Photosynthetic assimilation of CO2 regulates TOR activity

The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin–Benson–Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii. Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.

Quantification and surface localization of the hemolysin A type 1 secretion system at the endogenous level and under conditions of overexpression

Secretion systems are essential for Gram-negative bacteria as these nanomachineries allow a communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type one secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli (E. coli) , which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC and the substrate HlyA, a member of the family of RTX (repeats in toxins) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression (T7 expression system, BL21(DE3)-BD). The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by super-resolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence the polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS cluster at the outer membrane generating domains of so far not described identity. Importance Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide representing a global burden to the healthcare system. UPEC secrete many virulence factors among these the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the super-resolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

Effect of Cadmium on the Level of Isoprenoid-Derived Phytohormones in Duckweed Wolffia arrhiza

Wolffia arrhiza (L.) Horkel ex Wimm. is an aquatic plant belonging to the Lemnaceae family. It does not have leaves, stems, and roots, flowers rarely occur, while body size can reach 1 mm of width and 1.3 mm of length. The present study demonstrates the endogenous level of isoprenoid-derived phytohormones and their changes under the influence of different cadmium (Cd) concentrations (0.1, 1, 10, and 100 µM). A liquid chromatography quadrupole-time-of-flight mass spectrometry analysis indicated the presence of abscisic acid, eight brassinosteroids (6-deoxocastasterone, 6-deoxotyphasterol, cathasterone, typhasterol, castasterone, 24-epicastasterone, brassinolide, and 28-homobrassinolide), seven free bases of cytokinins [trans-zeatin (tZ), cis-zeatin (cZ), dihydrozeatin (DHZ), N6-isopentenyladenine, N6-isopentenyladenosine, ortho-topolin, and meta-topolin], eight conjugates of cytokinins (tZ riboside, tZ-9-glucoside, tZ-7-glucoside, tZ-O-glucoside riboside, cZ-9-glucoside, DHZ riboside, DHZ-O-glucoside, and N6-isopentenyladenosine-7-glucoside) and gibberellic acid (GA3) in this duckweed. The level of phytohormones in plants treated with Cd has changed, e.g., the ABA level increased while GA3 decreased. Whereas the amount of BRs and CKs was different in Cd dose-dependent manner. Besides, it is worth noting that the distribution of 25 various phytohormones in the Wolffia arrhiza is reported for the first time.

A regulatory interface on RIPK2 is required for XIAP binding and NOD signaling activity

Signaling via the intracellular pathogen receptors Nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2 requires Receptor Interacting Kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG-tagged RIPK2 knock-in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice we were able to generate a detailed map of post-translational modifications on RIPK2 during NOD signaling and we identified a new regulatory interface on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP.

Lack of RAN-mediated toxicity in Huntington’s disease knock-in mice

Identification of repeat-associated non-AUG (RAN) translation in trinucleotide (CAG) repeat diseases has led to the emerging concept that CAG repeat diseases are caused by nonpolyglutamine products. Nonetheless, the in vivo contribution of RAN translation to the pathogenesis of CAG repeat diseases remains elusive. Via CRISPR/Cas9-mediated genome editing, we established knock-in mouse models that harbor expanded CAG repeats in the mouse huntingtin gene to express RAN-translated products with or without polyglutamine peptides. We found that RAN translation is not detected in the knock-in mouse models when expanded CAG repeats are expressed at the endogenous level. Consistently, the expanded CAG repeats that cannot be translated into polyglutamine repeats do not yield the neuropathological and behavioral phenotypes that were found in knock-in mice expressing expanded polyglutamine repeats. Our findings suggest that RAN-translated products do not play a major role in the pathogenesis of CAG repeat diseases and underscore the importance in targeting polyglutamine repeats for therapeutics.

Localization of the signal of dystonia-associated protein torsinA near the Golgi apparatus in cultured central neurons

ABSTRACTA single in-frame deletion of a codon for a glutamic acid residue within the TOR1A gene is linked to the autosomal-dominant movement disorder DYT1 dystonia, a condition characterized by involuntary muscle contractions that cause abnormal posture. This gene encodes the protein torsinA, and the functions of both wild-type and mutant (ΔE-torsinA) forms remain poorly understood. Previous studies based on overexpression systems indicated that wild-type torsinA resides mainly in the endoplasmic reticulum but that ΔE-torsinA is localized to the nuclear envelope or intracellular inclusions. This mutation-associated mis-localization has been proposed to underlie at least a part of the pathophysiology of DYT1 dystonia. However, the subcellular localization of torsinA has not been extensively studied when expressed at the endogenous level. Here we report an immunocytochemical analysis of torsinA proteins in cultured mouse neurons from a ΔE-torsinA knock-in model of DYT1 dystonia, where torsinA proteins are not upregulated. In all examined neurons of wild-type, heterozygous and homozygous mice, torsinA signal was found mainly near the Golgi apparatus, and only weakly in the endoplasmic reticulum and nuclear envelope. These results suggest that, in the absence of overexpression, torsinA proteins are localized near the Golgi apparatus and may influence cellular function involving the organelle.

CGGBP1 regulates CTCF occupancy at repeats

Background CGGBP1 is a repeat-binding protein with diverse functions in the regulation of gene expression, cytosine methylation, repeat silencing and genomic integrity. CGGBP1 has also been identified as a cooperator of histone-modifying enzymes and as a component of CTCF-containing complexes that regulate the enhancer–promoter looping. CGGBP1–CTCF cross talk in chromatin regulation has been hitherto unknown. Results Here, we report that the occupancy of CTCF at repeats depends on CGGBP1. Using ChIP-sequencing for CTCF, we describe its occupancy at repetitive DNA. Our results show that endogenous level of CGGBP1 ensures CTCF occupancy preferentially on repeats over canonical CTCF motifs. By combining CTCF ChIP-sequencing results with ChIP sequencing for three different kinds of histone modifications (H3K4me3, H3K9me3 and H3K27me3), we show that the CGGBP1-dependent repeat-rich CTCF-binding sites regulate histone marks in flanking regions. Conclusion CGGBP1 affects the pattern of CTCF occupancy. Our results posit CGGBP1 as a regulator of CTCF and its binding sites in interspersed repeats.