Identification and Functional Analysis of Lysine 2-Hydroxyisobutyrylation in Cyanobacteria.

Cyanobacteria are the oldest prokaryotic photoautotrophic microorganisms and have evolved complicated post-translational modification (PTM) machinery to respond to environmental stress. Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified PTM that is reported to play important roles in diverse biological processes, however, its distribution and function in cyanobacteria have not been reported. Here, we performed the first systematic studies of Khib in a model cyanobacterium Synechococcus sp. strain PCC 7002 (Syn7002) using peptide prefractionation, pan-Khib antibody enrichment, and high-accuracy mass spectrometry (MS) analysis. A total of 1875 high-confidence Khib sites on 618 proteins were identified, and a large proportion of Khib sites are present on proteins in the cellular metabolism, protein synthesis, and photosynthesis pathways. Using site-directed mutagenesis and functional studies, we showed that Khib of glutaredoxin (Grx) affects the efficiency of the PS II reaction center and H2O2 resistance in Syn7002. Together, this study provides novel insights into the functions of Khib in cyanobacteria and suggests that reversible Khib may influence the stress response and photosynthesis in both cyanobacteria and plants.

Size Effects of Au/Ni-Coated Polymer Particles on the Electrical Performance of Anisotropic Conductive Adhesive Films under Flexible Mechanical Conditions.

In soft electronics, anisotropic conductive adhesive films (ACFs) are the trending interconnecting approach due to their substantial softness and superior bondability to flexible substrates. However, low bonding pressure (≤1 MPa) and fine-pitch interconnections of ACFs become challenging while being extended in advanced device developments such as wafer-level packaging and three-dimensional multi-layer integrated circuit board assembly. To overcome these difficulties, we studied two types of ACFs with distinct conductive filler sizes (ACF-1: ~20 µm and ACF-2: ~5 µm). We demonstrated a low-pressure thermo-compression bonding technique and investigated the size effect of conductive particles on ACF's mechanical properties in a customized testing device, which consists of flexible printing circuits and Flex on Flex assemblies. A consistency of low interconnection resistance (<1 Ω) after mechanical stress (cycling bending test up to 600 cycles) verifies the assembly's outstanding electrical reliability and mechanical stability and thus validates the great effectiveness of the ACF bonding technique. Additionally, in numerical studies using the finite element method, we developed a generic model to disclose the size effect of Au/Ni-coated polymer fillers in ACF on device reliability under mechanical stress. For the first time, we confirmed that ACFs with smaller filler particles are more prone to coating fracture, leading to deteriorated electrical interconnections, and are more likely to peel off from substrate electrode pads resulting in electrical faults. This study provides guides for ACF design and manufacturing and would facilitate the advancement of soft wearable electronic devices.

Interactome Analysis of ClpX Reveals Its Regulatory Role in Metabolism and Photosynthesis in Cyanobacteria.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

Association between the dietary inflammatory index and all-cause and cardiovascular mortality in patients with atherosclerotic cardiovascular disease.

BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death worldwide. Inflammation is pivotal in atherosclerosis development. The dietary inflammatory index (DII) is a tool to quantify the overall inflammatory potential of diet. The association between DII and all-cause and cardiovascular mortality in ASCVD patients remains undetermined. METHODS AND RESULTS: Participants included individuals with ASCVD from the National Health and Nutrition Examination Survey from 1999 to 2018. A total of 5006 participants were included, during a median of 6.6 years of follow-up, of which 2220 (44.4 %) were dead. The Cox proportional hazard model evaluated the association between DII and all-cause and cardiovascular mortality. Participants in the higher DII quartile exhibited a higher mortality of ASCVD. Compared with the patients in quartile 1, those in quartile 4 had a 34 % increased risk for all-cause mortality (HR = 1.34, 95 % CI = 1.21-1.61, p = 0.001). Cardiovascular mortality showed a similar trend, however the correlation is not significant. The restricted cubic spline (RCS) showed that the relationship between DII and all-cause and cardiovascular mortality was linear. Subgroup analysis revealed a persistently positive association between DII and all-causemortality across population subgroups. However, an interaction was detected between DII and alcohol history in relation to cardiovascular mortality. CONCLUSION: DII was positively correlated with the all-cause mortality of ASCVD patients. The intake of a pro-inflammatory diet may increase mortality in ASCVD patients.

Expression and Significance of Inflammatory Factors in Idiopathic Spinal Epidural Lipomatosis (Grade III).

BACKGROUND: Idiopathic spinal epidural lipomatosis (iSEL) is frequently associated with the utilization of steroids, endocrine disorders, obesity, and surgical interventions. Nevertheless, the pathogenesis of iSEL remains inadequately understood. The study aimed to investigate the contribution of inflammatory factors to idiopathic epidural lipomatosis. METHODS: Thirteen patients with iSEL (Grade III, iSEL group) and 12 patients with lumbar disc herniation (control group) who underwent unilateral biportal endoscopy from March 2020 to April 2023 were enrolled. Histological examination of adipose tissue was the performed to analyze expressions of pro-inflammatory cytokines (TNF-α, IL-1ß), and anti-inflammatory factors (arginase-1, IL-10) in serum and epidural adipose cells. RESULTS: Compared with the control group, the number of inflammatory cell infiltrations per field in HE-stained sections was significantly elevated, TNF-α and IL-1ß expression in adipocytes of epidural adipose tissue were markedly higher, and arginase-1 and IL-10 expression were significantly lower in the iSEL group (all p < 0.001). However, no statistically significant differences were observed in the serum level of TNF-α, IL-1ß, arginase-1, and IL-10 between the two groups (p = 0.963). In addition, there was also no significant disparity in adipocyte size between the two groups (p = 0.739). CONCLUSION: iSEL demonstrated elevated inflammatory cells and imbalance towards proinflammatory cytokines in adipocytes of epidural adipose tissue that may be associated with the pathogenesis of symptomatic iSEL. These data suggest that inflammatory response could be one of the mechanisms of iSEL. However, further multicenter epidemiological investigations and rigorous basic and clinical research are warranted to elucidate the specific etiology of iSEL.

Designing and preparing supramolecular encapsulation systems based on fraxetin and cyclodextrins for highly selective detection of nicotine.

Herein, a series of water-soluble supramolecular inclusion complexes (ICs) probes were prepared using cyclodextrins (CDs) and fraxetin (FRA) to detect nicotine (NT) with high selectivity in vitro and in vivo. The FRA/CD ICs prepared through the saturated solution method exhibited excellent water solubility, stability, and biocompatibility. A clear host-guest inclusion model was provided by the theoretical calculations. The investigation revealed that NT was able to enter into the cavities of FRA/ß-CD IC and FRA/γ-CD IC, and further formed charge transfer complexes with FRA in the CD cavities, resulting in a rapid and highly selective fluorescence-enhanced response with the lowest detection limits of 1.9 × 10-6 M and 9.7 × 10-7 M, and the linear response ranged from 0.02 to 0.3 mM and 0.01-0.05 mM, respectively. The IC probes showed good anti-interference performance to common interferents or different pH environments, with satisfactory reproducibility and repeatability of response to NT. Furthermore, the potentiality of the probes was confirmed through fluorescence imaging experiments using human lung cancer cells and the lung tissue of mice. This study offers a fresh perspective for detecting NT in environmental and biomedical analysis.

Deciphering the structure, function, and mechanism of lysine acetyltransferase cGNAT2 in cyanobacteria.

Lysine acetylation is a conserved regulatory posttranslational protein modification that is performed by lysine acetyltransferases (KATs). By catalyzing the transfer of acetyl groups to substrate proteins, KATs play critical regulatory roles in all domains of life; however, no KATs have yet been identified in cyanobacteria. Here, we tested all predicted KATs in the cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) and demonstrated that A1596, which we named cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2), can catalyze lysine acetylation in vivo and in vitro. Eight amino acid residues were identified as the key residues in the putative active site of cGNAT2, as indicated by structural simulation and site-directed mutagenesis. The loss of cGNAT2 altered both growth and photosynthetic electron transport in Syn7002. In addition, quantitative analysis of the lysine acetylome identified 548 endogenous substrates of cGNAT2 in Syn7002. We further demonstrated that cGNAT2 can acetylate NAD(P)H dehydrogenase J (NdhJ) in vivo and in vitro, with the inability to acetylate K89 residues, thus decreasing NdhJ activity and affecting both growth and electron transport in Syn7002. In summary, this study identified a KAT in cyanobacteria and revealed that cGNAT2 regulates growth and photosynthesis in Syn7002 through an acetylation-mediated mechanism.

A bacteria-derived tetramerized protein ameliorates nonalcoholic steatohepatitis in mice via binding and relocating acetyl-coA carboxylase.

Increased de novo lipogenesis (DNL) is a major feature of nonalcoholic steatohepatitis (NASH). None of the drugs targeting the catalytic activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in the DNL process, have been approved by the FDA. Whether cytosolic ACC1 can be regulated spatially remains to be explored. Herein, we find that streptavidin (SA), which is a bacterium-derived tetrameric protein, forms cytosolic condensates and efficiently induces a spatial re-localization of ACC1 in liver cells, concomitant with inhibited lipid accumulation. Both SA tetrameric structure and multivalent protein interaction are required for condensate formation. Interestingly, the condensates are further characterized as gel-like membraneless organelle (SAGMO) and significantly restrict the cytosolic dispersion of ACC1 and fatty acid synthase. Notably, AAV-mediated delivery of SA partially blocks mouse liver DNL and ameliorates NASH without eliciting hypertriglyceridemia. In summary, our study shows that insulating lipogenesis-related proteins by SAGMO might be effective for NASH treatment.

PP2C phosphatases Ptc1 and Ptc2 dephosphorylate PGK1 to regulate autophagy and aflatoxin synthesis in the pathogenic fungus Aspergillus flavus.

IMPORTANCE: Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Improving the genome and proteome annotations of the marine model diatom Thalassiosira pseudonana using a proteogenomics strategy.

Diatoms are unicellular eukaryotic phytoplankton that account for approximately 20% of global carbon fixation and 40% of marine primary productivity; thus, they are essential for global carbon biogeochemical cycling and climate. The availability of ten diatom genome sequences has facilitated evolutionary, biological and ecological research over the past decade; however, a complimentary map of the diatom proteome with direct measurements of proteins and peptides is still lacking. Here, we present a proteome map of the model marine diatom Thalassiosira pseudonana using high-resolution mass spectrometry combined with a proteogenomic strategy. In-depth proteomic profiling of three different growth phases and three nutrient-deficient samples identified 9526 proteins, accounting for ~ 81% of the predicted protein-coding genes. Proteogenomic analysis identified 1235 novel genes, 975 revised genes, 104 splice variants and 234 single amino acid variants. Furthermore, our quantitative proteomic analysis experimentally demonstrated that a considerable number of novel genes were differentially translated under different nutrient conditions. These findings substantially improve the genome annotation of T. pseudonana and provide insights into new biological functions of diatoms. This relatively comprehensive diatom proteome catalog will complement available diatom genome and transcriptome data to advance biological and ecological research of marine diatoms. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00161-y.

Research on high efficiency and high dynamic optimal matching of the electro-hydraulic servo pump control system based on NSGA-II.

An electro-hydraulic servo pump control system (hereinafter referred to as EHSPCS) is a volume servo control unit that is highly integrated with servo motors, fixed-displacement pumps, hydraulic cylinders and functional valve groups. Because of its unique volume direct-drive control mode, the dynamic performance of the system is limited, and the thermal power loss is large, which seriously restricts the improvement of the working quality of the system. To improve the dynamic performance of the system and reduce the thermal power loss to the maximum extent, a multi-objective optimization design method for the EHSPCS is proposed by comprehensively considering the dynamic and efficient energy-saving characteristics of the system. The evaluation model of the dynamic period of the hydraulic cylinder and the thermal power loss of the servo motor are given. Parameters such as the electromagnetic torque of the servo motor, displacement of the hydraulic pump, and working area of the hydraulic cylinder are intelligently optimized by a non-dominated sorting genetic algorithm with elite strategy (NSGA-II). The Pareto front of multi-objective optimization and the corresponding Pareto solution set are obtained; thus, the optimal matching of the system characteristics is realized. Finally, the relevant theory of the multi-objective optimization algorithm is applied to optimize the performance parameters of the hydraulic servo motor, and the prototype is tested in engineering. The experimental results show that the dynamic period of the hydraulic servo motor is accelerated after optimization, and the thermal power loss is significantly reduced. The dynamic and efficient energy-saving characteristics of the system are improved, which further verifies the feasibility of the proposed theory.

cKMT1 is a New Lysine Methyltransferase That Methylates the Ferredoxin-NADP(+) Oxidoreductase and Regulates Energy Transfer in Cyanobacteria.

Lysine methylation is a conserved and dynamic regulatory posttranslational modification performed by lysine methyltransferases (KMTs). KMTs catalyze the transfer of mono-, di-, or tri-methyl groups to substrate proteins and play a critical regulatory role in all domains of life. To date, only one KMT has been identified in cyanobacteria. Here, we tested all of the predicted KMTs in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and we biochemically characterized sll1526 that we termed cKMT1 (cyanobacterial lysine methyltransferase 1) and determined that it can catalyze lysine methylation both in vivo and in vitro. Loss of cKMT1 alters photosynthetic electron transfer in Synechocystis. We analyzed cKMT1-regulated methylation sites in Synechocystis using a timsTOF Pro instrument. We identified 305 class I lysine methylation sites within 232 proteins, and of these, 80 methylation sites in 58 proteins were hypomethylated in ΔcKMT1 cells. We further demonstrated that cKMT1 could methylate ferredoxin-NADP(+) oxidoreductase (FNR) and its potential sites of action on FNR were identified. Amino acid residues H118 and Y219 were identified as key residues in the putative active site of cKMT1 as indicated by structure simulation, site-directed mutagenesis, and KMT activity measurement. Using mutations that mimic the unmethylated forms of FNR, we demonstrated that the inability to methylate K139 residues results in a decrease in the redox activity of FNR and affects energy transfer in Synechocystis. Together, our study identified a new KMT in Synechocystis and elucidated a methylation-mediated molecular mechanism catalyzed by cKMT1 for the regulation of energy transfer in cyanobacteria.

iGDP: An integrated genome decontamination pipeline for wild ciliated microeukaryotes.

Ciliates are a large group of ubiquitous and highly diverse single-celled eukaryotes that play an essential role in the functioning of microbial food webs. However, their genomic diversity is far from clear due to the need to develop cultivation methods for most species, so most research is based on wild organisms that almost invariably contain contaminants. Here we establish an integrated Genome Decontamination Pipeline (iGDP) that combines homology search, telomere reads-assisted and clustering approaches to filter contaminated ciliate genome assemblies from wild specimens. We benchmarked the performance of iGDP using genomic data from a contaminated ciliate culture and the results showed that iGDP could recall 91.9% of the target sequences with 96.9% precision. We also used a synthetic dataset to offer guidelines for the application of iGDP in the removal of various groups of contaminants. Compared with several popular metagenome binning tools, iGDP could show better performance. To further validate the effectiveness of iGDP on real-world data, we applied it to decontaminate genome assemblies of three wild ciliate specimens and obtained their genomes with high quality comparable to that of previously well-studied model ciliate genomes. It is anticipated that the newly generated genomes and the established iGDP method will be valuable community resources for detailed studies on ciliate biodiversity, phylogeny, ecology and evolution. The pipeline (https://github.com/GWang2022/iGDP) can be implemented automatically to reduce manual filtering and classification and may be further developed to apply to other microeukaryotes.

Giant proteins in a giant cell: Molecular basis of ultrafast Ca2+-dependent cell contraction.

The giant single-celled eukaryote, Spirostomum, exhibits one of the fastest movements in the biological world. This ultrafast contraction is dependent on Ca2+ rather than ATP and therefore differs to the actin-myosin system in muscle. We obtained the high-quality genome of Spirostomum minus from which we identified the key molecular components of its contractile apparatus, including two major Ca2+ binding proteins (Spasmin 1 and 2) and two giant proteins (GSBP1 and GSBP2), which act as the backbone and allow for the binding of hundreds of spasmins. The evidence suggests that the GSBP-spasmin protein complex is the functional unit of the mesh-like contractile fibrillar system, which, coupled with various other subcellular structures, provides the mechanism for repetitive ultrafast cell contraction and extension. These findings improve our understanding of the Ca2+-dependent ultrafast movement and provide a blueprint for future biomimicry, design, and construction of this kind of micromachine.

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus.

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.

Proteomic analysis of the regulatory networks of ClpX in a model cyanobacterium Synechocystis sp. PCC 6803.

Protein homeostasis is tightly regulated by protein quality control systems such as chaperones and proteases. In cyanobacteria, the ClpXP proteolytic complex is regarded as a representative proteolytic system and consists of a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. However, the functions and molecular mechanisms of ClpX in cyanobacteria remain unclear. This study aimed to decipher the unique contributions and regulatory networks of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We showed that the interruption of clpX led to slower growth, decreased high light tolerance, and impaired photosynthetic cyclic electron transfer. A quantitative proteomic strategy was employed to globally identify ClpX-regulated proteins in Synechocystis cells. In total, we identified 172 differentially expressed proteins (DEPs) upon the interruption of clpX. Functional analysis revealed that these DEPs are involved in diverse biological processes, including glycolysis, nitrogen assimilation, photosynthetic electron transport, ATP-binding cassette (ABC) transporters, and two-component signal transduction. The expression of 24 DEPs was confirmed by parallel reaction monitoring (PRM) analysis. In particular, many hypothetical or unknown proteins were found to be regulated by ClpX, providing new candidates for future functional studies on ClpX. Together, our study provides a comprehensive ClpX-regulated protein network, and the results serve as an important resource for understanding protein quality control systems in cyanobacteria.

Polarization of Tumor-Associated Macrophages Promoted by Vitamin C-Loaded Liposomes for Cancer Immunotherapy.

While checkpoint blockade immunotherapy as a promising clinical modality has revolutionized cancer treatment, it is of benefit to only a subset of patients because of the tumor immunosuppressive microenvironment. Herein, we report that the specified delivery of vitamin C at the tumor site by responsive lipid nanoparticles can efficiently induce oxidative toxicity and the polarization of M1 macrophages, promoting the infiltration of activating cytotoxic T lymphocytes in the tumor microenvironment for intensive immune checkpoint blocking therapy. Both in vitro and in vivo assays demonstrate successful vitamin C-induced polarization of M2 macrophages to M1 macrophages. In vivo transcriptome analysis also reveals the activation mechanism of vitamin C immunity. More importantly, the combination approach displays much better immune response and immune process within the tumor microenvironment than clinical programmed cell death ligand 1 (Anti-PD-L1) alone. This work provides a powerful therapeutic application of vitamin C to amplify Anti-PD-L1 immunotherapy in cancer treatment, which brings hope to patients with clinically insensitive immunity.

Systematic Survey of the Regulatory Networks of the Long Noncoding RNA BANCR in Cervical Cancer Cells.

Long noncoding RNAs (lncRNAs) are increasingly recognized as functional regulators of human cancers. BRAF-activated noncoding RNA (BANCR), an oncogenic lncRNA, has a carcinogenic effect on many types of cancers. However, the clinical significance and molecular mechanisms of BANCR in cervical cancer are still unclear. Here, we generated BANCR knockout cell lines via CRISPR/Cas9 editing and revealed that BANCR plays roles in the apoptosis, proliferation, and migration of HeLa cells. A quantitative proteomics strategy was employed to globally identify BANCR-regulated proteins in HeLa cells. In total, we identified 569 differentially expressed proteins (DEPs) upon knockout of BANCR in HeLa cells. Bioinformatic analysis revealed that these DEPs were involved in diverse cellular pathways. Functional studies revealed that BANCR exerts its effects on the proliferation and apoptosis of HeLa cells through the regulation of cAMP-responsive element binding protein 1 (CREB1) expression. Mechanistically, BANCR could inhibit the expression of miR-582-5p, and CREB1 is a direct target of miR-582-5p; therefore, BANCR may exert its function by regulating CREB1 expression via targeting miR-582-5p in cervical cancer cells. Collectively, this study established a proteome-wide BANCR regulatory network, which provides novel insight into the molecular pathogenesis of cervical cancer and can serve as a basis for the development of targeted therapies.

Proteogenomic Analysis Provides Novel Insight into Genome Annotation and Nitrogen Metabolism in Nostoc sp. PCC 7120.

Cyanobacteria, capable of oxygenic photosynthesis, play a vital role in nitrogen and carbon cycles. Nostoc sp. PCC 7120 (Nostoc 7120) is a model cyanobacterium commonly used to study cell differentiation and nitrogen metabolism. Although its genome was released in 2002, a high-quality genome annotation remains unavailable for this model cyanobacterium. Therefore, in this study, we performed an in-depth proteogenomic analysis based on high-resolution mass spectrometry (MS) data to refine the genome annotation of Nostoc 7120. We unambiguously identified 5,519 predicted protein-coding genes and revealed 26 novel genes, 75 revised genes, and 27 different kinds of posttranslational modifications in Nostoc 7120. A subset of these novel proteins were further validated at both the mRNA and peptide levels. Functional analysis suggested that many newly annotated proteins may participate in nitrogen or cadmium/mercury metabolism in Nostoc 7120. Moreover, we constructed an updated Nostoc 7120 database based on our proteogenomic results and presented examples of how the updated database could be used to improve the annotation of proteomic data. Our study provides the most comprehensive annotation of the Nostoc 7120 genome thus far and will serve as a valuable resource for the study of nitrogen metabolism in Nostoc 7120. IMPORTANCE Cyanobacteria are a large group of prokaryotes capable of oxygenic photosynthesis and play a vital role in nitrogen and carbon cycles on Earth. Nostoc 7120 is a commonly used model cyanobacterium for studying cell differentiation and nitrogen metabolism. In this study, we presented the first comprehensive draft map of the Nostoc 7120 proteome and a wide range of posttranslational modifications. In addition, we constructed an updated database of Nostoc 7120 based on our proteogenomic results and presented examples of how the updated database could be used for system-level studies of Nostoc 7120. Our study provides the most comprehensive annotation of Nostoc 7120 genome and a valuable resource for the study of nitrogen metabolism in this model cyanobacterium.

Long noncoding RNA HOTAIR interacts with Y-Box Protein-1 (YBX1) to regulate cell proliferation.

HOTAIR is a long noncoding RNA (lncRNA) which serves as an important factor regulating diverse processes linked with cancer development. Here, we used comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) to explore the HOTAIR-protein interactome. We were able to identify 348 proteins interacting with HOTAIR, allowing us to establish a heavily interconnected HOTAIR-protein interaction network. We further developed a novel near-infrared fluorescent protein (iRFP)-trimolecular fluorescence complementation (TriFC) system to assess the interaction between HOTAIR and its interacting proteins. Then, we determined that HOTAIR specifically binds to YBX1, promotes YBX1 nuclear translocation, and stimulates the PI3K/Akt and ERK/RSK signaling pathways. We further demonstrated that HOTAIR exerts its effects on cell proliferation, at least in part, through the regulation of two YBX1 downstream targets phosphoenolpyruvate carboxykinase 2 (PCK2) and platelet derived growth factor receptor ß. Our findings revealed a novel mechanism, whereby an lncRNA is able to regulate cell proliferation via altering intracellular protein localization. Moreover, the imaging tools developed herein have excellent potential for future in vivo imaging of lncRNA-protein interaction.