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【Objective】This study is aimed to construct a konjac cDNA library and screen for interacting proteins of the transcription factor WRKY72 in order to elucidate the molecular mechanism underlying WRKY72-mediated konjac resistance against diseases.【Method】Konjac plants at different stages of bacterial soft rot infection were used as experimental materials. Total RNA was extracted, pooled in equal amounts, and used to construct a konjac cDNA library. The bait vector containing the transcription factor WRKY72 was constructed using the homologous recombination method, and a yeast two-hybrid screen was performed to identify candidate target proteins, which were further verified individually.【Result】The yeast library titer was 1.6×107 CFU/mL, and the average length of the inserted fragments was around 1 000 bp. The bait vector was verified to have no self-activation in yeast. The bait vector pGBKT7-WRKY72 was used to screen library and 41 positive interacting proteins were obtained, including transport proteins, channel proteins, heat shock proteins, chlorophyll-binding proteins, and peroxidases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed on these interacting proteins, and 12 of them were further verified by one-on-one yeast two-hybrid assays.【Conclusion】A high-quality konjac cDNA library was successfully constructed, and the interacting proteins of the transcription factor WRKY72 were screened, providing a theoretical basis for elucidating the mechanism of resistance against soft rot disease in konjac. [ABSTRACT FROM AUTHOR]

Abstract Ubiquitination is a prevalent post-translational modification that plays a crucial role in a wide range of pathophysiological processes, including cell proliferation, apoptosis, autophagy, immune response, and DNA damage repair. Among the enzymes involved in ubiquitination, E3 ubiquitin ligases are particularly significant, serving as key regulators of numerous diseases, including tumours. This review focuses on HECW2 (HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2, also known as NEDL2), providing a comprehensive overview of its interactors and its pathological roles in tumorous cancer and other diseases. The insights gained from this review may contribute to the development of novel treatment strategies for various diseases, particularly tumours.

Ubiquitination is a prevalent post-translational modification that plays a crucial role in a wide range of pathophysiological processes, including cell proliferation, apoptosis, autophagy, immune response, and DNA damage repair. Among the enzymes involved in ubiquitination, E3 ubiquitin ligases are particularly significant, serving as key regulators of numerous diseases, including tumours. This review focuses on HECW2 (HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2, also known as NEDL2), providing a comprehensive overview of its interactors and its pathological roles in tumorous cancer and other diseases. The insights gained from this review may contribute to the development of novel treatment strategies for various diseases, particularly tumours. [ABSTRACT FROM AUTHOR]

The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley. [ABSTRACT FROM AUTHOR]

FOXM1 is a key transcriptional regulator involved in various biological processes in mammals, including carbohydrate and lipid metabolism, aging, immune regulation, development, and disease. Early studies have shown that FOXM1 acts as an oncogene by regulating cell proliferation, cell cycle, migration, metastasis, and apoptosis, as well as genes related to diagnosis, treatment, chemotherapy resistance, and prognosis. Researchers are increasingly focusing on FOXM1 functions in tumor microenvironment, epigenetics, and immune infiltration. However, researchers have not comprehensively described FOXM1's involvement in tumor microenvironment shaping, epigenetics, and immune cell infiltration. Here we review the role of FOXM1 in the formation and development of malignant tumors, and we will provide a comprehensive summary of the role of FOXM1 in transcriptional regulation, interacting proteins, tumor microenvironment, epigenetics, and immune infiltration, and suggest areas for further research. [ABSTRACT FROM AUTHOR]

Carotenoids are widely distributed and structurally diverse, which have significant roles in the photosynthesis of plants. As a precursor of vitamin A, carotenoids are also antioxidants that reduce various chronic diseases, which are beneficial for human health. Currently, the existing studies concerned the biological roles of APETALA2 (AP2)/ethylene-responsive factor (ERF) genes originated from higher plants. The AP2 superfamily of the transcriptional regulator was identified in higher plants, which was related to growth, development, carotenoid metabolism, and responses to various stresses. However, the regulatory mechanisms of the AP2-modulating carotenoid metabolism have not been reported in microalgae, which remain to be elucidated. Dunaliella parva AP2 (i.e., DpAP2), an important transcription factor, promotes carotenoid accumulation by binding to the promoter of target gene. Here, we identified an important AP2/ERF transcription factor, DpAP2, which could promote carotenoid accumulation by binding to the promoter of target gene. To demonstrate the function of DpAP2, the interacting proteins were identified by the yeast two-hybrid system. The results showed that DpAP2 could interact with three proteins with different activities (DNAbinding transcription factor activity, protein kinase activity, and alpha-Dphosphohexomutase activity); these proteins may be associated with multiple biological processes. This paper laid a good foundation for a deep understanding of the regulatory mechanisms of DpAP2 and genetic engineering breeding in D. parva. [ABSTRACT FROM AUTHOR]

Despite extensive research highlighting the pivotal role of MYB transcription factors in regulating anthocyanin biosynthesis, the interactive regulatory network involving these MYB factors in pear fruits remains inadequately characterized. In this study, the anthocyanin-regulatory gene PbrMYB114 was successfully cloned from 'Yuluxiang' pear (Pyrus bretschneideri) fruits, and its influence on anthocyanin accumulation was confirmed through transient expression assays. Specifically, the co-transformation of PbrMYB114 with its partner PbrbHLH3 in pears served to validate the functional role of PbrMYB114. Subsequently, PbrMYB114 was employed as bait in a yeast two-hybrid screening assay, using a 'Yuluxiang' pear protein library, which led to the identification of 25 interacting proteins. Further validation of the interactions between PbrMYB114 and PbrMT2/PbrMT3 was conducted. Investigations into the role of PbrMT2 and PbrMT3 in 'Duli' seedlings (Pyrus betulaefolia) revealed their potential to enhance anthocyanin accumulation. The outcomes of these studies provide novel insights into the protein network that regulates pear anthocyanin biosynthesis, particularly the functional interactions among PbrMYB114 and associated proteins. [ABSTRACT FROM AUTHOR]

Despite extensive research highlighting the pivotal role of MYB transcription factors in regulating anthocyanin biosynthesis, the interactive regulatory network involving these MYB factors in pear fruits remains inadequately characterized. In this study, the anthocyanin-regulatory gene PbrMYB114 was successfully cloned from ‘Yuluxiang’ pear (Pyrus bretschneideri) fruits, and its influence on anthocyanin accumulation was confirmed through transient expression assays. Specifically, the co-transformation of PbrMYB114 with its partner PbrbHLH3 in pears served to validate the functional role of PbrMYB114. Subsequently, PbrMYB114 was employed as bait in a yeast two-hybrid screening assay, using a ‘Yuluxiang’ pear protein library, which led to the identification of 25 interacting proteins. Further validation of the interactions between PbrMYB114 and PbrMT2/PbrMT3 was conducted. Investigations into the role of PbrMT2 and PbrMT3 in ‘Duli’ seedlings (Pyrus betulaefolia) revealed their potential to enhance anthocyanin accumulation. The outcomes of these studies provide novel insights into the protein network that regulates pear anthocyanin biosynthesis, particularly the functional interactions among PbrMYB114 and associated proteins.

BackgroundLeptin Receptor (LEPR) has been suggested to have several roles in cancer metastasis. However, the role of LEPR and its underlying mechanisms in lymphatic metastasis of hepatocarcinoma have not yet been studied.MethodsWe performed bioinformatics analysis, qRT-PCR, western blotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent, coimmunoprecipitation assays and a series of functional assays to investigate the roles of LEPR in hepatocellular carcinoma.ResultsWe discovered that LEPR was highly expressed in liver cancer tissues, and the expression of LEPR in Hca-F cells was higher than that in Hca-P cells. Furthermore, LEPR promotes the proliferation, migration and invasion and inhibits the apoptosis of hepatocarcinoma lymphatic metastatic cells. Further studies indicated that LEPR interacts with ANXA7. Mechanistically, LEPR regulated ERK1/2 and JAK2/STAT3 expression via ANXA7 regulation.ConclusionsThese findings unveiled a previously unappreciated role of LEPR in the regulation of lymphatic metastatic hepatocellular carcinoma, assigning ANXA7-LEPR as a promising therapeutic target for liver cancer treatments.

Abstract Background Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated. Methods We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays. Results A total of 74 ARID1A-interacting proteins were identified. Protein–protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and β-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays. Conclusions We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and β-actin. However, the role of ARID1A deficiency in angiogenesis is independent of β-actin.

The protein phosphatase PP2C plays an important role in plant responses to stress. Therefore, the identification of maize PP2C genes that respond to drought stress is particularly important for the improvement and creation of new drought-resistant assortments of maize. In this study, we identified 102 ZmPP2C genes in maize at the genome-wide level. We analyzed the physicochemical properties of 102 ZmPP2Cs and constructed a phylogenetic tree with Arabidopsis. By analyzing the gene structure, conserved protein motifs, and synteny, the ZmPP2Cs were found to be strongly conserved during evolution. Sixteen core genes involved in drought stress and rewatering were screened using gene co-expression network mapping and expression profiling. The qRT-PCR results showed 16 genes were induced by abscisic acid (ABA), drought, and NaCl treatments. Notably, ZmPP2C15 exhibited a substantial expression difference. Through genetic transformation, we overexpressed ZmPP2C15 and generated the CRISPR/Cas9 knockout maize mutant zmpp2c15. Overexpressing ZmPP2C15 in Arabidopsis under drought stress enhanced growth and survival compared with WT plants. The leaves exhibited heightened superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) activities, elevated proline (Pro) content, and reduced malondialdehyde (MDA) content. Conversely, zmpp2c15 mutant plants displayed severe leaf dryness, curling, and wilting under drought stress. Their leaf activities of SOD, POD, APX, and CAT were lower than those in B104, while MDA was higher. This suggests that ZmPP2C15 positively regulates drought tolerance in maize by affecting the antioxidant enzyme activity and osmoregulatory substance content. Subcellular localization revealed that ZmPP2C15 was localized in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments demonstrated ZmPP2C15's interaction with ZmWIN1, ZmADT2, ZmsodC, Zmcab, and ZmLHC2. These findings establish a foundation for understanding maize PP2C gene functions, offering genetic resources and insights for molecular design breeding for drought tolerance. [ABSTRACT FROM AUTHOR]

Copyright of Bulletin of Botanical Research is the property of Bulletin of Botanical Research Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated. Methods: We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays. Results: A total of 74 ARID1A-interacting proteins were identified. Protein–protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and β-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays. Conclusions: We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and β-actin. However, the role of ARID1A deficiency in angiogenesis is independent of β-actin. [ABSTRACT FROM AUTHOR]

Background: Previously, we reported that cysteine‐rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine‐rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. Objectives: This study aimed to identify cysteine‐rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non‐capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187‐induced acrosome reaction. Materials and Methods: The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography–mass spectrometry (LC‐MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC‐MS for protein identification. The most prominent cystein‐rich secretory protein 2 interacting proteins that appeared in both independent LC‐MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein‐rich secretory protein 2 in pig sperm. Results: Blue native gel electrophoresis and native immunoblots revealed that cystein‐rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein‐rich secretory‐protein 2‐immunoreactive bands from blue native gels as well as cystein‐rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein‐rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co‐immunoprecipitation and immunoblotting indicated that cystein‐rich secretory protein 2 interacted with pro‐acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein‐rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein‐rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein‐rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post‐acrosomal sheath region upon capacitation. Discussion and Conclusion: These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Chinese Institute of Food Science & Technology / Zhongguo Shipin Xuebao is the property of Journal of Chinese Institute of Food Science & Technology Periodical Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

S-adenosylmethionine synthase (SAMS) is the only enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM) from methionine and ATP. Research shows that SAMS participates in lignin biosynthesis. In this study, we analyzed the expression patterns and protein interaction networks of the SAMS gene family identified from ’Huangdan’ tea plant (Camellia sinensis) to explore candidate genes of CsSAMS that may be involved in lignin synthesis. Then we identified the members of CsSAMS gene family by bioinformatics based on the genome of ’Huangdan’, and further analyzed their protein physicochemical properties, phylogenetic tree, chromosome location, gene structure,protein structure and expression pattern. We also studied the protein interaction network by yeast two-hybrid technology (Y2H). Finally, we determined the lignin contents of the first bud and the two leaves of ’Haungdan’, ’Tieguanyin’, ’Jinguanyin’ and ’Fuding Dahao’ by UV spectrophotometer. The results of bioinformatics analysis showed that 4 CsSAMS gene family members were identified in ’Huangdan’.The number of encoded amino acids was 345-519, and the isoelectric point ranged from 6.12 to 6.47. The prediction results of subcellular localization revealed that CsSAMS1 was located in the chloroplast, CsSAMS2 and CsSAMS3 were located in the cytoplasm, and CsSAMS4 was located in the cytoskeleton. Expression of CsSAMS and lignin content detection of different tea varieties indicated that CsSAMS2, CsSAMS3 and CsSAMS4 might potentially regulate lignin content. In addition, Y2H results showed that CsSAMS4 formed homodimers with itself. In this study,the physicochemical properties of four CsSAMS members are identified and analyzed, and their functions are predicted. The expression patterns of CsSAMS genes in different tissue sites, under nitrogen and fluoride treatments, as well as the potential involvement of CsSAMS in lignin synthesis process, are clarified. [ABSTRACT FROM AUTHOR]

Our group screened out two genes (CSS0013384 and CSS0036305) related to the albino leaf color of Camellia sinensis Baijiguan through the transcriptome, in order to explore the expression patterns of CSS0013384 and CSS0036305 in albino tea plants interacting proteins. Using cultivar Baijiguan as material, the full-length cDNA sequences of CSS0013384 and CSS0036305 were cloned. Bioinformatics, yeast one-hybrid and yeast two-hybrid were used to analyze the protein physicochemical properties, the phylogenetic tree, the chromosome location, the gene structure, the protein structure, the protein regulation and interaction network, and gene expression pattern. Bioinformatics analysis showed that CSS0013384 and CSS0036305 belonged to the pentatricopeptide repeat protein (PPR) and chaperone (CPN60-like) families, respectively. The lengths of their protein coding sequence (CDS) was 1 893 bp and 1 752 bp, the number of encoded amino acids was 631 and 575, the protein mass was 71.87 kD and 60.79 kD, the isoelectric points were 8.93 and 6.21, the subcellular localization prediction results indicated that CSS0013384 was localized in chloroplast, and CSS0036305 was localized in mitochondria. The RT-qPCR results of the second leaf of Baijiguan under shading and restored light treatment and tea plant varieties with different leaf colors revealed that CSS0013384 and CSS0036305 were highly expressed in albino leaves. CSS0002807 belonged to the PIF transcription factor family, and the yeast one-hybrid results indicated that CSS0002807 bound to the CSS0013384 promoter. CSS0013384 and CSS0036305 may be involved in the development of chloroplast and mitochondria in albino tea leaves, and play an important role in the process of leaf albino. The results may provide a reference for further research on the mechanism of tea leaf albino. [ABSTRACT FROM AUTHOR]

[Objectives]The aim of this study was to identify the interacting proteins of MaMADS2, a key regulator of banana fruit ripening, and reveal the molecular mechanism of MaMADS2 regulating fruit ripening. [Methods]Using MaMADS2 as a “bait”, the candidate MaMADS2-interacting proteins were identified by yeast two-hybrid library, and its coding sequence of interacting proteins was cloned for confirming its interaction by one-to-one yeast two-hybrid assay. The expression of interacting protein related genes during fruit ripening was analyzed by transcriptome. The physical interactions between important interacting proteins and MaMADS2 were identified by bimolecular fluorescent complimentary(BiFC)assay. Finally, the expression of MaMADS2 protein during fruit ripening was analyzed by Western-blot assay. [Results]A cDNA library was successfully constructed with ethephon-treated banana fruit as materials. The capacities of the primary and secondary libraries were 2.9×106 and 3.0×106 CFU·mL-1, respectively, and the average size of inserts was above 1 200 bp in the cDNA library, which could meet the requirements of subsequent yeast two-hybrid screening. Finally seven interacting proteins were determined, including two MADS-box transcription factor homologs, three E3 ubiquitin ligases, and two proteins with transcriptional inhibitory activity. Transcriptome data indicated that ethephon inhibited the expression of two MADS-box genes, while 1-MCP induced their expression. BiFC assay results showed that MaMADS2 and MADS-box transcription factor Ma04_P30020.1 could interact with each other. Moreover, Western-blot assay analysis indicated that the protein level of MaMADS2 decreased gradually during fruit ripening, which was consistent with the fact that E3 ubiquitin ligases could interact with it. [Conclusions]By constructing high-quality fruit cDNA library and yeast two-hybrid screening library technology, several proteins interacting with MaMADS2 were identified, and the interaction between MaMADS2 and another MADS-box transcription factor Ma04_P30020.1 was demonstrated. [ABSTRACT FROM AUTHOR]

The protein phosphatase PP2C plays an important role in plant responses to stress. Therefore, the identification of maize PP2C genes that respond to drought stress is particularly important for the improvement and creation of new drought-resistant assortments of maize. In this study, we identified 102 ZmPP2C genes in maize at the genome-wide level. We analyzed the physicochemical properties of 102 ZmPP2Cs and constructed a phylogenetic tree with Arabidopsis. By analyzing the gene structure, conserved protein motifs, and synteny, the ZmPP2Cs were found to be strongly conserved during evolution. Sixteen core genes involved in drought stress and rewatering were screened using gene co-expression network mapping and expression profiling. The qRT-PCR results showed 16 genes were induced by abscisic acid (ABA), drought, and NaCl treatments. Notably, ZmPP2C15 exhibited a substantial expression difference. Through genetic transformation, we overexpressed ZmPP2C15 and generated the CRISPR/Cas9 knockout maize mutant zmpp2c15. Overexpressing ZmPP2C15 in Arabidopsis under drought stress enhanced growth and survival compared with WT plants. The leaves exhibited heightened superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) activities, elevated proline (Pro) content, and reduced malondialdehyde (MDA) content. Conversely, zmpp2c15 mutant plants displayed severe leaf dryness, curling, and wilting under drought stress. Their leaf activities of SOD, POD, APX, and CAT were lower than those in B104, while MDA was higher. This suggests that ZmPP2C15 positively regulates drought tolerance in maize by affecting the antioxidant enzyme activity and osmoregulatory substance content. Subcellular localization revealed that ZmPP2C15 was localized in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments demonstrated ZmPP2C15’s interaction with ZmWIN1, ZmADT2, ZmsodC, Zmcab, and ZmLHC2. These findings establish a foundation for understanding maize PP2C gene functions, offering genetic resources and insights for molecular design breeding for drought tolerance.

Background: This study aimed to explore the relationship between MALAT1 and the prognosis of patients with hepatocellular carcinoma (HCC).Methods: We constructed a MALAT1 protein-protein interaction network using the STRING database and a network of competing endogenous RNAs (ceRNAs) using the StarBase database. Using data from the GEPIA2 database, we studied the association between genes in these networks and survival of patients with HCC. The potential mechanisms underlying the relationship between MALAT1 and HCC prognosis were studied using combined data from RNA sequencing, DNA methylation, and somatic mutation data from The Cancer Genome Atlas (TCGA) liver cancer cohort. Tumor tissues and 19 paired adjacent non-tumor tissues (PANTs) from HCC patients who underwent radical resection were analyzed for MALAT1 mRNA levels using real-time PCR, and associations of MALAT1 expression with clinicopathological features or prognosis of patients were analyzed using log-rank test and Gehan-Breslow-Wilcoxon test.Results: Five interacting proteins and five target genes of MALAT1 in the ceRNA network significantly correlated with poor survival of patients with HCC (p < 0.05). High MALAT1 expression was associated with mutations in two genes leading to poor prognosis and may upregulate some prognostic risk genes through methylation. MALAT1 was significantly co-expressed with various signatures of genes involved in HCC progression, including the cell cycle, DNA damage repair, mismatch repair, homologous recombination, molecular cancer m6A, exosome, ferroptosis, infiltration of lymphocyte (p < 0.05). The expression of MALAT1 was markedly upregulated in HCC tissues compared with PANTs. In Kaplan-Meier analysis, patients with high MALAT1 expression had significantly shorter progression-free survival (PFS) (p = 0.033) and overall survival (OS) (p = 0.023) than those with low MALAT1 expression. Median PFS was 19.2 months for patients with high MALAT1 expression and 52.8 months for patients with low expression, while the corresponding median OS was 40.5 and 78.3 months. In subgroup analysis of patients with vascular invasion, cirrhosis, and HBsAg positive or AFP positive, MALAT1 overexpression was significantly associated with shorter PFS and OS. Models for predicting PFS and OS constructed based on MALAT1 expression and clinicopathological features had moderate predictive power, with areas under the receiver operating characteristic curves of 0.661–0.731. Additionally, MALAT1 expression level was significantly associated with liver cirrhosis, vascular invasion, and tumor capsular infiltration (p < 0.05 for all).Conclusion:MALAT1 is overexpressed in HCC, and higher expression is associated with worse prognosis. MALAT1 mRNA level may serve as a prognostic marker for patients with HCC after hepatectomy.

This document is a corrigendum for an article titled "Identification of interacting proteins of transcription factor DpAP2 related to carotenoid biosynthesis from marine microalga Dunaliella parva." The corrigendum addresses an error in the funding statement of the original article, where the grant number for the National Training Program of Innovation and Entrepreneurship for Undergraduates was incorrect. The correct grant number is provided, and the authors state that this error does not affect the scientific conclusions of the article. The corrigendum also includes the contact information for the corresponding author and the publication details of the original article. [Extracted from the article]

Metabotropic glutamate receptor Type 3 (mGlu3) controls the sleep/wake architecture, which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptor expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that this scaffolding protein is important for mGlu3 surface expression and function in hippocampal primary cultures. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors in neurons. We next investigated the impact of disrupting the mGlu3-PICK1 interaction on hippocampal theta oscillations in vitro and in vivo in WT male mice. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what was previously observed in mGlu3 KO mice. In addition, hippocampal theta power was reduced during rapid eye movement sleep, non-rapid eye movement (NREM) sleep, and wake states after intraventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia. [ABSTRACT FROM AUTHOR]

SELF-PRUNING 6A (SP6A), a homolog of FLOWERING LOCUS T (FT), has been identified as tuberigen in potato. StSP6A is a mobile signal synthesized in leaves and transmitted to the stolon through phloem, and plays multiple roles in the growth and development of potato. However, the global StSP6A protein interaction network in potato remains poorly understood. In this study, BK-StSP6A was firstly used as the bait to investigate the StSP6A interaction network by screening the yeast two-hybrid (Y2H) library of potato, resulting in the selection of 200 independent positive clones and identification of 77 interacting proteins. Then, the interaction between StSP6A and its interactors was further confirmed by the Y2H and BiFC assays, and three interactors were selected for further expression analysis. Finally, the expression pattern of Flowering Promoting Factor 1.1 (StFPF1.1), No Flowering in Short Days 1 and 2 (StNFL1 and StNFL2) was studied. The three genes were highly expressed in flowers or flower buds. StFPF1.1 exhibited an expression pattern similar to that of StSP6A at the stolon swelling stages. StPHYF-silenced plants showed up-regulated expression of StFPF1.1 and StSP6A, while expression of StNFL1 and StNFL2 was down-regulated in the stolon. The identification of these interacting proteins lays a solid foundation for further functional studies of StSP6A. [ABSTRACT FROM AUTHOR]

Key message: Heterologous expression of VaMYB44 gene in Arabidopsis and V. vinifera cv. 'Thompson Seedless' increases cold sensitivity, which is mediated by the interaction of VaMYC2 and VaTIFY5A with VaMYB44 MYB transcription factors play critical roles in plant stress response. However, the function of MYB44 under low temperature stress is largely unknown in grapes. Here, we isolated a VaMYB44 gene from Chinese wild Vitis amurensis acc. 'Shuangyou' (cold-resistant). The VaMYB44 is expressed in various organs and has lower expression levels in stems and young leaves. Exposure of the cold-sensitive V. vinifera cv. 'Thompson Seedless' and cold-resistant 'Shuangyou' grapevines to cold stress (−1 °C) resulted in differential expression of MYB44 in leaves with the former reaching 14 folds of the latter after 3 h of cold stress. Moreover, the expression of VaMYB44 was induced by exogenous ethylene, abscisic acid, and methyl jasmonate in the leaves of 'Shuangyou'. Notably, the subcellular localization assay identified VaMYB44 in the nucleus. Interestingly, heterologous expression of VaMYB44 in Arabidopsis and 'Thompson Seedless' grape increased freezing-induced damage compared to their wild-type counterparts. Accordingly, the transgenic lines had higher malondialdehyde content and electrolyte permeability, and lower activities of superoxide dismutase, peroxidase, and catalase. Moreover, the expression levels of some cold resistance-related genes decreased in transgenic lines. Protein interaction assays identified VaMYC2 and VaTIFY5A as VaMYB44 interacting proteins, and VaMYC2 could bind to the VaMYB44 promoter and promote its transcription. In conclusion, the study reveals VaMYB44 as the negative regulator of cold tolerance in transgenic Arabidopsis and transgenic grapes, and VaMYC2 and VaTIFY5A are involved in the cold sensitivity of plants by interacting with VaMYB44. [ABSTRACT FROM AUTHOR]

Cysteine proteinases (thiol) carry out diverse and critical functions in plants through their ability to hydrolyze peptide bonds in target proteins. Here, we cloned a cysteine proteinase gene designated VaCP17 from a highly cold-resistant wild Vitis amurensis accession 'Shuangyou', and then its potential function in cold resistance was investigated. The results showed that the CDS of VaCP17 is 1404 bp, encoding 467 amino acids, the VaCP17 protein localized to the cell membrane. Expression of CP17 was highly distinctive among different structures of 'Shuangyou' and the cold-sensitive Vitis vinifera cultivar 'Red Globe', with the highest expression in the stem of 'Shuangyou' and the flower of 'Red Globe'. Arabidopsis plants constitutively expressing a VaCP17-GREEN FLUORESCENT PROTEIN fusion (35S::VaCP17-GFP) showed increased survival after transient exposure to freezing (-6 °C), and showed lower electrolyte leakage and MDA content, higher soluble sugar content and SOD, POD and CAT activities, as compared with non-transgenic Arabidopsis controls. The expression of nine cold-resistance related genes (CBF1, CBF2, CBF3, RD29A, COR15A, KIN1, NCED3, AOC1 and JAZ10) in 35S::VaCP17-GFP plants was increased under cold treatment at 4 °C, relative to control plants. Using a yeast two-hybrid system, we identified VaNAC72, VaCAM7 and VaDi19 as potential interactors of VaCP17, and their interactions were demonstrated by a bimolecular fluorescence complementation assay. In conclusion, we revealed that VaCP17 can enhance cold resistance by influencing physiology and biochemistry and the expression of cold resistance related genes under cold stress. Key message: VaCP17 gene was cloned from the high cold-resistant wild Vitis amurensis acc. 'Shuangyou'. The heterologous expression of VaCP17 can enhance the cold tolerance of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Carotenoids are widely distributed and structurally diverse, which have significant roles in the photosynthesis of plants. As a precursor of vitamin A, carotenoids are also antioxidants that reduce various chronic diseases, which are beneficial for human health. Currently, the existing studies concerned the biological roles of APETALA2 (AP2)/ethylene-responsive factor (ERF) genes originated from higher plants. The AP2 superfamily of the transcriptional regulator was identified in higher plants, which was related to growth, development, carotenoid metabolism, and responses to various stresses. However, the regulatory mechanisms of the AP2-modulating carotenoid metabolism have not been reported in microalgae, which remain to be elucidated. Dunaliella parva AP2 (i.e., DpAP2), an important transcription factor, promotes carotenoid accumulation by binding to the promoter of target gene. Here, we identified an important AP2/ERF transcription factor, DpAP2, which could promote carotenoid accumulation by binding to the promoter of target gene. To demonstrate the function of DpAP2, the interacting proteins were identified by the yeast two-hybrid system. The results showed that DpAP2 could interact with three proteins with different activities (DNA-binding transcription factor activity, protein kinase activity, and alpha-D-phosphohexomutase activity); these proteins may be associated with multiple biological processes. This paper laid a good foundation for a deep understanding of the regulatory mechanisms of DpAP2 and genetic engineering breeding in D. parva.

Abstract Background Leptin Receptor (LEPR) has been suggested to have several roles in cancer metastasis. However, the role of LEPR and its underlying mechanisms in lymphatic metastasis of hepatocarcinoma have not yet been studied. Methods We performed bioinformatics analysis, qRT-PCR, western blotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent, coimmunoprecipitation assays and a series of functional assays to investigate the roles of LEPR in hepatocellular carcinoma. Results We discovered that LEPR was highly expressed in liver cancer tissues, and the expression of LEPR in Hca-F cells was higher than that in Hca-P cells. Furthermore, LEPR promotes the proliferation, migration and invasion and inhibits the apoptosis of hepatocarcinoma lymphatic metastatic cells. Further studies indicated that LEPR interacts with ANXA7. Mechanistically, LEPR regulated ERK1/2 and JAK2/STAT3 expression via ANXA7 regulation. Conclusions These findings unveiled a previously unappreciated role of LEPR in the regulation of lymphatic metastatic hepatocellular carcinoma, assigning ANXA7-LEPR as a promising therapeutic target for liver cancer treatments.

Improving tolerance to low-temperature stress during the rice seedling stage is of great significance in agricultural science. In this study, using the low silicon gene 1 (Lsi1)-overexpressing (Dular-OE) and wild-type rice (Dular-WT), we showed that Lsi1 overexpression enhances chilling tolerance in Dular-OE. The overexpression of the Lsi1 increases silicon absorption, but it was not the main reason for chilling tolerance in Dular-OE. Instead, our data suggest that the overexpression of a Lsi1-encoding NIP and its interaction with key proteins lead to chilling tolerance in Dular-OE. Additionally, we show that the high-mobility group protein (HMG1) binds to the promoter of Lsi1, positively regulating its expression. Moreover, Nod26-like major intrinsic protein (NIP)'s interaction with α and β subunits of ATP synthase and the 14-3-3f protein was validated by co-immunoprecipitation (Co-IP), bimolecular fluorescent complementary (BiFC), and GST-pulldown assays. Western blotting revealed that the overexpression of NIP positively regulates the ATP-synthase subunits that subsequently upregulate calcineurin B-like interacting protein kinases (CIPK) negatively regulating 14-3-3f. Overall, these NIP-mediated changes trigger corresponding pathways in an orderly manner, enhancing chilling tolerance in Dular-OE. [ABSTRACT FROM AUTHOR]

Late embryogenesis-abundant (LEA) proteins are critical in helping plants cope with salt stress. "Y1805" is a salt-tolerant Tritipyrum. We identified a "Y1805"-specific LEA gene that was expressed highly and sensitively under salt stress using transcriptome analysis. The novel group 2 LEA gene (TtLEA2-1) was cloned from "Y1805." TtLEA2-1 contained a 453 bp open reading frame encoding an 151-amino-acid protein that showed maximum sequence identity (77.00%) with Thinopyrum elongatum by phylogenetic analysis. It was mainly found to be expressed highly in the roots by qRT-PCR analysis and was located in the whole cell. Forty-eight candidate proteins believed to interact with TtLEA2-1 were confirmed by yeast two-hybrid analysis. These interacting proteins were mainly enriched in "environmental information processing," "glycan biosynthesis and metabolism," and "carbohydrate metabolism." Protein-protein interaction analysis indicated that the translation-related 40S ribosomal protein SA was the central node. An efficient wheat transformation system has been established. A coleoptile length of 2 cm, an Agrobacteria cell density of 0.55–0.60 OD600, and 15 KPa vacuum pressure were ideal for common wheat transformation, with an efficiency of up to 43.15%. Overexpression of TaLEA2-1 in wheat "1718" led to greater height, stronger roots, and higher catalase activity than in wild type seedlings. TaLEA2-1 conferred enhanced salt tolerance in transgenic wheat and may be a valuable gene for genetic modification in crops. [ABSTRACT FROM AUTHOR]

Late embryogenesis-abundant (LEA) proteins are critical in helping plants cope with salt stress. “Y1805” is a salt-tolerant Tritipyrum. We identified a “Y1805”-specific LEA gene that was expressed highly and sensitively under salt stress using transcriptome analysis. The novel group 2 LEA gene (TtLEA2-1) was cloned from “Y1805.” TtLEA2-1 contained a 453 bp open reading frame encoding an 151-amino-acid protein that showed maximum sequence identity (77.00%) with Thinopyrum elongatum by phylogenetic analysis. It was mainly found to be expressed highly in the roots by qRT-PCR analysis and was located in the whole cell. Forty-eight candidate proteins believed to interact with TtLEA2-1 were confirmed by yeast two-hybrid analysis. These interacting proteins were mainly enriched in “environmental information processing,” “glycan biosynthesis and metabolism,” and “carbohydrate metabolism.” Protein-protein interaction analysis indicated that the translation-related 40S ribosomal protein SA was the central node. An efficient wheat transformation system has been established. A coleoptile length of 2 cm, an Agrobacteria cell density of 0.55–0.60 OD600, and 15 KPa vacuum pressure were ideal for common wheat transformation, with an efficiency of up to 43.15%. Overexpression of TaLEA2-1 in wheat “1718” led to greater height, stronger roots, and higher catalase activity than in wild type seedlings. TaLEA2-1 conferred enhanced salt tolerance in transgenic wheat and may be a valuable gene for genetic modification in crops.

Rapeseed is one important oil crop in China. However, its planting benefit is frequently affected by environmental stresses such as drought in the northwest region of China. The abscisic acid(ABA) signaling pathway plays an important role in plant abiotic stress response and tolerance, and ABFs/AREBs(ABA-responsive element binding factors/ABA-responsive element binding proteins) are the core transcription factors that regulate the expression of ABA-responsive genes. To dissect the key transcription factors mediated abiotic stress, we mainly characterized abscisic acid insensitive 5(BnaABI5) in rapeseed, including its subcellular localization, expression pattern in response to various stress and tissue-specific expression analysis, transcriptional activity analysis as well as interaction screening with BnaMPKs(mitogen-activated protein kinases). Our results showed that the BnaABI5-GFP fusion protein was localized in the nucleus, and its transcript level is induced by drought stress and was mainly expressed in the roots of rapeseed. Furthermore, BnaABI5 showed transcriptional activation activity through a yeast transactivation assay and it also activated the promoter activity of EM6 target gene in the transient expression system in tobacco leaves. Moreover, BnaABI5 interacted with BnaMPK6 and BnaMPK13 through BiFC and Y2H analysis. This study preliminarily explored the expression characteristics of transcription factor BnaABI5 and its interaction with BnaMPKs, which might help us for further understanding the function of BnaABI5.

The protein arginine methyltransferases (PRMTs) are involved in such biological processes as transcription regulation, DNA repair, RNA splicing, and signal transduction, etc. In this study, we mainly focused on PRMT5, a member of the type II PRMTs, which functions mainly alongside other interacting proteins. PRMT5 has been shown to be overexpressed in a wide variety of cancers and other diseases, and is involved in the regulation of Epstein-Barr virus infection, viral carcinogenesis, spliceosome, hepatitis B, cell cycles, and various signaling pathways. We analyzed the regulatory roles of PRMT5 and interacting proteins in various biological processes above-mentioned, to elucidate for the first time the interaction between PRMT5 and its interacting proteins. This systemic analysis will enrich the biological theory and contribute to the development of novel therapies.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (Et CDPK4), a putative interactor, translation initiation factor eIF-5A (Et eIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between Et eIF-5A and Et CDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of Et eIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of Et eIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of Et eIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the Et eIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, Et eIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that Et eIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-r Et eIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%. [ABSTRACT FROM AUTHOR]

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Nrf2 (NFE2L2 – nuclear factor (erythroid-derived 2)-like 2) is a transcription factor, which is repressed by interaction with a redox-sensitive protein Keap1 (Kelch-like ECH-associated protein 1). Deregulation of Nrf2 transcriptional activity has been described in the pathogenesis of multiple diseases, and the Nrf2/Keap1 axis has emerged as a crucial modulator of cellular homeostasis. Whereas the significance of Nrf2 in the modulation of biological processes has been well established and broadly discussed in detail, the focus on Keap1 rarely goes beyond the regulation of Nrf2 activity and redox sensing. However, recent studies and scrutinized analysis of available data point to Keap1 as an intriguing and potent regulator of cellular function. This review aims to shed more light on Keap1 structure, interactome, regulation and non-canonical functions, thereby enhancing its significance in cell biology. We also intend to highlight the impact of balance between Keap1 and Nrf2 in the maintenance of cellular homeostasis. Image 1 • In this review we emphasize the non-canonical functions of Keap1. • We summarize numerous post-translational modifications on Keap1 and its rich interactome. • The modes of Keap1 regulation are presented. • The impact of balance between Keap1 and Nrf2 in the maintenance of cellular homeostasis is highlighted. [ABSTRACT FROM AUTHOR]

The anatomic gene expression atlas (AGEA) of the adult mouse brain of the Allen Institute for Brain Science is a transcriptome-based atlas of the adult C57Bl/6 J mouse brain, based on the extensive in situ hybridization dataset of the Institute. This spatial mapping of the gene expression levels of mice under baseline conditions could assist in the formation of new, reasonable transcriptome-derived hypotheses on brain structure and underlying biochemistry, which could also have functional implications. The aim of this work is to use the data of the AGEA (in combination with Tabula Muris, a compendium of single cell transcriptome data collected from mice, enabling direct and controlled comparison of gene expression among cell types) to provide further insights into the physiology of TAG-1/Contactin-2 and its interactions, by presenting the expression of the corresponding gene across the adult mouse brain under baseline conditions and to investigate any spatial genomic correlations between TAG-1/Contactin-2 and its interacting proteins and markers of mature and immature oligodendrocytes, based on the pre-existing experimental or bibliographical evidence. The across-brain correlation analysis on the gene expression intensities showed a positive spatial correlation of TAG-1/Contactin-2 with the gene expression of Plp1, Myrf, Mbp, Mog, Cldn11, Bace1, Kcna1, Kcna2, App and Nfasc and a negative spatial correlation with the gene expression of Cspg4, Pdgfra, L1cam, Ncam1, Ncam2 and Ptprz1. Spatially correlated genes are mainly expressed by mature oligodendrocytes (like Cntn2), while spatially anticorrelated genes are mainly expressed by oligodendrocyte precursor cells. According to the data presented in this work, we propose that even though Contactin-2 expression during development correlates with high plasticity events, such as neuritogenesis, in adulthood it correlates with pathways characterized by low plasticity. [ABSTRACT FROM AUTHOR]

Agouti signaling protein (ASP) is a secreted paracrine protein that has been widely reported to function in melanogenesis and obesity and could potentially be a core protein that regulates the color and fatty phenotype of P. sinensis. In this study, we screened out interacting proteins of ASP by combined co-immunoprecipitation mass spectrometry (CoIP-MS), yeast two hybrid (Y2H) analysis, and computational predictions. We performed docking of ASP with its well-known receptor melanocortin receptor 4 (MC4R) to predict the binding capacity and to screen out actual ASP interacting proteins, CoIP-MS was performed where identified 32 proteins that could bind with ASP and Y2H confirmed seven proteins binding with ASP directly. CoIP-MS and Y2H screening results including PPI prediction revealed that vitronectin (VTN), apolipoprotein A1 (APOA1), apolipoprotein B (APOB), and filamin B (FLNB) were the key interacting proteins of ASP. VTN, APOA1, and APOB are functional proteins in lipid metabolism and various skin disorders, suggesting ASP may function in lipid metabolism through these partners. This study provided protein-protein interaction information of ASP, and the results will promote further research into the diverse roles of ASP, as well as its binding partners, and their function in different strains of P. sinensis. [ABSTRACT FROM AUTHOR]

The (asymptotic) degree distributions of the best-known "scale-free'' network models are all similar and are independent of the seed graph used; hence, it has been tempting to assume that networks generated by these models are generally similar. In this paper, we observe that several key topological features of such networks depend heavily on the specific model and the seed graph used. Furthermore, we show that starting with the "right" seed graph (typically a dense subgraph of the protein-protein interaction network analyzed), the duplication model captures many topological features of publicly available protein-protein interaction networks very well.

Mycoplasma genitalium is a newly recognized pathogen associated with sexually transmitted diseases (STDs). MgPa, the adhesion protein of Mycoplasma genitalium, is the main adhesin and the key factor for M. genitalium interacting with host cells. Currently, the long-term survival mechanism of M. genitalium in the host is not clear. In this study, a T7 phage-displayed human urothelial cell (SV-HUC-1) cDNA library was constructed, and the interaction of MgPa was screened from this library using the recombinant MgPa (rMgPa) as a target molecule. We verified that 60S ribosomal protein L35 (RPL35) can interact with MgPa using far-Western blot and co-localization analysis. According to the results of tandem mass tag (TMT) labeling and proteome quantitative analysis, there were altogether 407 differentially expressed proteins between the pcDNA3.1(+)/MgPa-transfected cells and non-transfected cells, of which there were 6 downregulated proteins and 401 upregulated proteins. The results of qRT-PCR demonstrated that interaction between rMgPa and RPL35 could promote the expressions of EIF2, SRP68, SERBP1, RPL35A, EGF, and TGF-β. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide bromide (MTT) assays corroborated that the interaction between rMgPa and RPL35 could promote SV-HUC-1 cell proliferation. Therefore, our findings indicated that the interaction between rMgPa and RPL35 can enhance the expressions of transcription-initiation and translation-related proteins and thus promote cell proliferation. This study elucidates a new biological function of MgPa and can explain this new mechanism of M. genitalium in the host.

Leucine aminopeptidase of Taenia pisiformis (TpLAP) belonging to the M17 peptidase family has been implicated as a stage-differentially expressed protein in the adult stage of T. pisiformis. In order to further dissect the biological functions of TpLAP in the growth and development of adult worms, TpLAP-interacting partners were investigated. In this study, a yeast two-hybrid (Y2H) cDNA library from adult T. pisiformis was constructed. Using pGBKT7-TpLAP as bait, proteins interacting with TpLAP were screened by Y2H system and positive preys were sequenced and analyzed using the Basic Local Alignment Search Tool (BLAST). Our results showed that six genuine TpLAP-interacting proteins, including LAP, dynein light chain (DLC), SUMO-conjugating enzyme (UBC9), histone-lysine n-methyltransferase, trans-acting transcriptional, and one unknown protein, were identified via Y2H assay. Furthermore, the interaction between TpLAP and UBC9 of T. pisiformis (TpUBC9), an important protein involved in SUMOylation pathway, was further validated by one-to-one Y2H assay, co-immunoprecipitation, and confocal analysis. These findings provide a deeper understanding of the biological functions of TpLAP and offer the first clue that TpLAP may act as a novel SUMOylated substrate, suggesting that the SUMO modification pathway plays an important role in regulation of adult worm growth and development. [ABSTRACT FROM AUTHOR]

The influence of pure transgenic wheat strain 1477 and Zhengmai 9023 were used to explore the effect of exogenous RsRHA2b gene introduction on the activity of key enzymes for starch synthesis and protein synthesis and the expression of related genes in transgenic wheat during filling period. The results showed that the introduction of exogenous RsRHA2b increased the activity of adenosine diphosphate glucose pyrophosphorylase (AGPP), granular starch synthase (GBSS), starch branching enzyme (SBE), soluble starch synthase (SSS) and glutamate synthase (GOGAT), and decreased the activity of glutamyl ammonia synthase (GS). Exogenous RsRHA2b promoted the expression of YTH311, YTH611, YTH1065, YTH2437, YTH2438, YTH2456, YTH2496 and YTH3049, and inhibited the expression of YTH2433. These results indicated that the change in carbon and nitrogen metabolic key enzyme activity and the effect on related gene expression caused by the introduction of exogenous RsRHA2b gene may be the main cause of the increased resistance to ear germination of RsRHA2b transgenic wheat. [ABSTRACT FROM AUTHOR]

Hemocyanin is the main component of hemolymph plasma proteins and possesses diverse immunological properties and immunomodulatory functions. However, the interacting networks of hemocyanin in shrimp immune response remain poorly understood. In this study, 39 potential hemocyanin interacting partners were identified from Litopenaeus vannamei plasma by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Gene Ontology (GO) analysis showed that most of the identified interactors were cell proteins involved in metabolic process and binding. Among these identified proteins, transglutaminase (TGase), a crucial regulator in hemolymph clotting cascade, was chosen for further studies. Far-Western blot and His-pull down assays revealed that hemocyanin directly interacted with TGase. Further analysis demonstrated that hemocyanin and TGase followed similar expression patterns upon pathogen infection. Moreover, in vivo knockdown of hemocyanin led to a significant decrease in TGase expression, as well as inhibited hemolymph clotting. Taken together, these data suggest that hemocyanin might positively regulate hemolymph clotting by modulating TGase in shrimp. The interaction networks among immune-related factors is critical for the innate immune response in invertebrates. We report for the first time, proteins that potentially interact with hemocyanin, which led to the identification of 39 possible hemocyanin-proteins including the clotting-related factor TGase. Further studies demonstrated that hemocyanin directly interacted with TGase and modulated its expression, therefore affecting the formation of hemolymph clotting. These findings not only extend our knowledge of the immune interaction networks but also contribute to shrimp disease control and prevention. Unlabelled Image • We identified 39 potential interacting partners in Litopenaeus vannamei plasma. • Hemocyanin was demonstrated to directly interact with TGase. • Hemocyanin could modulate TGase expression and affect the formation of hemolymph clotting. [ABSTRACT FROM AUTHOR]

Anti-depressant treatment has been found to be associated with the development of Brugada syndrome (BrS) through poorly defined mechanisms. Herein, this study aimed to explore the molecular basis for amitriptyline-induced BrS. The effects of long-term treatments of amitriptyline on Nav1.5 were investigated using neonatal rat ventricular myocytes. The electrophysiological properties, expression and distribution of Nav1.5 were studied using the patch clamp, Western blot and confocal laser microscopy assays. Interactions between Nav1.5 and its interacting proteins, including ankyrin-G and dystrophin, were evaluated by co-immunoprecipitation. A larger decrease in the peak INa occurred after long-term treatments to amitriptyline (56.64%) than after acute exposure to amitriptyline (28%). Slow recovery from inactivation of Nav1.5 was observed after acute or long-term treatments to amitriptyline. The expression of Nav1.5 on the cell membrane showed a larger decrease by long-term treatments to amitriptyline than by acute exposure to amitriptyline. After long-term treatments to amitriptyline, we observed reduced Nav1.5 proteins on the cell membrane and the disrupted co-localization of Nav1.5 and ankyrin-G or dystrophin. Co-immunoprecipitation experiments further testified that the combination of Nav1.5 and ankyrin-G or dystrophin was severely weakened after long-term treatments to amitriptyline, implying the failed interaction between Nav1.5 and ankyrin-G or dystrophin. Our data suggest that the long-term effect of amitriptyline serves as an important contribution to BrS induced by amitriptyline. The mechanisms of BrS induced by amitriptyline were related to Nav1.5 trafficking and could be explained by the disrupted interaction of ankyrin-G, dystrophin and Nav1.5.

Protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in eukaryotes. Protein tyrosine phosphorylation and dephosphorylation are catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. The combinatorial action of both PTKs and PTPs is essential for properly maintaining cellular functions. In this unit, we discuss different novel methods to identify PTP substrates. PTPs depend on specific invariant residues that enable binding to tyrosine-phosphorylated substrates and aid catalytic activity. Identifying PTP substrates has paved the way to understanding their role in distinct intracellular signaling pathways. Due to their high specific activity, the interaction between PTPs and their substrates is transient; therefore, identifying the physiological substrates of PTPs has been challenging. To identify the physiological substrates of PTPs, various PTP mutants have been generated. These PTP mutants, named "substrate-trapping mutants," lack catalytic activity but bind tightly to their tyrosine-phosphorylated substrates. Identifying the substrates for the PTPs will provide critical insight into the function of physiological and pathophysiological signal transduction. In this chapter, we describe interaction assays used to identify the PTP substrates., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)