Proteome analysis of response to different spectral light irradiation in Synechocystis sp. PCC 6803.

In cyanobacteria, it is known that the excitation ratios of photosystem (PS) I and PSII changes with the wavelength of irradiated light due to mobile phycobilisome (PBS) and spillover, affecting the photosynthetic ATP/NADPH synthesis ratio and metabolic flux state. However, the mechanisms by which these changes are controlled have not been well studied. In this study, we performed a targeted proteomic analysis of Synechocystis sp. PCC 6803 under different spectral light conditions to clarify the regulation mechanisms of mobile PBS, spillover and metabolisms under different light qualities at the protein level. The results showed an increase in the amount of proteins mainly involved in CO2 fixation under Red1 light conditions with a high specific growth rate, suggesting that the rate of intracellular metabolism is controlled by the rate of carbon uptake, not by changes in the amount of each enzyme. Correlation analysis between protein levels and PSI/PSII excitation ratios revealed that PsbQUY showed high correlations and significantly increased under Blue and Red2 light conditions, where the PSI/PSII excitation ratio was higher due to spillover. In the strains lacking the genes encoding these proteins, a decrease in the PSI/PSII excitation ratio was observed, suggesting that PsbQUY contribute to spillover occurrence. SIGNIFICANCE: In cyanobacteria, the photosynthetic apparatus's responses, such as state transition [mobile PBS and spillover], occur due to the intensity and wavelength of irradiated light, resulting in changes in photosynthetic electron transport and metabolic flux states. Previous studies have analyzed the response of Synechocystis sp. PCC 6803 to light intensity from various directions, but only spectroscopic analysis of the photosynthetic apparatus has been done on the response to changes in the wavelength of irradiated light. This study analyzed the response mechanisms of mobile PBS, spillover, photosynthetic, and metabolic systems in Synechocystis sp. PCC 6803 under six different spectral light conditions by a targeted proteomic analysis. As a result, many proteins were successfully quantified, and the metabolic enzymes and photosynthetic apparatus were analyzed using an integrated approach. Principal component and correlation analyses and volcano plots revealed that the PSII subunits PsbQ, PsbU, and PsbY have a strong correlation with the PSI/PSII excitation ratio and contribute to spillover occurrence. Thus, statistical analysis based on proteome data revealed that PsbQ, PsbU, and PsbY are involved in spillover, as revealed by spectroscopic analysis.

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms.

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.

Metabolic abnormalities in adult T-cell leukemia/lymphoma and induction of specific leukemic cell death using photodynamic therapy.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm caused by human T-cell leukemia virus type I (HTLV-I). Therapeutic interventions have not been associated with satisfactory outcomes. We showed that the porphyrin metabolic pathway preferentially accumulates the endogenous photosensitive metabolite, protoporphyrin IX (PpIX) in ATL, after a short-term culture with 5-aminolevulinic acid (ALA). PpIX accumulated 10-100-fold more in ATL leukemic cells when compared to healthy peripheral blood mononuclear cells (PBMCs). Patient specimens showed dynamic changes in flow cytometry profiles during the onset and progression of ATL. Furthermore, 98.7% of ATL leukemic cell death in the ATL patient specimens could be induced with 10 min of visible light exposure, while 77.5% of normal PBMCs survived. Metabolomics analyses revealed that a specific stage of the metabolic pathway progressively deteriorated with HTLV-I infection and at the onset of ATL. Therefore, this method will be useful in diagnosing and identifying high-risk HTLV-I carriers with single cell resolutions. Photodynamic therapy in the circulatory system may be a potential treatment due to its highly-specific, non-invasive, safe, simultaneous, and repeatedly-treatable modalities.