Shigella effector IpaH4.5 targets 19S regulatory particle subunit RPN13 in the 26S proteasome to dampen cytotoxic T lymphocyte activation

Subversion of antigen‐specific immune responses by intracellular pathogens is pivotal for successful colonisation. Bacterial pathogens, including Shigella, deliver effectors into host cells via the type III secretion system (T3SS) in order to manipulate host innate and adaptive immune responses, thereby promoting infection. However, the strategy for subverting antigen‐specific immunity is not well understood. Here, we show that Shigella flexneri invasion plasmid antigen H (IpaH) 4.5, a member of the E3 ubiquitin ligase effector family, targets the proteasome regulatory particle non‐ATPase 13 (RPN13) and induces its degradation via the ubiquitin–proteasome system (UPS). IpaH4.5‐mediated RPN13 degradation causes dysfunction of the 19S regulatory particle (RP) in the 26S proteasome, inhibiting guidance of ubiquitinated proteins to the proteolytically active 20S core particle (CP) of 26S proteasome and thereby suppressing proteasome‐catalysed peptide splicing. This, in turn, reduces antigen cross‐presentation to CD8+ T cells via major histocompatibility complex (MHC) class I in vitro. In RPN13 knockout mouse embryonic fibroblasts (MEFs), loss of RPN13 suppressed CD8+ T cell priming during Shigella infection. Our results uncover the unique tactics employed by Shigella to dampen the antigen‐specific cytotoxic T lymphocyte (CTL) response.     

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase C?-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-17, interferon-γ, IL-1β, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-α in BCECs and consequently repairs TNF-α-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cϵ (PKCϵ) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCϵ inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCϵ-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.     

Absorption spectrum of allophycocyanin isolated from Anabaena cylindrica: variation of the absorption spectrum induced by changes of the physico-chemical environment

The absorption spectrum of allophycocyanin of Anabaena cylindrica was studied. The extinctions of the main absorption bands (650 and 620 nm) varied depending on the protein concentration, ionic strength, and pH. At higher protein concentrations or higher ionic strength, the 650 nm band became stronger and the 620 nm band became weaker. At pH values lower than 6.0, reverse changes occurred in association with protein dissociation into monomer. Similar spectral variation was also induced by sugars and polyols. Glucose, sucrose, or glycerol (1-5 M) induced an increase in the 650 nm band and a decrease in the 620 nm band without causing any changes in protein conformation. Propylene glycol and ethylene glycol showed a reverse effect and caused protein dissociation into monomer. The difference spectra of all spectral changes were identical, consisting of a sharp and strong peak at 650 nm and a broad and weak one in the reverse direction at a wavelength below 620 nm. The spectral variation probably results from shifts of the electronic state of phycocyanobilin. We postulated that a protein field favorable to the state producing the 650 nm band is established around phycocyanobilin when the protein takes a "tight state" through protein association or by the action of sugar in aqueous environment; in a "relaxed state" in the monomer, the state of phycocyanobilin similar to that in phycocyanin becomes dominant.     

Multiple pathways of excitation energy flow in the photosynthetic pigment system of a cryptophyte,Cryptomonas sp. (CR-1)*

Energy transfer pathways in a cryptophyte, Cryptomonas sp. (CR-1 strain) were investigated mainly by the steady state fluorescence spectroscopy and the time-resolved spectrum. Cryptomonas sp. (CR-1) contains chlorophyll (Chi) a, Chi c₂, carotenoids and cryptomonad phycoer-ythrin (Cr-PE₅₆₅), the last of which is known to be located in the lumenal side of the thyiakoid membranes. The spectral heterogeneity cf pigments was resolved by fluorescence spectra; there were at least five emission bands of Chi a at -196°C. Chlorophyll C₂ and carotenoids transferred independently to the common Chi a form (Chi a₆₆₃), and Cr-PE₅₆₅, to the different form (Chi a₆₈₂). Chlorophyll c₂ was not an intermediary component of energy transfer from carotenoid to Ch a; this is a common phenomenon to green algae and brown algae. The Chi a₆₆₃ and Chi a₆₈₂ are postulated to be located in the light-harvesting chlorophyll protein (LHC) II; thus, the energy is accumulated on Chi a₆₈₂‘n LHC II. The energy transfer step in Cr-PE_;565, was short, which was shown by a small, time-dependent red-shift of the emission. In the photosystem (PS) II core, two fluorescence components were resolved at 688 and 696 nm. The former was the trap at cryogenic temperatures. A large red-shift induced by the low temperature was explained by an equilibrium between Chi a₆₈₂ in LHC II and Chi a₆₈₈ in PS II core. The presence of Chi a₆₈₂ emission at physiological temperature is a unique feature of this alga. This was also reported in dinophyceae, which contain peridinin-ChI a-protein in the lumenal side of the thyiakoid membrane. Thus, this modification might be common in systems where the antenna complexes bind to the LHC II on the lumenal side. Based on the spectral data, we proposed a model for the molecular organization of PS II and the energy transfer pathways in cryptophyceae.     

Identification of the Terminal Energy Donor to the Reaction Center in the Pigment System of Green Plants by Time-Resolved Fluorescence Spectroscopy at - 196C

A specific antenna component in the photosynthetic pigment system,the direct energy donor to the reaction center (RC), was identifiedin PS I and II in green plants by time-resolved fluorescencespectroscopy at – 196C. The fluorescence components weredetected at 707 nm and 685 nm for PS I and II, respectively,and these antennae are hereafter referred to as the terminalenergy donors. In PS II, component that fluoresces at 695 nmalso functions as an energy pool; its rise time is slow (50ps), suggesting that it represents a side path in the flow ofenergy. The energy levels of these direct donors were lowerthan, or almost the same as that of the primary electron donorsin RC I (P700) and II (P680), respectively. The similaritiesand differences between these donors are discussed in relationto the structural and energetic properties of the antenna componentsin PS I and II core complexes. ¹This paper is dedicated to Prof. Y. Fujita on the occationof the 60th anniversary of his birth.     

Establishment of the forward genetic analysis of the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017 by applying in vivo transposon mutagenesis system

Photosynthesis Research - Acaryochloris marina MBIC 11017 possesses chlorophyll (Chl) d as a major Chl, which enables this organism to utilize far-red light for photosynthesis. Thus, the adaptation...     

Studies on excitation energy flow in the photosynthetic pigment system; Structure and energy transfer mechanism

Excitation energy flow in photosynthetic pigment systems is discussed in relation to structure of the system and transfer mechanism for each elementary process. Three typical examples for actual transfer processes are shown for the phycobilin system in cyanobacteria, the antenna system of photosynthetic bacteria and the Chla/c antenna system of brown algae. The main analytical method was the time-resolved fluorescence spectroscopy in the picosecond time range. In general, static optical charactersitics are not the main reason for the transfer efficiency, but the structure of the system is a prerequisite for the transfer process. On the phycobilin system, theoretical investigation was compared with experimental analysis, which leads to the essential understanding of the transfer process in terms of quantum mechanics. Recipient of the Botanical Society Award for Young Scientists, 1989.