin that can dimerize with ALG-2 seems to be restricted in mammalian cells.

Apoptosis Regulatory Proteins↗