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A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites designated by UAG stop codons. This site-specific Uaa incorporation can be used as a controller of target gene expression at the translational level by conditional read-through of internal UAG stop codons. Recent advances in optimization of site-specific Uaa incorporation for translational regulation have enabled more precise control over a wide range of novel important applications, such as Uaa-auxotrophy-based biological containment, live-attenuated vaccine, and high-yield zero-leakage expression systems, in which Uaa translational control is exclusively used as an essential genetic element. This review summarizes the history and recent advance of the translational control by conditional stop codon read-through, especially focusing on the methods using the site-specific Uaa incorporation.

Protein homeostasis in the thylakoid membranes is dependent on protein quality control mechanisms, which are necessary to remove photodamaged and misfolded proteins. An ATP-dependent zinc metalloprotease, FtsH, is the major thylakoid membrane protease. FtsH proteases in the thylakoid membranes of form a hetero-hexameric complex consisting of four FtsH subunits, which are divided into two types: type A (FtsH1 and FtsH5) and type B (FtsH2 and FtsH8). An increasing number of studies have identified the critical roles of FtsH in the biogenesis of thylakoid membranes and quality control in the photosystem II repair cycle. Furthermore, the involvement of FtsH proteolysis in a singlet oxygen- and EXECUTER1-dependent retrograde signaling mechanism has been suggested recently. FtsH is also involved in the degradation and assembly of several protein complexes in the photosynthetic electron-transport pathways. In this minireview, we provide an update on the functions of FtsH in thylakoid biogenesis and describe our current understanding of the D1 degradation processes in the photosystem II repair cycle. We also discuss the regulation mechanisms of FtsH protease activity, which suggest the flexible oligomerization capability of FtsH in the chloroplasts of seed plants.

Chloroplasts originated from the endosymbiosis of ancestral cyanobacteria and maintain transcription and translation machineries for around 100 proteins. Most endosymbiont genes, however, have been transferred to the host nucleus, and the majority of the chloroplast proteome is composed of nucleus-encoded proteins that are biosynthesized in the cytosol and then imported into chloroplasts. How chloroplasts and the nucleus communicate to control the plastid proteome remains an important question. Protein-degrading machineries play key roles in chloroplast proteome biogenesis, remodeling, and maintenance. Research in the past few decades has revealed more than 20 chloroplast proteases, which are localized to specific suborganellar locations. In particular, two energy-dependent processive proteases of bacterial origin, Clp and FtsH, are central to protein homeostasis. Processing endopeptidases such as stromal processing peptidase and thylakoidal processing peptidase are involved in the maturation of precursor proteins imported into chloroplasts by cleaving off the amino-terminal transit peptides. Presequence peptidases and organellar oligopeptidase subsequently degrade the cleaved targeting peptides. Recent findings have indicated that not only intraplastidic but also extraplastidic processive protein-degrading systems participate in the regulation and quality control of protein translocation across the envelopes. In this review, we summarize current knowledge of the major chloroplast proteases in terms of type, suborganellar localization, and diversification. We present details of these degradation processes as case studies according to suborganellar compartment (envelope, stroma, and thylakoids). Key questions and future directions in this field are discussed.

We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl). Although ZK is non-toxic for normal organisms, the growth of Escherichia coli carrying the ZK incorporation system was inhibited in a ZK concentration-dependent manner without causing rapid bacterial death, presumably due to generation of non-functional or toxic proteins. The extent of growth inhibition strongly depended on the anticodon sequence of the tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl gene. The results suggest that the ZK-regulated growth controller is a simple, target-specific, environmental noise-resistant and titratable system. This technique may be applicable to a wide variety of organisms because the growth inhibitory effects are caused by “informational disturbance”, in which the highly conserved system for transmission of information from DNA to proteins is perturbed.

Various oscillatory phenomena occur in the world. Because some are associated with abnormal states (e.g. epilepsy), it is important to establish ways to terminate oscillations by external stimuli. However, despite the prior development of techniques for stabilizing unstable oscillations, relatively few studies address the transition from oscillatory to resting state in nonlinear dynamics. This study mainly analyzes the oscillation-quenching of metronomes on a platform as an example of such transitions. To facilitate the analysis, we describe the impulsive force (escapement mechanism) of a metronome by a fifth-order polynomial. By performing both averaging approximation and numerical simulation, we obtain a phase diagram for synchronization and oscillation quenching. We find that quenching occurs when the feedback to the oscillator increases, which will help explore the general principle regarding the state transition from oscillatory to resting state. We also numerically investigate the bifurcation of out-of-phase synchronization and beat-like solution. Despite the simplicity, our model successfully reproduces essential phenomena in interacting mechanical clocks, such as the bistability of in-phase and anti-phase synchrony and oscillation quenching occurring for a large mass ratio between the oscillator and the platform. We believe that our simple model will contribute to future analyses of other dynamics of mechanical clocks.

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.

Specific degradation of photodamaged D1, the photosystem II (PSII) reaction center protein, is a crucial step in the PSII repair cycle to maintain photosynthesis activity. Processive proteolysis by the FtsH protease is fundamental to cooperative D1 degradation. Here, we attempted to purify the FtsH complex to elucidate its regulation mechanisms and substrate recognition in Arabidopsis (Arabidopsis thaliana). Unlike previously reported prokaryotic and mitochondrial FtsHs, the Arabidopsis chloroplastic FtsH does not appear to form a megacomplex with prohibition-like proteins but instead accumulates as smaller complexes. The copurified fraction was enriched with a partial PSII intermediate presumably undergoing repair, although its precise properties were not fully clarified. In addition, we copurified a bacteria-type GTPase localized in chloroplasts, EngA, and confirmed its interaction with FtsH by subsequent pull-down and bimolecular fluorescence complementation assays. While the engA mutation is embryo lethal, the transgenic lines overexpressing EngA (EngA-OX) showed leaf variegation reminiscent of the variegated mutant lacking FtsH2. EngA-OX was revealed to accumulate more cleaved D1 fragments and reactive oxygen species than the wild type, indicative of compromised PSII repair. Based on these results and the fact that FtsH becomes more stable in EngA-OX, we propose that EngA negatively regulates FtsH stability. We demonstrate that proper FtsH turnover is crucial for PSII repair in the chloroplasts of Arabidopsis. Consistent with the increased turnover of FtsH under high-light conditions in Chlamydomonas reinhardtii, our findings underline the rapid turnover of not only D1 but also FtsH proteases in the PSII repair cycle.

Biological containment is a genetic technique that programs dangerous organisms to grow only in the laboratory and to die in the natural environment. Auxotrophy for a substance not found in the natural environment is an ideal biological containment. Here, we constructed an Escherichia coli strain that cannot survive in the absence of the unnatural amino acid 3-iodo-L-tyrosine. This synthetic auxotrophy was achieved by conditional production of the antidote protein against the highly toxic enzyme colicin E3. An amber stop codon was inserted in the antidote gene. The translation of the antidote mRNA was controlled by a translational switch using amber-specific 3-iodo-L-tyrosine incorporation. The antidote is synthesized only when 3-iodo-L-tyrosine is present in the culture medium. The viability of this strain rapidly decreased with less than a 1 h half-life after removal of 3-iodo-L-tyrosine, suggesting that the decay of the antidote causes the host killing by activated colicin E3 in the absence of this unnatural amino acid. The contained strain grew 1.5 times more slowly than the parent strains. The escaper frequency was estimated to be 1.4 mutations (95% highest posterior density 1.1-1.8) per 10(5) cell divisions. This containment system can be constructed by only plasmid introduction without genome editing, suggesting that this system may be applicable to other microbes carrying toxin-antidote systems similar to that of colicin E3.

Diagnosis using artificial intelligence (AI) with deep learning could be useful in endoscopic examinations. We investigated the ability of AI to detect superficial esophageal squamous cell carcinoma (ESCC) from esophagogastroduodenoscopy (EGD) videos. We retrospectively collected 8428 EGD images of esophageal cancer to develop a convolutional neural network through deep learning. We evaluated the detection accuracy of the AI diagnosing system compared with that of 18 endoscopists. We used 144 EGD videos for the two validation sets. First, we used 64 EGD observation videos of ESCCs using both white light imaging (WLI) and narrow-band imaging (NBI). We then evaluated the system using 80 EGD videos from 40 patients (20 with superficial ESCC and 20 with non-ESCC). In the first set, the AI system correctly diagnosed 100% ESCCs. In the second set, it correctly detected 85% (17/20) ESCCs. Of these, 75% (15/20) and 55% (11/22) were detected by WLI and NBI, respectively, and the positive predictive value was 36.7%. The endoscopists correctly detected 45% (25–70%) ESCCs. With AI real-time assistance, the sensitivities of the endoscopists were significantly improved without AI assistance (p < 0.05). AI can detect superficial ESCCs from EGD videos with high sensitivity and the sensitivity of the endoscopist was improved with AI real-time support.

FtsH is an essential ATP-dependent metalloprotease for protein quality control in the thylakoid membrane of Arabidopsis thaliana chloroplasts. It is required for chloroplast development during leaf growth, and particularly for the specific degradation of photo-damaged D1 protein in the photosystem II (PSII) complex to maintain photosynthesis activity. In the thylakoid membrane, the reversible phosphorylation of proteins is known to control the activity and remodeling of photosynthetic complexes, and previous studies implicate that FtsH is also phosphorylated. We therefore assessed the phosphorylation status of FtsH and its possible role in the regulatory mechanism in this study. The phosphorylation level of FtsHs that compose the FtsH heterohexameric complex was investigated by phosphate-affinity gel electrophoresis using a Phos-Tag molecule. Phos-tag SDS-PAGE of thylakoid proteins and subsequent immunoblot analysis showed that both type A (FtsH1/5) and type B (FtsH2/8) subunits were separable into phosphorylated and non-phosphorylated forms. Neither different light conditions nor the lack of two major thylakoid kinases, STN7 and STN8, resulted in any clear difference in FtsH phosphorylation, suggesting that this process is independent of the light-dependent regulation of photosynthesis-related proteins. Site-directed mutagenesis of putatively phosphorylated Ser or Thr residues into Ala demonstrated that Ser-212 may play a role in FtsH stability in the thylakoid membranes. Different phosphorylation status of FtsH oligomers analyzed by two-dimensional clear-native/Phos-tag SDS-PAGE implied that phosphorylation partially affects FtsH complex formation or its stability.