Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster

Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior.     

Comparative biochemical studies of placental alkaline phosphatase in animals (author's transl)]

Biochemical properties of alkaline phosphatase (ALP) from placenta were compared between man, dog, cat, rabbit and cattle. 1) Optimum pH of the enzyme was essentially identical through the species of the animals but the inhibition of L-phenylalanine was clearly demonstrable with human ALP but little with that of other animals. 2) ALP of human placenta was not inactivated by heating at 65 degrees C for 15 min. but one of the other animals was thermolabile. Such thermostability of human placental ALP almost disappeared after treatment with EDTA, suggesting that the divalent metal ions were required for the thermostability. 3) Activities of placental ALP were inhibited by cationsurfactants in human and rat but not in the other animals, while the inhibition by DOC-Na, an anion-surfactant, was seen only in human. 4) The affinity to beta-glycerophosphae of placental ALP was seen only in human.     

Serotonin attenuates biotic stress and leads to lesion browning caused by a hypersensitive response to Magnaporthe oryzae penetration in rice

The hypersensitive response (HR) of plants is one of the earliest responses to prevent pathogen invasion. A brown dot lesion on a leaf is visual evidence of the HR against the blast fungus Magnaporthe oryzae in rice, but tracking the browning process has been difficult. In this study, we induced the HR in rice cultivars harboring the blast resistance gene Pit by inoculation of an incompatible M. oryzae strain, which generated a unique resistance lesion with a brown ring (halo) around the brown fungal penetration site. Inoculation analysis using a plant harboring Pit but lacking an enzyme that catalyzes tryptamine to serotonin showed that high accumulation of the oxidized form of serotonin was the cause of the browning at the halo and penetration site. Our analysis of the halo browning process in the rice leaf revealed that abscisic acid enhanced biosynthesis of serotonin under light conditions, and serotonin changed to the oxidized form via hydrogen peroxide produced by light. The dramatic increase in serotonin, which has a high antioxidant activity, suppressed leaf damage outside the halo, blocked expansion of the browning area and attenuated inhibition of plant growth. These results suggest that serotonin helps to reduce biotic stress in the plant by acting as a scavenger of oxygen radicals to protect uninfected tissues from oxidative damage caused by the HR. The deposition of its oxide at the HR lesion is observed as lesion browning.     

Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation

Ferroptosis is a form of regulated non-apoptotic cell death that has been implicated in several disease contexts. A better understanding of the ferroptotic death mechanism could lead to the development of new therapeutics for degenerative diseases, and a better understanding of how to induce ferroptosis in specific tumor contexts. We performed an unbiased genome-wide siRNA screen to find genetic suppressors of ferroptosis. We determined that loss of CARS, the cysteinyl-tRNA synthetase, suppresses ferroptosis induced by erastin, which inhibits the cystine–glutamate antiporter known as system x_c⁻. Knockdown of CARS inhibited erastin-induced death by preventing the induction of lipid reactive oxygen species, without altering iron homeostasis. Knockdown of CARS led to the accumulation of cystathionine, a metabolite on the transsulfuration pathway, and upregulated genes associated with serine biosynthesis and transsulfuration. In addition, inhibition of the transsulfuration pathway resensitized cells to erastin, even after CARS knockdown. These studies demonstrate a new mechanism of resistance to ferroptosis and may lead to strategies for inducing and suppressing ferroptosis in diverse contexts.Precise regulation of cell death is essential for tissue homeostasis. Dysregulation of cell death processes is implicated in a variety of pathological conditions, such as ischemia and neurodegenerative diseases, providing a rationale for exploring cell-death-modulating compounds as potential therapeutics.¹ However, an incomplete understanding of cell death mechanisms in specific disease contexts has hindered efforts to develop therapeutics. Mechanistic analyses of cell death processes in disease contexts may uncover new strategies for drug discovery. Ferroptosis, a form of oxidative, non-apoptotic cell death, has recently been described and implicated in several pathological conditions, including Huntington''s disease (HD), periventricular leukomalacia (PVL) and kidney dysfunction.^{2, 3, 4} Ferroptotic cell death can be induced through perturbation of redox homeostasis maintained by glutathione, a key regulator of the intracellular redox state.Glutathione (GSH) is a tripeptide, the synthesis of which is dependent on the availability of the amino acid cysteine. A substantial fraction of extracellular cysteine exists as its oxidized disulfide form, cystine, because of the oxidative extracellular environment.⁵ Some cells primarily obtain cysteine by importing extracellular cystine through system x_c⁻, the cystine–glutamate antiporter. Cystine is then reduced to cysteine inside cells, fueling GSH synthesis. GSH maintains redox homeostasis by acting as a reductive substrate for reactive oxygen species (ROS)-detoxifying enzymes. As one example, glutathione peroxidase 4 (GPX4) uses GSH to reduce lipid hydroperoxides and organic hydroperoxides to alcohols, serving a critical role in lipid repair and detoxification. GPX4 was recently shown to be a central regulator of ferroptosis.⁶Ferroptosis can be induced by two classes of compounds, exemplified by erastin and (1 S, 3 R)-RSL3.^{6, 7, 8, 9} These two compounds target different parts of the ferroptotic pathway. Erastin inhibits system x_c⁻ to deplete GSH, which effectively inactivates all cellular glutathione peroxidases, including GPX4. RSL3, on the other hand, acts downstream, inhibiting GPX4 directly. In both cases, the loss of GPX4 activity causes accumulation of lipid peroxides, and ultimately, cell death. Recently, the FDA-approved drugs sorafenib and sulfasalazine were also found to induce ferroptosis through inhibition of system x_c⁻ activity,^{10, 11} although these lower-potency compounds may also activate other competing processes at similar or slightly higher concentrations. A specific inhibitor of ferroptosis, ferrostatin-1, and its analogs have been shown to suppress cell death in several degenerative disease models, including HD, PVL and kidney dysfunction, as well as in a model of glutamate toxicity, suggesting the involvement of ferroptosis in these conditions.^{4, 12} Collectively, these findings suggest that modulation of ferroptosis is of potential therapeutic relevance in several pathological conditions.Given the involvement of ferroptosis in these different contexts, we sought to identify specific features and regulators of ferroptosis. Ferroptosis is biochemically and morphologically distinct from necrosis and apoptosis.¹² Genetic analysis of ferroptosis has been performed using a limited set of genes related to mitochondrial function.¹² This previous analysis revealed that ferroptosis requires a distinct set of genes compared with apoptosis. However, this analysis cast a relatively narrow net; therefore, we sought to extend our understanding of the genetic regulation of ferroptosis further to identify essential genes and pathways using a genome-wide siRNA screen. Such genes may illuminate novel targets whose inhibition could be therapeutic in disease conditions involving aberrant activation of ferroptosis, or suggest strategies for inducing ferroptosis in specific tumor contexts.     

Molecular Characterization of an Intact p53 Pathway Subtype in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive histological type of epithelial ovarian cancer, which is characterized by a high frequency of somatic TP53 mutations. We performed exome analyses of tumors and matched normal tissues of 34 Japanese patients with HGSOC and observed a substantial number of patients without TP53 mutation (24%, 8/34). Combined with the results of copy number variation analyses, we subdivided the 34 patients with HGSOC into subtypes designated ST1 and ST2. ST1 showed intact p53 pathway and was characterized by fewer somatic mutations and copy number alterations. In contrast, the p53 pathway was impaired in ST2, which is characterized by abundant somatic mutations and copy number alterations. Gene expression profiles combined with analyses using the Gene Ontology resource indicate the involvement of specific biological processes (mitosis and DNA helicase) that are relevant to genomic stability and cancer etiology. In particular we demonstrate the presence of a novel subtype of patients with HGSOC that is characterized by an intact p53 pathway, with limited genomic alterations and specific gene expression profiles.     

Molecular Evolution of the Domain Structures of Protein Disulfide Isomerases

Protein disulfide isomerase (PDI) is an enzyme that promotes protein folding by catalyzing disulfide bridge isomerization. PDI and its relatives form a diverse protein family whose members are characterized by thioredoxin-like (TX) domains in the primary structures. The family was classified into four classes by the number and the relative positions of the TX domains. To investigate the evolution of the domain structures, we aligned the amino acid sequences of the TX domains, and the molecular phylogeny was examined by the NJ and ML methods. We found that all of the current members of the PDI family have evolved from an ancestral enzyme, which has two TX domains in the primary structure. The diverse domain structures of the members have been generated through domain duplications and deletions.     

Latent infection with Corynebacterium kutscheri in mice after peroral inoculation and its provocation by cortisone (author's transl)]

Latent infection with Corynebacterium kutscheri in mice and its provocation by cortisone were studied with a rifampicin-resistant strain of the organism. Mice having been infected perorally began to excrete the organisms in feces within 6 hours, and most of them were found to be carrying the organisms in the intestine, especially in the cecum even 90 days after infection. In such state of latency, however, no organisms were detected in other main organs, and neither visible lesions nor serum agglutinin was detectable. The latent infection with excretion of the organisms in feces after peroral infection was shown to become overt and fatal by cortisone treatment made even 90 days after infection. In infected mice excreting no organisms in feces and having bites on their skin, the wounds became severe ulcers after cortisone treatment resulting in septicemia.     

Potential roles for ubiquitin and the proteasome during ribosome biogenesis

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.