Identification of a highly efficient chloroplast-targeting peptide for plastid engineering.

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.

Poly(A) Tail Length of Messenger RNA Regulates Translational Efficiency of the Mitochondria-Targeting Delivery System.

Mitochondria are essential for cellular functions, such as energy production. Human mitochondrial DNA (mtDNA), encoding 13 distinct genes, two rRNA, and 22 tRNA, is crucial for maintaining vital functions, along with nuclear-encoded mitochondrial proteins. However, mtDNA is prone to somatic mutations due to replication errors and reactive oxygen species exposure. These mutations can accumulate, leading to heteroplasmic conditions associated with severe metabolic diseases. Therefore, developing methodologies to improve mitochondrial health is highly demanded. Introducing nucleic acids directly into mitochondria is a promising strategy to control mitochondrial gene expression. Messenger RNA (mRNA) delivery especially offers several advantages such as faster gene expression and reduced risk of genome integration if accidentally delivered to the cell nucleus. In this study, we investigated the effect of the poly(A) tail length of mRNA on the mitochondrial translation to achieve efficient expression. We used a peptide-based mitochondrial targeting system, mitoNEET-(RH)9, comprising a mitochondria-targeting sequence (MTS) and a cationic sequence, to deliver mRNA with various poly(A) tails into the mitochondria. The poly(A) tail length significantly affected translational efficiency, with a medium length of 60 nucleotides maximizing protein expression in various cell lines due to enhanced interaction with mitochondrial RNA-binding proteins. Our findings highlight the importance of optimizing poly(A) tail length for efficient mitochondrial mRNA translation, providing a potential strategy for improving mitochondrial gene therapy. These results pave the way for further exploration of the mechanisms and clinical applications of mitochondrial mRNA delivery systems.

Ampholytic Peptides Consisting of an Alternating Lysine/Glutamic Acid Sequence for the Simultaneous Formation of Polyion Complex Vesicles.

Nanoarchitectures such as micelles and vesicles that self-assemble via electrostatic interactions between their charged polymeric components have been widely used as material delivery platforms. In this work, ampholytic peptides with a sequence of alternating lysine and glutamic acid residues were designed and synthesized via chemoenzymatic polymerization. This alternating sequence was achieved by trypsin-catalyzed polymerization of a dipeptide monomer. Due to the electrostatic interaction between the anionic and cationic residues, the prepared ampholytic peptides spontaneously formed nanosized assemblies with a size of 100-200 nm in water. Modification with tetra(ethylene glycol) (TEG) at the N-terminus of these ampholytic alternating peptides resulted in the formation of stable nanosized assemblies, while peptides consisting of random sequences of lysine and glutamic acid formed large aggregates with deteriorated stability even with TEG modification. Morphological observations using a field-emission scanning electron microscope and an atomic force microscope revealed that the obtained assemblies were spherical and hollow, indicating the spontaneous formation of vesicles from the TEG-modified ampholytic alternating peptides. These vesicles were able to encapsulate a model fluorescent protein within their hollow structures without structural collapse causing loss of fluorescence, demonstrating the potential of these nanocarriers for use in material delivery systems.

Regiocontrol of the Bulk Polymerization of Lysine Ethyl Ester by the Selection of Suitable Immobilized Enzyme Catalysts.

The development of a green and facile method for the controlled synthesis of functional polypeptides is desired for sustainable material applications. In this study, the regioselective synthesis of poly(l-lysine) (polyLys) via enzyme-catalyzed aminolysis was achieved by bulk polymerization of l-lysine ethyl ester (Lys-OEt) using immobilized Candida antarctica lipase Novozym 435 (IM-lipase) or trypsin (IM-trypsin). Structural characterization of the obtained polyLys revealed that IM-lipase resulted solely in ε-linked amide bond formation, whereas IM-trypsin predominantly provided α-linked polyLys. Optimization of the conditions for the bulk polymerization using immobilized enzymes resulted in high monomer conversion and a high degree of polymerization, with excellent regioselectivity. Molecular docking simulations revealed different binding conformations of Lys-OEt to the catalytic pockets of lipase and trypsin, which putatively resulted in different amino moieties being used for amide bond formation. The immobilized enzymes were recovered and recycled for bulk polymerization, and the initial activity was maintained in the case of IM-trypsin. The obtained α- and ε-linked polyLys products exhibited different degradability against proteolysis, demonstrating the possibility of versatile applications as sustainable materials. This enzymatic regioregular control enabled the synthesis of well-defined polypeptide-based materials with a diverging structural variety.

Synthesis of All-Peptide-Based Rotaxane from a Proline-Containing Cyclic Peptide.

Rotaxane cross-linkers enhance the toughness of the resulting rotaxane cross-linked polymers through a stress dispersion effect, which is attributed to the mobility of the interlocked structure. To date, the compositional diversity of rotaxane cross-linkers has been limited, and the poor compatibility of these cross-linkers with peptides and proteins has made their use in such materials challenging. The synthesis of a rotaxane composed of peptides may result in a biodegradable cross-linker that is compatible with peptides and proteins, allowing the fortification of polypeptides and proteins and ultimately leading to the development of innovative materials that possess excellent mechanical properties and biodegradability. However, the chemical synthesis of all-peptide-based rotaxanes has remained elusive because of the absence of strong binding motifs in peptides, which prevents an axial peptide from penetrating a cyclic peptide. Here, we synthesized all-peptide-based rotaxanes using an active template method for proline-containing cyclic peptides. The results of molecular dynamics simulations suggested that cyclic peptides with an expansive inner cavity and carbonyl oxygens oriented toward the center are favorable for rotaxane synthesis. This rotaxane synthesis method is expected to accelerate the synthesis of peptides and proteins with mechanically interlocked structures, potentially leading to the development of peptide- and protein-based materials with unprecedented functionalities.

Tunable thermal diffusivity of silk protein assemblies based on their structural control and photo-induced chemical cross-linking.

Silk, which has excellent mechanical properties and is lightweight, serves as a structural material in natural systems. However, the structural and functional applications of silk in artificial systems have been limited due to the difficulty in controlling its properties. In this study, we demonstrate the tunable thermal diffusivity of silk-based assemblies (films) based on secondary structural control and subsequent cross-linking. We found that the thermal diffusivity of the silk film is increased by the formation of ß-sheet structures and dityrosine between Tyr residues adjacent to the ß-sheet structures. Our results demonstrate the applicability of silk proteins as material components for thermally conductive biopolymer-based materials.

Phenylboronic Acid-Functionalized Micelles Dual-Targeting Boronic Acid Transporter and Polysaccharides for siRNA Delivery into Brown Algae.

Brown algae play essential roles ecologically, practically, and evolutionarily because they maintain coastal areas, capture carbon dioxide, and produce valuable chemicals such as therapeutic drugs. To unlock their full potential, understanding the unique molecular biology of brown algae is imperative. Genetic engineering tools that regulate homeostasis in brown algae are essential for determining their biological mechanisms in detail. However, few methodologies have been developed to control gene expression due to the robust structural barriers of brown algae. To address this issue, we designed peptide-based, small interfering RNA (siRNA)-loaded micelles decorated with phenylboronic acid (PBA) ligands. The PBA ligands facilitated the cellular uptake of the micelles into a model brown alga, Ectocarpus siliculosus (E. Siliculosus), through chemical interaction with polysaccharides in the cell wall and biological recognition by boronic acid transporters on the plasma membrane. The micelles, featuring "kill two birds with one stone" ligands, effectively induced gene silencing related to auxin biosynthesis. As a result, the growth of E. siliculosus was temporarily inhibited without persistent genome editing. This study demonstrated the potential for exploring the characteristics of brown algae through a simple yet effective approach and presented a feasible system for delivering siRNA in brown algae.

Replicating shear-mediated self-assembly of spider silk through microfluidics.

The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of ß-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that ß-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a ß-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers.

NMR assignment and dynamics of the dimeric form of soluble C-terminal domain major ampullate spidroin 2 from Latrodectus hesperus.

Spider dragline silk has attracted great interest due to its outstanding mechanical properties, which exceed those of man-made synthetic materials. Dragline silk, which is composed of at least major ampullate spider silk protein 1 and 2 (MaSp1 and MaSp2), contains a long repetitive domain flanked by N-terminal and C-terminal domains (NTD and CTD). Despite the small size of the CTD, this domain plays a crucial role as a molecular switch that regulates and directs spider silk self-assembly. In this study, we report the 1H, 13C, and 15N chemical shift assignments of the Latrodectus hesperus MaSp2 CTD in dimeric form at pH 7. Our solution NMR data demonstrated that this protein contains five helix regions connected by a flexible linker.

Peroxisomes undergo morphological changes in a light-dependent manner with proximity to the nucleus.

The size and shape of organelles can influence the rate of biochemical reactions in cells. Previous studies have suggested that organelle morphology changes due to intra- and extracellular environmental responses, affecting the metabolic efficiency of and signal transduction emanating from neighboring organelles. In this study, we tested the possibility that intracellularly distributed organelles exhibit a heterogeneous response to intra- and extracellular environments. We detected a high correlation between peroxisome morphology and distance to the nucleus in light-exposed cells. Moreover, the proximity area between chloroplasts and peroxisomes varied with distance to the nucleus. These results indicate that peroxisome morphology varies with proximity to the nucleus, suggesting the presence of a nucleus-peroxisome signal transduction cascade mediated by chloroplasts.

C-Terminal Domain Controls Protein Quality and Secretion of Spider Silk in Tobacco Cells.

The remarkable mechanical strength and extensibility of spider dragline silk spidroins are attributed to the major ampullate silk proteins (MaSp). Although fragmented MaSp molecules have been extensively produced in various heterologous expression platforms for biotechnological applications, complete MaSp molecules are required to achieve instinctive spinning of spidroin fibers from aqueous solutions. Here, a plant cell-based expression platform for extracellular production of the entire MaSp2 protein is developed, which exhibits remarkable self-assembly properties to form spider silk nanofibrils. The engineered transgenic Bright-yellow 2 (BY-2) cell lines overexpressing recombinant secretory MaSp2 proteins yield 0.6-1.3  µg L-1 at 22 days post-inoculation, which is four times higher than those of cytosolic expressions. However, only 10-15% of these secretory MaSp2 proteins are discharged into the culture media. Surprisingly, expression of functional domain-truncated MaSp2 proteins lacking the C-terminal domain in transgenic BY-2 cells increases recombinant protein secretion incredibly, from 0.9 to 2.8 mg L-1 per day within 7 days. These findings demonstrate significant improvement in the extracellular production of recombinant biopolymers such as spider silk spidroins using plant cells. In addition, the results reveal the regulatory roles of the C-terminal domain of MaSp2 proteins in controlling their protein quality and secretion.

Effects of polycaprolactone degradation products on the water flea, Daphnia magna: Carbodiimide additives have acute and chronic toxicity.

Plastics have benefited our lives in many ways, but their long persistence in the environment causes serious problems. Rapid decomposition and detoxification of plastics after use are significant challenges. As a possible solution, biodegradable plastics have attracted attention, and for environmental risk assessment research on polymer toxicity, use of indicator organisms, like water fleas and fish, has increased globally. However, such research often focuses on standardized substances without considering changes in toxicity due to plastic degradation products. Additionally, tests generally focus on acute toxicity, while long-term effects on organismal reproduction and lifespan are largely unknown. Understanding the impact of degraded polymers on biological activities is crucial for accurate risk assessment. In this study, we investigated the biological toxicity of substances generated during degradation of polycaprolactone (PCL), a common biodegradable plastic, using the indicator organism, Daphnia magna. We examined PCL, oligocaprolactones (OCLs), and monomers resulting from polymer cleavage, as well as carbodiimides, added during polyester synthesis. As a result, PCL, which is insoluble in water, reduced individual survival and total number of offspring at an exposure concentration of 100 mg/L, while no toxicity was observed for water-soluble degradation products, OCLs, and monomers. Furthermore, carbodiimides, which are expected to be released during PCL degradation, showed strong toxicity, significantly reducing individual survival and total number of offspring at 0.1-10 mg/L. These findings suggest that changes in physical properties due to polymer degradation and release of additives can significantly alter their toxicity.

Bilirubin is produced nonenzymatically in plants to maintain chloroplast redox status.

Bilirubin, a potent antioxidant, is a product of heme catabolism in heterotrophs. Heterotrophs mitigate oxidative stress resulting from free heme by catabolism into bilirubin via biliverdin. Although plants also convert heme to biliverdin, they are generally thought to be incapable of producing bilirubin because they lack biliverdin reductase, the enzyme responsible for bilirubin biosynthesis in heterotrophs. Here, we demonstrate that bilirubin is produced in plant chloroplasts. Live-cell imaging using the bilirubin-dependent fluorescent protein UnaG revealed that bilirubin accumulated in chloroplasts. In vitro, bilirubin was produced nonenzymatically through a reaction between biliverdin and reduced form of nicotinamide adenine dinucleotide phosphate at concentrations comparable to those in chloroplasts. In addition, increased bilirubin production led to lower reactive oxygen species levels in chloroplasts. Our data refute the generally accepted pathway of heme degradation in plants and suggest that bilirubin contributes to the maintenance of redox status in chloroplasts.

Deuterium- and Alkyne-Based Bioorthogonal Raman Probes for In Situ Quantitative Metabolic Imaging of Lipids within Plants.

Plants can rapidly respond to different stresses by activating multiple signaling and defense pathways. The ability to directly visualize and quantify these pathways in real time using bioorthogonal probes would have practical applications, including characterizing plant responses to both abiotic and biotic stress. Fluorescence-based labels are widely used for tagging of small biomolecules but are relatively bulky and with potential effects on their endogenous localization and metabolism. This work describes the use of deuterium- and alkyne-derived fatty acid Raman probes to visualize and track the real-time response of plants to abiotic stress within the roots. Relative quantification of the respective signals could be used to track their localization and overall real-time responses in their fatty acid pools due to drought and heat stress without labor-intensive isolation procedures. Their overall usability and low toxicity suggest that Raman probes have great untapped potential in the field of plant bioengineering.

Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides.

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection.

Binding of Tau-derived peptide-fused GFP to plant microtubules in Arabidopsis thaliana.

Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro. However, there was no investigation of TPs on microtubules in planta. Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules.

Organelle-targeted gene delivery in plants by nanomaterials.

Genetic engineering of plants has revolutionized agriculture and has had a significant impact on our everyday life. It has allowed for the production of crops with longer shelf lives, enhanced yields and resistance to pests and disease. The application of nanomaterials in plant genetic engineering has further augmented these programs with higher delivery efficiencies, biocompatibility and the potential for plant regeneration. In particular, subcellular targeting using nanomaterials has recently become possible with the cutting-edge developments within nanomaterials, but remains challenging despite the promise in organellar engineering for the introduction of useful traits and the elucidation of subcellular interactions. This feature article provides an overview of nanomaterial delivery within plants and highlights the application of recent progress in nanomaterials for subcellular organelle-targeted delivery.

Effect of Oligomers Derived from Biodegradable Polyesters on Eco- and Neurotoxicity.

Biodegradable polymers are eco-friendly materials and have attracted attention for use in a sustainable society because they are not accumulated in the environment. Although the characteristics of biodegradable polymers have been assessed well, the effects of their degradation products have not. Herein, we comprehensively evaluated the chemical toxicities of biodegradable polyester, polycaprolactone (PCL), and synthetic oligocaprolactones (OCLs) with different degrees of polymerization. While the PCL did not show any adverse effects on various organisms, high levels of shorter OCLs and the monomer (1 µg/mL for freshwater microorganisms and 1 mg/mL for marine algae and mammalian cells) damaged the tested organisms, including freshwater microorganisms, marine algae, and mammalian cells, which indicated the toxicities of the degradation products under unnaturally high concentrations. These results highlight the need for a further understanding of the effects of the degradation products resulting from biodegradable polyesters to ensure a genuinely sustainable society.