Enterohemorrhagic E. coli (EHEC) infections are a leading cause of foodborne illness in the United States. Shiga-like toxins are produced that can cause hemorrhagic colitis and can lead to dangerous complications, such as acute kidney injury and hemolytic uremic syndrome (HUS). There are currently no specific treatments for HUS, and therefore more research into EHEC and HUS needs to be done. Our study focuses on Shiga-like toxin induction of endoplasmic reticulum (ER) stress in in vitro and in vivo systems, using human monocyte-like THP-1 cells and a non-human primate model of HUS. We used qPCR to determine the levels of ER stress marker expression induced by both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) challenges. We also looked at ER stress marker expression in non-human primates that survived a lethal Stx2 challenge after being given a Stx2 binding tetravalent peptide. We expected to see increased ER stress marker expression in THP-1 cells challenged with both Shiga-like toxins and in animals that received lethal doses of the toxins. Although results were inconclusive for THP-1 cell experiments, our preliminary non-human primate data suggest that the timing of ER stress marker production is important, and Shiga-like toxins may suppress the unfolded protein response (UPR) in some baboon tissues. We also show that the therapeutic peptide TVP may reverse this UPR suppression and relieve ER stress leading to animal survival. Our study, along with the current literature, shows that Shiga-like toxin induced ER stress is a promising area for future study.

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+.6,25 The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid15 or release of Ca2+ from intracellular stores such as mitochondria.1,8,18 Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose,2 so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10-4M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10-4M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.

The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.

CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36-/- mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36+/+ mice. CD36-/- mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36-/- mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB.

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23600号 / 理博第4762号 / 新制||理||1683(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 細川 暢子, 教授 杤尾 豪人, 教授 森 和俊 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The use of peptides in drug development has been hampered by their poor pharmaceutical properties, most notably their inability to reliably permeate biological membranes and lack of targeting. To overcome these disadvantages, the AMino acid Intracellular Delivery SysTem (AMIDST) was developed. This modular peptide-based delivery system confers cellular permeability and organelle-specific targeting for therapeutic peptides. As demonstrated in this study, the delivery of a HER2-binding peptide to the secretory organelles of breast cancer cells resulted in intracellular sequestration, a reduction in downstream signalling, and reduced viability compared to the delivery of a control peptide. Given its modular design and ease of production, AMIDST has the potential to enhance the use of peptides as therapeutic agents.

Excessive endoplasmic reticulum (ER) stress leads to cell loss in many diseases, e.g., contributing to endothelial cell loss after spinal cord injury. Here, we determined whether ER stress-induced mitochondrial dysfunction could be explained by interruption of the focal adhesion kinase (FAK)-mitochondrial STAT3 pathway we recently discovered. ER stress was induced in brain-derived mouse bEnd5 endothelial cells by thapsigargin or tunicamycin and caused apoptotic cell death over a 72 h period. In concert, ER stress caused mitochondrial dysfunction as shown by reduced bioenergetic function, loss of mitochondrial membrane potential and increased mitophagy. ER stress caused a reduction in mitochondrial phosphorylated S727-STAT3, known to be important for maintaining mitochondrial function. Normal activation or phosphorylation of the upstream cytoplasmic FAK was also reduced, through mechanisms that involve tyrosine phosphatases and calcium signaling, as shown by pharmacological inhibitors, bisperoxovanadium (bpV) and 2-aminoethoxydiphenylborane (APB), respectively. APB mitigated the reduction in FAK and STAT3 phosphorylation, and improved endothelial cell survival caused by ER stress. Transfection of cells rendered null for STAT3 using CRISPR technology with STAT3 mutants confirmed the specific involvement of S727-STAT3 inhibition in ER stress-mediated cell loss. These data suggest that loss of FAK signaling during ER stress causes mitochondrial dysfunction by reducing the protective effects of mitochondrial STAT3, leading to endothelial cell death. We propose that stimulation of the FAK-STAT3 pathway is a novel therapeutic approach against pathological ER stress.

Investigators have been attempting to identify the receptor for ribosomes on the rough endoplasmic reticulum (RER) for almost 20 years, yet the ribosome receptor has remained elusive. Rough microsomal membranes contain endogenous ribosomes bound in at least two types of interactions. Loosely associated ribosomes can be removed by extraction with a high ionic strength solution, but ribosomes that were actively engaged in translocation when the membranes were isolated remain tethered to the membrane by a nascent polypeptide (Adelman et al., 1973). The original assay for the ribosome receptor involved stripping all of the endogenous ribosomes off of intact membranes before adding back a quantitated amount of ribosomes. More recent assays have employed detergent solubilization of the membrane and then reconstitution of the membrane proteins into lipid vesicles before adding back ribosomes. In both cases ribosome binding to its receptor is measured in an assay that does not involve translation or translocation. We utilized a crosslinking assay to attempt to identify membrane proteins that function as a binding site for ribosomes engaged in protein translocation across the endoplasmic reticulum. In vivo bound ribosomes that remain associated with the membrane after extraction with a high ionic strength solution are likely to be bound to a functional translocation site. The water soluble, membrane impermeable, thiol-cleavable crosslinker 3,3'-dithiobis (sulfosuccinimidylpropionate) was selected to limit reaction to protein domains located on the cytoplasmic face of salt extracted microsomal membrane vesicles. A specific subset of RER proteins was reproducibly crosslinked to the endogenous ribosomes. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35 kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180 kD protein that had recently been proposed to be a ribosome receptor (Savitz and Meyer, 1990). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes in an in vitro binding assay to determine whether ribosome binding activity could be ascribed to the 180 kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180 kD protein, as determined by Coomassie blue staining of a polyacrylamide gel. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments and functional assays with proteoliposomes demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180 kD protein by chromatography. Taken together, the evidence indicates that the 180 kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor. To continue the investigation of ribosome binding, an assay was designed to characterize ribosome-nascent chain complexes bound to the microsomal membrane during translocation. A series of translocation intermediates consisting of discrete sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. Proteinase K was then used as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport. Only those partially translocated nascent chains of 100 amino acids or less were insensitive to protease digestion by externally added protease. It was concluded that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, shorter nascent polypeptides appear not to have lumenally exposed segments. Ultimately, a functional assay for the ribosome receptor should include binding studies conducted under physiological conditions. For this purpose, an assay was developed that allowed translation, translocation, and termination of a secretory protein to be monitored with probes designed to independently quantitate translating and non-translating ribosomes. A synchronized wheat-germ translation system was programmed with bovine preprolactin mRNA and aliquots were taken at various time points before and after adding membranes. The samples were then separated into membrane bound and soluble species by centrifugation. RNA was isolated from each supernatant and pellet sample and blotted onto nylon sheets. By probing the dot blots with probes that hybridize with either the 5S RNA of wheat germ ribosomes or the preprolactin transcript, the translating ribosomes could be monitored without the interference of the endogenous canine ribosomes on the membrane. By comparing the total amount of preprolactin transcript that bound to the membrane versus the total amount of wheat germ ribosomes bound to the membrane, it was discovered that the vast majority (>99%) of wheat germ ribosomes that bound to the microsomal membrane were non-translating ribosomes. In later experiments it was found that the non-translating ribosomes did not compete with the translating ribosomes; under all conditions tested, the translating ribosomes had access to translocation sites on the microsomal membrane. One interpretation of this data is that all ribosome binding sites are not identical. It may be that functional sites for translocation are a distinct subclass of total ribosome binding sites. Another interpretation is that a ribosome in a nascent chain-SRP complex has a much higher affinity for the ribosome receptor than nontranslating ribosomes or 60S subunits. Perhaps the non-translating ribosome can not compete with ribosomes engaged in translocation. As stated earlier, ribosomes do make at least two kinds of interactions with the microsomal membrane surface. This data may be indicative of those types of interactions.