Molecular Mechanism of Spectral Tuning by Chloride Binding in Monkey Green Sensitive Visual Pigment.

The visual pigments of the cones perceive red, green, and blue colors. The monkey green (MG) pigment possesses a unique Cl- binding site; however, its relationship to the spectral tuning in green pigments remains elusive. Recently, FTIR spectroscopy revealed the characteristic structural modifications of the retinal binding site by Cl- binding. Herein, we report the computational structural modeling of MG pigments and quantum-chemical simulation to investigate its spectral redshift and physicochemical relevance when Cl- is present. Our protein structures reflect the previously suggested structural changes. AlphaFold2 failed to predict these structural changes. Excited-state calculations successfully reproduced the experimental red-shifted absorption energies, corroborating our protein structures. Electrostatic energy decomposition revealed that the redshift results from the His197 protonation state and conformations of Glu129, Ser202, and Ala308; however, Cl- itself contributes to the blueshift. Site-directed mutagenesis supported our analysis. These modeled structures may provide a valuable foundation for studying cone pigments.

High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range of "Difficult-to-Express" Secretory Recombinant Proteins.

Yeast's extracellular expression provides a cost-efficient means of producing recombinant proteins of academic or commercial interests. However, depending on the protein to be expressed, the production occasionally results in a poor yield, which is frequently accompanied with a deteriorated growth of the host. Here we describe our simple approach, high cell-density expression, to circumvent the cellular toxicity and achieve the production of a certain range of "difficult-to-express" secretory protein in preparative amount. The system features an ease of performing: (a) pre-cultivate yeast cells to the stationary phase in non-inducing condition, (b) suspend the cells to a small aliquot of inducing medium to form a high cell-density suspension or "a phalanx," then (c) give a sufficient aeration to the phalanx. Factors and pitfalls that affect the system's performance are also described.

Endoplasmic Reticulum-Associated Degradation-Dependent Processing in Cross-Presentation and Its Potential for Dendritic Cell Vaccinations: A Review.

While the success of dendritic cell (DC) vaccination largely depends on cross-presentation (CP) efficiency, the precise molecular mechanism of CP is not yet characterized. Recent research revealed that endoplasmic reticulum (ER)-associated degradation (ERAD), which was first identified as part of the protein quality control system in the ER, plays a pivotal role in the processing of extracellular proteins in CP. The discovery of ERAD-dependent processing strongly suggests that the properties of extracellular antigens are one of the keys to effective DC vaccination, in addition to DC subsets and the maturation of these cells. In this review, we address recent advances in CP, focusing on the molecular mechanisms of the ERAD-dependent processing of extracellular proteins. As ERAD itself and the ERAD-dependent processing in CP share cellular machinery, enhancing the recognition of extracellular proteins, such as the ERAD substrate, by ex vivo methods may serve to improve the efficacy of DC vaccination.

Loss of poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster.

Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. Here we report a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25 degrees C. However, one-fourth of the mutants progress to the adult stage at 29 degrees C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADP-ribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimer's and Parkinson's diseases that are caused by abnormal accumulation of substances in nervous tissue.

Subcellular localization of poly(ADP-ribose) glycohydrolase in mammalian cells.

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.