Investigation of N-Glycan Functions in Receptor for Advanced Glycation End Products V Domain through Chemical Glycoprotein Synthesis.

The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized significance of N-glycosylation in the ligand-binding V domain (VD) of RAGE, a comprehensive understanding of the site-activity and structure-activity relationships is lacking due to the challenges in obtaining homogeneous glycoprotein samples through biological expression. Here, we combined chemical and chemoenzymatic approaches to synthesize RAGE-VD and its congeners with Asn3-glycosylation by incorporating precise N-glycan structures. Evaluation of these samples revealed that, in comparison to other RAGE-VD forms, α2,6-sialylated N-glycosylation at the Asn3 site results in more potent inhibition of HMGB1-induced nuclear factor-κB (NF-κB) expression in RAGE-overexpressing cells. Hydrogen/deuterium exchange-mass spectrum analysis revealed a sialylated RAGE-VD-induced interaction region within HMGB1. Conversely, Asn3 N-glycosylation in VD has negligible effects on RAGE-VD/S100B interactions. This study established an approach for accessing homogeneously glycosylated RAGE-VD and explored the modulatory effects of N-glycosylation on the interactions between RAGE-VD and its ligand proteins.

Discovery of endosomalytic cell-penetrating peptides based on bacterial membrane-targeting sequences.

Cell-penetrating peptides (CPPs) are prominent scaffolds for drug developments and related research, particularly the endocytic delivery of biomacromolecules. Effective cargo release from endosomes prior to lysosomal degradation is a crucial step, where the rational design and selection of CPPs remains a challenge and calls for deeper mechanistic understandings. Here, we have investigated a strategy of designing CPPs that selectively disrupt endosomal membranes based on bacterial membrane targeting sequences (MTSs). Six synthesized MTS peptides all exhibit cell-penetrating abilities, among which two d-peptides (d-EcMTS and d-TpMTS) are able to escape from endosomes and localize at ER after entering the cell. The utility of this strategy has been demonstrated by the intracellular delivery of green fluorescent protein (GFP). Together, these results suggest that the large pool of bacterial MTSs may be a rich source for the development of novel CPPs.

Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space.

Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.

Exploring the RNA Editing Events and Their Potential Regulatory Roles in Tea Plant (Camellia sinensis L.).

RNA editing is a post-transcriptional modification process that alters the RNA sequence relative to the genomic blueprint. In plant organelles (namely, mitochondria and chloroplasts), the most common type is C-to-U, and the absence of C-to-U RNA editing results in abnormal plant development, such as etiolation and albino leaves, aborted embryonic development and retarded seedling growth. Here, through PREP, RES-Scanner, PCR and RT-PCR analyses, 38 and 139 RNA editing sites were identified from the chloroplast and mitochondrial genomes of Camellia sinensis, respectively. Analysis of the base preference around the RNA editing sites showed that in the -1 position of the edited C had more frequent occurrences of T whereas rare occurrences of G. Three conserved motifs were identified at 25 bases upstream of the RNA editing site. Structural analyses indicated that the RNA secondary structure of 32 genes, protein secondary structure of 37 genes and the three-dimensional structure of 5 proteins were altered due to RNA editing. The editing level analysis of matK and ndhD in six tea cultivars indicated that matK-701 might be involved in the color change of tea leaves. Furthermore, 218 PLS-CsPPR proteins were predicted to interact with the identified RNA editing sites. In conclusion, this study provides comprehensive insight into RNA editing events, which will facilitate further study of the RNA editing phenomenon of the tea plant.

A novel amphiphilic motif at the C-terminus of FtsZ1 facilitates chloroplast division.

In bacteria and chloroplasts, the GTPase filamentous temperature-sensitive Z (FtsZ) is essential for division and polymerizes to form rings that mark the division site. Plants contain two FtsZ subfamilies (FtsZ1 and FtsZ2) with different assembly dynamics. FtsZ1 lacks the C-terminal domain of a typical FtsZ protein. Here, we show that the conserved short motif FtsZ1Carboxyl-terminus (Z1C) (consisting of the amino acids RRLFF) with weak membrane-binding activity is present at the C-terminus of FtsZ1 in angiosperms. For a polymer-forming protein such as FtsZ, this activity is strong enough for membrane tethering. Arabidopsis thaliana plants with mutated Z1C motifs contained heterogeneously sized chloroplasts and parallel FtsZ rings or long FtsZ filaments, suggesting that the Z1C motif plays an important role in regulating FtsZ ring dynamics. Our findings uncover a type of amphiphilic beta-strand motif with weak membrane-binding activity and point to the importance of this motif for the dynamic regulation of protein complex formation.

Essential Roles of the Linker Sequence Between Tetratricopeptide Repeat Motifs of Ethylene Overproduction 1 in Ethylene Biosynthesis.

Ethylene Overproduction 1 (ETO1) is a negative regulator of ethylene biosynthesis. However, the regulation mechanism of ETO1 remains largely unclear. Here, a novel eto1 allele (eto1-16) was isolated with typical triple phenotypes due to an amino acid substitution of G480C in the uncharacterized linker sequence between the TPR1 and TPR2 motifs. Further genetic and biochemical experiments confirmed the eto1-16 mutation site. Sequence analysis revealed that G480 is conserved not only in two paralogs, EOL1 and EOL2, in Arabidopsis, but also in the homologous protein in other species. The glycine mutations (eto1-11, eto1-12, and eto1-16) do not influence the mRNA abundance of ETO1, which is reflected by the mRNA secondary structure similar to that of WT. According to the protein-protein interaction analysis, the abnormal root phenotype of eto1-16 might be caused by the disruption of the interaction with type 2 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs) proteins. Overall, these data suggest that the linker sequence between tetratricopeptide repeat (TPR) motifs and the glycine in TPR motifs or the linker region are essential for ETO1 to bind with downstream mediators, which strengthens our knowledge of ETO1 regulation in balancing ACSs.

Genome-Wide Identification and Characterization of the CsFHY3/FAR1 Gene Family and Expression Analysis under Biotic and Abiotic Stresses in Tea Plants (Camellia sinensis).

The FHY3/FAR1 transcription factor family, derived from transposases, plays important roles in light signal transduction, and in the growth and development of plants. However, the homologous genes in tea plants have not been studied. In this study, 25 CsFHY3/FAR1 genes were identified in the tea plant genome through a genome-wide study, and were classified into five subgroups based on their phylogenic relationships. Their potential regulatory roles in light signal transduction and photomorphogenesis, plant growth and development, and hormone responses were verified by the existence of the corresponding cis-acting elements. The transcriptome data showed that these genes could respond to salt stress and shading treatment. An expression analysis revealed that, in different tissues, especially in leaves, CsFHY3/FAR1s were strongly expressed, and most of these genes were positively expressed under salt stress (NaCl), and negatively expressed under low temperature (4 °C) stress. In addition, a potential interaction network demonstrated that PHYA, PHYC, PHYE, LHY, FHL, HY5, and other FRSs were directly or indirectly associated with CsFHY3/FAR1 members. These results will provide the foundation for functional studies of the CsFHY3/FAR1 family, and will contribute to the breeding of tea varieties with high light efficiency and strong stress resistance.

Probing N-Glycan Functions in Human Interleukin-17A Based on Chemically Synthesized Homogeneous Glycoforms.

N-Glycosylation represents an essential type of posttranslational modification for proteins. However, deciphering the functions of N-glycosylation remains a challenge due to the lack of analytical and biochemical methods to accurately differentiate the protein glycoforms with various intact glycans. Here we report our synthesis and evaluation of homogeneously glycosylated interleukin-17A (IL-17A), based on a synthetic approach combining solid-phase synthesis of (glyco)peptides, chemoenzymatic glycan modification on segments, and chemical ligations. The obtained homogeneous glycoproteins allow for the demonstration of the stabilizing role of N-glycans during the folding step. A comparison of three IL-17A glycoforms in a normal human dermal fibroblast (NHDF) assay reveals dose-dependent interleukin-6-inducing activities in all cases, wherein the glycoform with sialyl undecasaccharides displays much weaker stimulatory effect than that of the GlcNAc- or GlcNAc(ß1→4)GlcNAc-modified proteins. Further surface plasmon resonance (SPR) and hydrogen/deuterium exchange mass spectroscopic experiments confirm that the evaluated complex type N-glycan impedes the binding between IL-17A and its receptor IL-17RA. This structure-activity relationship study on glycoproteins highlights the viability of applying the de novo approach to probe the roles of N-glycans.

Self-Assembling Glycopeptide Conjugate as a Versatile Platform for Mimicking Complex Polysaccharides.

Polysaccharides are a class of carbohydrates that play pivotal roles in living systems such as being chemical messengers in many vital biological pathways. However, the complexity and heterogeneity of these natural structures have posed daunting challenges on their production, characterization, evaluation, and applications. While there have been various types of synthetic skeletons that could mimic some biological aspects of polysaccharides, a safer and more easily accessed system is still desired to avoid the unnatural components and difficulties in modifying the structures. In this work, conveniently accessible self-assembling glycopeptide conjugates are developed, where the natural O-glycosidic linkages and phosphoryl modifications assist the self-assembly and concurrently reduce the risk of toxicity. The generated nanoparticles in aqueous solution offer a multivalent display of structurally controllable carbohydrates as mimics of polysaccharides, among which a mannosylated version exhibits immunostimulatory effects in both cellular assays and vaccination of mice. The obtained results demonstrate the potential of this glycopeptide conjugate-derived platform in exploiting the intriguing properties of carbohydrates in a more structurally maneuverable fashion.

The Putative Smallest Introns in the Arabidopsis Genome.

Most eukaryotic genes contain introns, which are noncoding sequences that are removed during premRNA processing. Introns are usually preserved across evolutionary time. However, the sizes of introns vary greatly. In Arabidopsis, some introns are longer than 10 kilo base pairs (bp) and others are predicted to be shorter than 10 bp. To identify the shortest intron in the genome, we analyzed the predicted introns in annotated version 10 of the Arabidopsis thaliana genome and found 103 predicted introns that are 30 bp or shorter, which make up only 0.08% of all introns in the genome. However, our own bioinformatics and experimental analyses found no evidence for the existence of these predicted introns. The predicted introns of 30-39 bp, 40-49 bp, and 50-59 bp in length are also rare and constitute only 0.07%, 0.2%, and 0.28% of all introns in the genome, respectively. An analysis of 30 predicted introns 31-59 bp long verified two in this range, both of which were 59 bp long. Thus, this study suggests that there is a limit to how small introns in A. thaliana can be, which is useful for the understanding of the evolution and processing of small introns in plants in general.

Large Introns of 5 to 10 Kilo Base Pairs Can Be Spliced out in Arabidopsis.

Most of the eukaryotic genes contain introns, which are removed from the pre-RNA during RNA processing. In contrast to the introns in animals, which are usually several kilo base pairs (kb), those in plants generally are very small, which are mostly from dozens of base pairs (bp) to a few hundred bp. According to annotation version 10.0 of the genome of Arabidopsis thaliana, there are 127,854 introns in the nuclear genes; 99.23% of them are less than 1 kb, and only 16 introns are annotated to be larger than 5 kb, which are extremely large introns (ELI) in Arabidopsis. To learn whether these introns are true introns or not and how large introns could be in Arabidopsis, RT-PCR analysis of genes containing these ELIs were carried out. The results indicated that some of these putative introns are indeed ELIs. These ELIs are mainly composed of transposons or transposable elements (TE), excepting one, whose counterparts are also very long in diverse plant species. Thus, this study confirms the existence of introns larger than 5 kb or even 10 kb in Arabidopsis.

Structural insights into the coordination of plastid division by the ARC6-PDV2 complex.

Chloroplasts divide by binary fission, which is accomplished by the simultaneous constriction of the FtsZ ring on the stromal side of the inner envelope membrane, and the ARC5 ring on the cytosolic side of the outer envelope membrane. The two rings are connected and coordinated mainly by the interaction between the inner envelope membrane protein ARC6 and the outer envelope membrane protein PDV2 in the intermembrane space. The underlying mechanism of this coordination is unclear to date. Here, we solved the crystal structure of the intermembrane space region of the ARC6-PDV2 complex. The results indicated that PDV2 inserts its carboxy terminus into a pocket formed in ARC6, and this interaction further induces the dimerization of the intermembrane space regions of two ARC6 molecules. A pdv2 mutant attenuating PDV2-induced ARC6 dimerization showed abnormal morphology of ARC6 rings and compromised chloroplast division in plant cells. Together, our data reveal that PDV2-induced dimerization of ARC6 plays a critical role in chloroplast division and provide insights into the coordination mechanism of the internal and external plastid division machineries.

Alternative splicing affects the targeting sequence of peroxisome proteins in Arabidopsis.

KEY MESSAGE: A systematic analysis of the Arabidopsis genome in combination with localization experiments indicates that alternative splicing affects the peroxisomal targeting sequence of at least 71 genes in Arabidopsis. Peroxisomes are ubiquitous eukaryotic cellular organelles that play a key role in diverse metabolic functions. All peroxisome proteins are encoded by nuclear genes and target to peroxisomes mainly through two types of targeting signals: peroxisomal targeting signal type 1 (PTS1) and PTS2. Alternative splicing (AS) is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants, often encoding proteins with functional differences. However, the effects of AS on the PTS1 or PTS2 and the targeting of the protein were rarely studied, especially in plants. Here, we systematically analyzed the genome of Arabidopsis, and found that the C-terminal targeting sequence PTS1 of 66 genes and the N-terminal targeting sequence PTS2 of 5 genes are affected by AS. Experimental determination of the targeting of selected protein isoforms further demonstrated that AS at both the 5' and 3' region of a gene can affect the inclusion of PTS2 and PTS1, respectively. This work underscores the importance of AS on the global regulation of peroxisome protein targeting.

Arabidopsis FRS4/CPD25 and FHY3/CPD45 work cooperatively to promote the expression of the chloroplast division gene ARC5 and chloroplast division.

ARC5 is a dynamin-related GTPase essential for the division of chloroplasts in plants. The arc5 mutant frequently exhibits enlarged, dumbbell-shaped chloroplasts, indicating a role for ARC5 in the constriction of the chloroplast division site. In a screen for chloroplast division mutants with a phenotype similar to arc5, two mutants, cpd25 and cpd45, were obtained. CPD45 was identified as being the same gene as FHY3, a key regulator of far-red light signaling recently shown to be involved in the regulation of ARC5. CPD25 was previously named FRS4 and is homologous to FHY3. We found that CPD25 is also required for the expression of ARC5, suggesting that its function is not redundant to that of FHY3. Moreover, cpd25 does not have the far-red light-sensing defect present in fhy3 and far1. Both FRS4/CPD25 and FHY3/CPD45 could bind to the FBS-like 'ACGCGC' motifs in the promoter region of ARC5, and the binding efficiency of FRS4/CPD25 was much higher than that of FHY3/CPD45. Unlike FHY3/CPD45, FRS4/CPD25 has no ARC5 activation activity. Our data suggest that FRS4/CPD25 and FHY3/CPD45 function as a heterodimer that cooperatively activates ARC5, that FRS4/CPD25 plays the major role in promoter binding, and that FHY3/CPD45 is largely responsible for the gene activation. This study not only provides insight into the mechanisms underlying the regulation of chloroplast division in higher plants, but also suggests a model that shows how members of a transcription factor family can evolve to have different DNA-binding and gene activation features.