Biomedical subjects

Yuichi Fujita

Publications and source records attributed to Yuichi Fujita.

8 recordsLinked to original sources

PubMed2006

Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr(138) predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.

Animals↗

PubMed2006

Nitrogenase Fe protein-like Fe-S cluster is conserved in L-protein (BchL) of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide reductase (DPOR) in bacteriochlorophyll biosynthesis is a nitrogenase-like enzyme consisting of L-protein (BchL-dimer) as a reductase component and NB-protein (BchN-BchB-heterotetramer) as a catalytic component. Metallocenters of DPOR have not been identified. Here we report that L-protein has an oxygen-sensitive [4Fe-4S] cluster similar to nitrogenase Fe protein. Purified L-protein from Rhodobacter capsulatus showed absorption spectra and an electron paramagnetic resonance signal indicative of a [4Fe-4S] cluster. The activity quickly disappeared upon exposure to air with a half-life of 20s. These results suggest that the electron transfer mechanism is conserved in nitrogenase Fe protein and DPOR L-protein.

Bacterial Proteins↗

PubMed2006

Differential operation of dual protochlorophyllide reductases for chlorophyll biosynthesis in response to environmental oxygen levels in the cyanobacterium Leptolyngbya boryana.

Most oxygenic phototrophs, including cyanobacteria, have two structurally unrelated protochlorophyllide (Pchlide) reductases in the penultimate step of chlorophyll biosynthesis. One is light-dependent Pchlide reductase (LPOR) and the other is dark-operative Pchlide reductase (DPOR), a nitrogenase-like enzyme assumed to be sensitive to oxygen. Very few studies have been conducted on how oxygen-sensitive DPOR operates in oxygenic phototrophic cells. Here, we report that anaerobic conditions are required for DPOR to compensate for the loss of LPOR in cyanobacterial cells. An LPOR-lacking mutant of the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) failed to grow in high light conditions and this phenotype was overcome by cultivating it under anaerobic conditions (2% CO(2)/N(2)). The critical oxygen level enabling the mutant to grow in high light was determined to be 3% (v/v). Oxygen-sensitive Pchlide reduction activity was successfully detected as DPOR activity in cell-free extracts of anaerobically grown mutants, whereas activity was undetectable in the wild type. The content of two DPOR subunits, ChlL and ChlN, was significantly increased in mutant cells compared with wild type. This suggests that the increase in subunits stimulates the DPOR activity that is protected efficiently from oxygen by anaerobic environments, resulting in complementation of the loss of LPOR. These results provide important concepts for understanding how dual Pchlide reductases operate differentially in oxygenic photosynthetic cells grown under natural environments where oxygen levels undergo dynamic changes. The evolutionary implications of the coexistence of two Pchlide reductases are discussed.

Anaerobiosis↗

PubMed2006

A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis: reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus.

In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase (DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase (COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm (P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenase-like enzymes to determine the ring structure of (bacterio)chlorophyll pigments are discussed.

Adenosine Triphosphate↗

PubMed2005

Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide oxidoreductase (DPOR) plays a crucial role in light-independent (bacterio)chlorophyll biosynthesis in most photosynthetic organisms. However, the biochemical properties of DPOR are still largely undefined. Here, we constructed an overexpression system of two separable components of DPOR, L-protein (BchL) and NB-protein (BchN-BchB), in the broad-host-range vector pJRD215 in Rhodobacter capsulatus. We established a stable DPOR assay system by mixing crude extracts from the two transconjugants under anaerobic conditions. Using this assay system, we demonstrated some basic properties of DPOR. The Km value for protochlorophyllide was 10.6 muM. Ferredoxin functioned as an electron donor to DPOR. Elution profiles in gel filtration chromatography indicated that L-protein and NB-protein are a homodimer [(BchL)(2)] and a heterotetramer [(BchN)(2)(BchB)(2)], respectively. These results provide a framework for the characterization of these components in detail, and further support a nitrogenase model of DPOR.

Base Sequence↗

PubMed2004

A case of Kuttner tumor of the submandibular gland.

Kuttner tumor (known as chronic sclerosing sialadenitis) is a totally benign inflammatory lesion and to date there has been no report of malignancy. However, because of its clinical features, the clinical diagnosis is often that of a salivary gland neoplasm. We report a case of Kuttner tumor and discuss its differential diagnosis, especially the pathological diagnosis. Kuttner tumor may be mainly due to T lymphocyte immune reaction and of itself is a benign lesion, but it may provide a state in which a malignant lesion arises.

Aged↗

PubMed2003

Developmental regulation of the proteolysis of the p35 cyclin-dependent kinase 5 activator by phosphorylation.

Cyclin-dependent kinase 5 (Cdk5), a cdc2-related kinase expressed in postmitotic neurons, is activated by association with a brain-specific activator, p35. It has been suggested that the conversion of p35 to p25 by the protease calpain is involved in neuronal cell death. However, p35 protein is turned over rapidly via proteasomal degradation in living neurons. In this study we show that the phosphorylation of p35 by Cdk5 suppresses the cleavage to p25 by calpain, whereas phosphorylation facilitates the proteasomal degradation of p35. The phosphorylation site in p35 that might be involved in preventing calpain cleavage was distinct from the phosphorylation site involved in facilitating proteasomal degradation. A phosphorylated form of p35 that was resistant to cleavage by calpain was more prevalent in the fetal brain, whereas the unphosphorylated form of p35 occurred in the adult brain. These results suggest that the phosphorylation of p35 serves as a protective mechanism that suppresses the generation of p25 in developing brains.

Age Factors↗

PubMed2003

Arrest of chlorophyll synthesis and differential decrease of Photosystems I and II in a cyanobacterial mutant lacking light-independent protochlorophyllide reductase.

The chlL gene encodes one subunit of the light-independent protochlorophyllide reductase. A chlL-lacking mutant of the cyanobacterium Plectonema boryanum is unable to synthesize chlorophyll (Chl) in the dark, causing Chl synthesis to become light-dependent as in angiosperms. When the mutant cells were cultivated heterotrophically in the dark, Chl synthesis was arrested and the Chl content decreased exponentially in reverse profile to cell propagation, indicating that most of the pre-existing Chl was recruited for daughter cells. During this 'etiolating' process the Chl content became less than 0.5% of the original level. In parallel to this there was a decrease in the activity of Photosystem I (PSI), the amount of its core Chl-binding subunits, PsaA/PsaB, and a peripheral subunit, PsaC. Levels of transcripts for these subunits were not significantly changed upon the arrest of Chl synthesis. In contrast, Photosystem II (PSII) was maintained to a significant extent in terms of activity and protein levels of D1 and CP47 until a late stage of the etiolation, implying that PSII is newly synthesized though Chl synthesis was arrested. Low-temperature (77 K) fluorescence spectral analysis supported a selective decrease in Chl associated with PSI. Taken together, it is suggested that the pre-existing Chl molecules in periphery of PSI could be released and re-distributed for PSII biosynthesis in the etiolating cyanobacterial cells.

Bacterial Proteins↗