PTMdyna: exploring the influence of post-translation modifications on protein conformational dynamics.

Protein post-translational modifications (PTM) play vital roles in cellular regulation, modulating functions by driving changes in protein structure and dynamics. Exploring comprehensively the influence of PTM on conformational dynamics can facilitate the understanding of the related biological function and molecular mechanism. Currently, a series of excellent computation tools have been designed to analyze the time-dependent structural properties of proteins. However, the protocol aimed to explore conformational dynamics of post-translational modified protein is still a blank. To fill this gap, we present PTMdyna to visually predict the conformational dynamics differences between unmodified and modified proteins, thus indicating the influence of specific PTM. PTMdyna exhibits an AUC of 0.884 tested on 220 protein-protein complex structures. The case of heterochromatin protein 1α complexed with lysine 9-methylated histone H3, which is critical for genomic stability and cell differentiation, was used to demonstrate its applicability. PTMdyna provides a reliable platform to predict the influence of PTM on protein dynamics, making it easier to interpret PTM functionality at the structure level. The web server is freely available at http://ccbportal.com/PTMdyna.

De Novo Designed Ru(II) Metallacycle as a Microenvironment-Adaptive Sonosensitizer and Sonocatalyst for Multidrug-Resistant Biofilms Eradication.

Albeit sonodynamic therapy (SDT) has achieved encouraging progress in microbial sterilization, the scarcity of guidelines for designing highly effective sonosensitizers and the intricate biofilm microenvironment (BME), substantially hamper the therapeutic efficacy against biofilm infections. To address the bottlenecks, we innovatively design a Ru(II) metallacycle-based sonosensitizer/sonocatalyst (named Ru-A3-TTD) to enhance the potency of sonotherapy by employing molecular engineering strategies tailored to BME. Our approach involves augmenting Ru-A3-TTD's production of ultrasonic-triggered reactive oxygen species (ROS), surpassing the performance of commercial sonosensitizers, through a straightforward but potent π-expansion approach. Within the BME, Ru-A3-TTD synergistically amplifies sonotherapeutic efficacy via triple-modulated approaches: (i) effective alleviation of hypoxia, leading to increased ROS generation, (ii) disruption of the antioxidant defense system, which shields ROS from glutathione consumption, and (iii) enhanced biofilm penetration, enabling ROS production in deep sites. Notably, Ru-A3-TTD sono-catalytically oxidizes NADPH, a critical coenzyme involved in antioxidant defenses. Consequently, Ru-A3-TTD demonstrates superior biofilm eradication potency against multidrug-resistant Escherichia coli compared to conventional clinical antibiotics, both in vitro and in vivo. To our knowledge, this study represents the pioneering instance of a supramolecular sonosensitizer/sonocatalyst. It provides valuable insights into the structure-activity relationship of sonosensitizers and paves a promising pathway for the treatment of biofilm infections.

Rapid response of midgut bacteria in Bactrocera tau (Walker) (Diptera: Tephritidae) to lambda-cyhalothrin- and spinosad-induced stress.

In recent decades, the increasingly widespread application of chemical pesticides has exacerbated the emergence of insecticide resistance among insect pests. In this study, we examined the rapid response of bacteria in the midgut of the fruit fly Bactrocera tau (Walker) (Diptera: Tephritidae) to stress induced by the insecticides lambda-cyhalothrin and spinosad by analyzing the bacterial community structure and diversity in the midguts of 4-day-old B. tau. The results revealed that 4-day-old B. tau females were more resistant to lambda-cyhalothrin and spinosad than their 4-day-old male counterparts. Alpha- and beta-diversity analyses revealed no significant differences between male and female B. tau with respect to the diversity and richness of gut bacteria in response to the same treatments. In response to treatment with lambda-cyhalothrin and spinosad at lethal concentration 50 (LC50), we detected significant changes in the structure and diversity of the bacterial community in the midguts of both male and female B. tau. Particularly among the dominant bacterial genera, there were decreases in the relative abundances of Citrobacter, Enterobacter, Klebsiella, and Pectobacterium. Increases were observed in the relative abundances of Dysgonomonas, Erwinia, and Providencia. Our findings provide a theoretical basis for gaining a better understanding of the relationships between midgut bacteria and the insecticide resistance of B. tau.

Phylogenetic Analysis of Spliceosome SF3a2 in Different Plant Species.

The formation of mature mRNA requires cutting introns and splicing exons. The occurrence of splicing involves the participation of the spliceosome. Common spliceosomes mainly include five snRNPs: U1, U2, U4/U6, and U5. SF3a2, an essential component of spliceosome U2 snRNP, participates in splicing a series of genes. There is no definition of SF3a2 in plants. The paper elaborated on SF3a2s from a series of plants through protein sequence similarity. We constructed the evolutionary relationship of SF3a2s in plants. Moreover, we analyzed the similarities and differences in gene structure, protein structure, the cis-element of the promoter, and expression pattern; we predicted their interacting proteins and constructed their collinearity. We have preliminarily analyzed SF3a2s in plants and clarified the evolutionary relationship between different species; these studies can better serve for in-depth research on the members of the spliceosome in plants.

HDAC1: An Essential and Conserved Member of the Diverse Zn2+-Dependent HDAC Family Driven by Divergent Selection Pressure.

Zn2+-dependent histone deacetylases (HDACs) are enzymes that regulate gene expression by removing acetyl groups from histone proteins. These enzymes are essential in all living systems, playing key roles in cancer treatment and as potential pesticide targets. Previous phylogenetic analyses of HDAC in certain species have been published. However, their classification and evolutionary origins across biological kingdoms remain unclear, which limits our understanding of them. In this study, we collected the HDAC sequences from 1451 organisms and performed analyses. The HDACs are found to diverge into three classes and seven subclasses under divergent selection pressure. Most subclasses show species specificity, indicating that HDACs have evolved with high plasticity and diversification to adapt to different environmental conditions in different species. In contrast, HDAC1 and HDAC3, belonging to the oldest class, are conserved and crucial in major kingdoms of life, especially HDAC1. These findings lay the groundwork for the future application of HDACs.

NIR-II Emissive Ru(II) Metallacycle Assisting Fluorescence Imaging and Cancer Therapy.

Despite the success of emissive Ruthenium (Ru) agents in biomedicine, problems such as the visible-light excitation/emission and single chemo- or phototherapy modality still hamper their applications in deep-tissue imaging and efficient cancer therapy. Herein, an second nearinfrared window (NIR-II) emissive Ru(II) metallacycle (Ru1000, λem  = 1000 nm) via coordination-driven self-assembly is reported, which holds remarkable deep-tissue imaging capability (≈6 mm) and satisfactory chemo-phototherapeutic performance. In vitro results indicate Ru1000 displays promising cellular uptake, good cancer-cell selectivity, attractive anti-metastasis properties, and remarkable anticancer activity against various cancer cells, including cisplatin-resistant A549 cells (IC50  = 3.4 × 10-6  m vs 92.8 × 10-6  m for cisplatin). The antitumor mechanism could be attributed to Ru1000-induced lysosomal membrane damage and mitochondrial-mediated apoptotic cell death. Furthermore, Ru1000 also allows the high-performance in vivo NIR-II fluorescence imaging-guided chemo-phototherapy against A549 tumors. This work may provide a paradigm for the development of long-wavelength emissive metallacycle-based agents for future biomedicine.

LARMD: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor.

Protein dynamics is central to all biological processes, including signal transduction, cellular regulation and biological catalysis. Among them, in-depth exploration of ligand-driven protein dynamics contributes to an optimal understanding of protein function, which is particularly relevant to drug discovery. Hence, a wide range of computational tools have been designed to investigate the important dynamic information in proteins. However, performing and analyzing protein dynamics is still challenging due to the complicated operation steps, giving rise to great difficulty, especially for nonexperts. Moreover, there is a lack of web protocol to provide online facility to investigate and visualize ligand-driven protein dynamics. To this end, in this study, we integrated several bioinformatic tools to develop a protocol, named Ligand and Receptor Molecular Dynamics (LARMD, http://chemyang.ccnu.edu.cn/ccb/server/LARMD/ and http://agroda.gzu.edu.cn:9999/ccb/server/LARMD/), for profiling ligand-driven protein dynamics. To be specific, estrogen receptor (ER) was used as a case to reveal ERß-selective mechanism, which plays a vital role in the treatment of inflammatory diseases and many types of cancers in clinical practice. Two different residues (Ile373/Met421 and Met336/Leu384) in the pocket of ERß/ERα were the significant determinants for selectivity, especially Met336 of ERß. The helix H8, helix H11 and H7-H8 loop influenced the migration of selective agonist (WAY-244). These computational results were consistent with the experimental results. Therefore, LARMD provides a user-friendly online protocol to study the dynamic property of protein and to design new ligand or site-directed mutagenesis.

AIMMS suite: a web server dedicated for prediction of drug resistance on protein mutation.

Drug resistance is one of the most intractable issues for successful treatment in current clinical practice. Although many mutations contributing to drug resistance have been identified, the relationship between the mutations and the related pharmacological profile of drug candidates has yet to be fully elucidated, which is valuable both for the molecular dissection of drug resistance mechanisms and for suggestion of promising treatment strategies to counter resistant. Hence, effective prediction approach for estimating the sensitivity of mutations to agents is a new opportunity that counters drug resistance and creates a high interest in pharmaceutical research. However, this task is always hampered by limited known resistance training samples and accurately estimation of binding affinity. Upon this challenge, we successfully developed Auto In Silico Macromolecular Mutation Scanning (AIMMS), a web server for computer-aided de novo drug resistance prediction for any ligand-protein systems. AIMMS can qualitatively estimate the free energy consequences of any mutations through a fast mutagenesis scanning calculation based on a single molecular dynamics trajectory, which is differentiated with other web services by a statistical learning system. AIMMS suite is available at http://chemyang.ccnu.edu.cn/ccb/server/AIMMS/.

Streptococcus humanilactis sp.nov., isolated from healthy nursing mother's breast milk.

Two bacterial strains were isolated from the breast milk of two healthy nursing mothers. The isolates were Gram-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of the 16S rRNA gene sequences of strain IMAU99125T shared 99.7 and 99.6% similarity with Streptococcus mitis ATCC 49456 T and Streptococcus pseudopneumoniae ATCC BAA-960 T, respectively. The nearly complete 16S rRNA gene sequences of IMAU99125T and IMAU99674 strains were very closely related (with only 0.06% difference between them). Sequence analysis of the gyrB and rpoB genes also indicated that IMAU99125T was closely related to S. mitis ATCC 49456 T (94.7% and 97.1%, respectively) and S. pseudopneumoniae ATCC BAA-960 T (94.4% and 97.1%, respectively). Average nucleotide identity (ANI) values between strain IMAU99125T and S. mitis ATCC 49456 T and S. pseudopneumoniae ATCC BAA-960 T, were 93.3% and 92.7%, respectively. Genome-to-genome distance (GGD) values between strain IMAU99125T and S. mitis ATCC 99125 T and S. pseudopneumoniae ATCC BAA-960 T were 53.4% (50.7-56.0) and 50.4% (47.7-53.0), respectively. The major fatty acids of the strain were C16:0 (51.4%). On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two strains be classified as representing a novel species of the genus Streptococcus, namely Streptococcus humanilactis sp.nov. The type strain is IMAU99125T (= GDMCC 1.1876 T = KCTC 21157 T). The genome of Streptococcus humanilactis sp. nov. is comprised of 2,027,143 bp. The DNA G + C content of the strain is 40.0 mol%.

Phylogenetic comparison of 5' splice site determination in central spliceosomal proteins of the U1-70K gene family, in response to developmental cues and stress conditions.

Intron-containing genes have the ability to generate multiple transcript isoforms by splicing, thereby greatly expanding the eukaryotic transcriptome and proteome. In eukaryotic cells, precursor mRNA (pre-mRNA) splicing is performed by a mega-macromolecular complex defined as a spliceosome. Among its splicing components, U1 small nuclear ribonucleoprotein (U1 snRNP) is the smallest subcomplex involved in early spliceosome assembly and 5'-splice site recognition. Its central component, named U1-70K, has been extensively characterized in animals and yeast. Very few investigations on U1-70K genes have been conducted in plants, however. To this end, we performed a comprehensive study to systematically identify 115 U1-70K genes from 67 plant species, ranging from algae to angiosperms. Phylogenetic analysis suggested that the expansion of the plant U1-70K gene family was likely to have been driven by whole-genome duplications. Subsequent comparisons of gene structures, protein domains, promoter regions and conserved splicing patterns indicated that plant U1-70Ks are likely to preserve their conserved molecular function across plant lineages and play an important functional role in response to environmental stresses. Furthermore, genetic analysis using T-DNA insertion mutants suggested that Arabidopsis U1-70K may be involved in response to osmotic stress. Our results provide a general overview of this gene family in Viridiplantae and will act as a reference source for future mechanistic studies on this U1 snRNP-specific splicing factor.

Rational Redesign of Enzyme via the Combination of Quantum Mechanics/Molecular Mechanics, Molecular Dynamics, and Structural Biology Study.

Increasing demands for efficient and versatile chemical reactions have prompted innovations in enzyme engineering. A major challenge in engineering α-ketoglutarate-dependent oxygenases is to develop a rational strategy which can be widely used for directly evolving the desired mutant to generate new products. Herein, we report a strategy for rational redesign of a model enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPPD), based on quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamic simulations. This strategy enriched our understanding of the HPPD catalytic reaction pathway and led to the discovery of a series of HPPD mutants producing hydroxyphenylacetate (HPA) as the alternative product other than the native product homogentisate. The predicted HPPD-Fe(IV)═O-HPA intermediate was further confirmed by the crystal structure of Arabidopsis thaliana HPPD/S267W complexed with HPA. These findings not only provide a good understanding of the structure-function relationship of HPPD but also demonstrate a generally applicable platform for the development of biocatalysts.

Structural dynamics and determinants of abscisic acid-receptor binding preference in different aggregation states.

In the 21st century, drought has been the main cause of shortages in world grain production and has created problems with food security. Abscisic acid (ABA) is a key plant hormone involved in the response to abiotic stress, especially drought. The pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory component of abscisic acid receptor (RCAR) family of proteins (simplified as PYLs) is a well-known ABA receptor family, which can be divided into dimeric and monomeric forms. PYLs can recognize ABA and activate downstream plant drought-resistance signals. However, the difference between monomeric and dimeric receptors in the mechanism of the response to ABA is unclear. Here, we reveal that monomeric receptors have a competitive advantage over dimeric receptors for binding to ABA, driven by the energy penalty resulting from dimer dissociation. ABA also plays different roles with the monomer and the dimer: in the monomer, it acts as a 'conformational stabilizer' for stabilizing the closed gate, whereas for the dimer, it serves as an 'allosteric promoter' for promoting gate closure, which leads to dissociation of the two subunits. This work illustrates how receptor oligomerization could modulate hormonal responses and provides a new concept for novel engineered plants based on ABA binding of monomers.

PIIMS Server: A Web Server for Mutation Hotspot Scanning at the Protein-Protein Interface.

Protein-protein interactions (PPIs) play vital roles in regulating biological processes, such as cellular and signaling pathways. Hotspots are certain residues located at protein-protein interfaces that contribute more in protein-protein binding than other residues. Research on the mutational effects of hotspots is important for understanding basic aspects of protein association. Hence, various computational tools have been developed to explore the impact of mutation hotspots, which will allow a better understanding of the forces that drive PPIs. However, tools that may provide comprehensive substitutions at hotspots are still rare. Hence, there is a strong need for a new free web server to explore mutational effects of hotspots. Herein we introduce a web server named PIIMS that integrates molecular dynamics simulation and one-step free energy perturbation. It contains two main computational functions: (1) computational alanine scanning analysis to identify hotspots and (2) full mutation scanning analysis to evaluate the effects of hotspot mutations. We rigidly validated its ability to predict binding free energy changes by using large and diverse datasets including 1,341 mutations from 50 PPIs with the correlation coefficient R = 0.75. The difference from the existing tools is that PIIMS can perform further evaluation of hotspot residues with regard to their different mutations. The PIIMS web server (accessible at http://chemyang.ccnu.edu.cn/ccb/server/PIIMS/index.php) is free and open to all users without login requirements.

Systematic characterization of the branch point binding protein, splicing factor 1, gene family in plant development and stress responses.

BACKGROUND: Among eukaryotic organisms, alternative splicing is an important process that can generate multiple transcripts from one same precursor messenger RNA, which greatly increase transcriptome and proteome diversity. This process is carried out by a super-protein complex defined as the spliceosome. Specifically, splicing factor 1/branchpoint binding protein (SF1/BBP) is a single protein that can bind to the intronic branchpoint sequence (BPS), connecting the 5' and 3' splice site binding complexes during early spliceosome assembly. The molecular function of this protein has been extensively investigated in yeast, metazoa and mammals. However, its counterpart in plants has been seldomly reported. RESULTS: To this end, we conducted a systematic characterization of the SF1 gene family across plant lineages. In this work, a total of 92 sequences from 59 plant species were identified. Phylogenetic relationships of these sequences were constructed, and subsequent bioinformatic analysis suggested that this family likely originated from an ancient gene transposition duplication event. Most plant species were shown to maintain a single copy of this gene. Furthermore, an additional RNA binding motif (RRM) existed in most members of this gene family in comparison to their animal and yeast counterparts, indicating that their potential role was preserved in the plant lineage. CONCLUSION: Our analysis presents general features of the gene and protein structure of this splicing factor family and will provide fundamental information for further functional studies in plants.

Genome-wide phylogenetic and structural analysis reveals the molecular evolution of the ABA receptor gene family.

The plant hormone abscisic acid (ABA) plays a crucial role during the plant life cycle as well as in adaptive responses to environmental stresses. The core regulatory components of ABA signaling in plants are the pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor family (PYLs), which comprise the largest plant hormone receptor family known. They act as negative regulators of members of the protein phosphatase type 2C family. Due to the biological importance of PYLs, many researchers have focused on their genetic redundancy and consequent functional divergence. However, little is understood of their evolution and its impact on the generation of regulatory diversity. In this study, we identify positive selection and functional divergence in PYLs through phylogenetic reconstruction, gene structure and expression pattern analysis, positive selection analysis, functional divergence analysis, and structure comparison. We found the correlation of desensitization of PYLs under specific modifications in the molecular recognition domain with functional diversification. Hence, an interesting antagonistic co-evolutionary mechanism is proposed for the functional diversification of ABA receptor family proteins. We believe a compensatory evolutionary pathway may have occurred.

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands.

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure-activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 µM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.

Identification, evolution and alternative splicing profile analysis of the splicing factor 30 (SPF30) in plant species.

MAIN CONCLUSION: The work offers a comprehensive evaluation on the phylogenetics and conservation of splicing patterns of the plant SPF30 splicing factor gene family. In eukaryotes, one pre-mRNA can generate multiple mRNA transcripts by alternative splicing (AS), which expands transcriptome and proteome diversity. Splicing factor 30 (SPF30), also known as survival motor neuron domain containing protein 1 (SMNDC1), is a spliceosomal protein that plays an essential role in spliceosomal assembly. Although SPF30 genes have been well characterised in human and yeast, little is known about their homologues in plants. Here, we report the genome-wide identification and phylogenetic analysis of SPF30 genes in the plant kingdom. In total, 82 SPF30 genes were found in 64 plant species from algae to land plants. Alternative transcripts were found in many SPF30 genes and splicing profile analysis revealed that the second intron in SPF30 genome is frequently associated with AS events and contributed to the birth of novel exons in a few SPF30 members. In addition, different conserved sequences were observed at these putative splice sites among moss, monocots and dicots, respectively. Our findings will facilitate further functional characterization of plant SPF30 genes as putative splicing factors.

Genome-wide identification and functional analysis of the splicing component SYF2/NTC31/p29 across different plant species.

MAIN CONCLUSION: This study systematically identifies plant SYF2/NTC31/p29 genes from 62 plant species by a combinatory bioinformatics approach, revealing the importance of this gene family in phylogenetics, duplication, transcriptional, and post-transcriptional regulation. Alternative splicing is a post-transcriptional regulatory mechanism, which is critical for plant development and stress responses. The entire process is strictly attenuated by a complex of splicing-related proteins, designated splicing factors. Human p29, also referred to as synthetic lethal with cdc forty 2 (SYF2) or the NineTeen complex 31 (NTC31), is a core protein found in the NTC complex of humans and yeast. This splicing factor participates in a variety of biological processes, including DNA damage repair, control of the cell cycle, splicing, and tumorigenesis. However, its function in plants has been seldom reported. Thus, we have systematically identified 89 putative plant SYF2s from 62 plant species among the deposited entries in the Phytozome database. The phylogenetic relationships and evolutionary history among these plant SYF2s were carefully examined. The results revealed that plant SYF2s exhibited distinct patterns regarding their gene structure, promoter sequences, and expression levels, suggesting their functional diversity in response to developmental cues or stress treatments. Although local duplication events, such as tandem duplication and retrotransposition, were found among several plant species, most of the plant species contained only one copy of SYF2, suggesting the existence of additional mechanisms to confer duplication resistance. Further investigation using the model dicot and monocot representatives Arabidopsis and rice SYF2s indicated that the splicing pattern and resulting protein isoforms might play an alternative role in the functional diversity.

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings.

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.