Protein Phosphorylation Research Articles

Prediction of SARS-CoV-2 Infection Phosphorylation Sites and Associations of these Modifications with Lung Cancer Development.

Since the emergence of SARS-CoV-2 viruses, multiple mutant strains have been identified. Infection with SARS-CoV-2 virus leads to alterations in host cell phosphorylation signal, which systematically modulates the immune response. Identification and analysis of SARS-CoV-2 virus infection phosphorylation sites enable insight into the mechanisms of viral infection and effects on host cells, providing important fundamental data for the study and development of potent drugs for the treatment of immune inflammatory diseases. In this paper, we have analyzed the SARS-CoV-2 virus-infected phosphorylation region and developed a transformer-based deep learning-assisted identification method for the specific identification of phosphorylation sites in SARS-CoV-2 virus-infected host cells. Furthermore, through association analysis with lung cancer, we found that SARS-CoV-2 infection may affect the regulatory role of the immune system, leading to an abnormal increase or decrease in the immune inflammatory response, which may be associated with the development and progression of cancer. We anticipate that this study will provide an important reference for SARS-CoV-2 virus evolution as well as immune-related studies and provide a reliable complementary screening tool for anti-SARS-CoV-2 virus drug and vaccine design.

Transcriptomic analysis provides insights into the molecular mechanism of melatonin-mediated cadmium tolerance in Medicago sativa L.

Cadmium (Cd), a toxic element, often makes a serious threat to plant growth and development. Previous studies found that melatonin (Mel) reduced Cd accumulation and reestablished the redox balance to alleviate Cd stress in Medicago sativa L., however, the complex molecular mechanisms are still elusive. Here, comparative transcriptome analysis and biochemical experiments were conducted to explore the molecular mechanisms of Mel in enhancing Cd tolerance. Results showed that 7237 differentially expressed genes (DEGs) were regulated by Mel pretreatment to Cd stress compared to the control condition in roots of Medicago sativa L. Besides, in comparison with Cd stress alone, Mel upregulated 1081 DEGs, and downregulated 1085 DEGs. These DEGs were mainly involved in the transcription and translation of genes and folding, sorting and degradation of proteins, carbohydrate metabolism, and hormone signal network. Application of Mel regulated the expression of several genes encoding ribosomal protein and E3 ubiquitin-protein ligase involved in folding, sorting and degradation of proteins. Moreover, transcriptomic analyse suggested that Mel might regulate the expression of genes encoding pectin lyase, UDP-glucose dehydrogenase, sucrose-phosphate synthase, hexokinase-1, and protein phosphorylation in the sugar metabolism. Therefore, these could promote sucrose accumulation and subsequently alleviate the Cd damage. In conclusion, above findings provided the mining of important genes and molecular basis of Mel in mitigating Cd tolerance and genetic cultivation of Medicago sativa L.

Screening and Activity Evaluation of Novel BCR-ABL/T315I Tyrosine Kinase Inhibitors.

Chronic myeloid leukemia (CML) is a kind of malignant tumor formed by the clonal proliferation of bone marrow hematopoietic stem cells. BCR-ABL fusion protein, found in more than 90% of patients, is a vital target for discovering anti-CML drugs. Up to date, imatinib is the first BCR-ABL tyrosine kinase inhibitor (TKI) approved by the FDA for treating CML. However, the drug resistance problems appeared for many reasons, especially the T135I mutation, a "gatekeeper" of BCR-ABL. Currently, there is no long-term effective and low side effect drug in clinical. This study intends to find novel TKIs targeting BCR-ABL with high inhibitory activity against T315I mutant protein by combining artificial intelligence technology and cell growth curve, cytotoxicity, flow cytometry and western blot experiments. The obtained compound was found to kill leukemia cells, which had good inhibitory efficacy in BaF3/T315I cells. Compound No 4 could induce cell cycle arrest, cause autophagy and apoptosis, and inhibit the phosphorylation of BCR-ABL tyrosine kinase, STAT5 and Crkl proteins. The results indicated that the screened compound could be used as a lead compound for further research to discover ideal chronic myeloid leukemia therapeutic drugs.

Biochanin A mitigates ulcerative colitis and intestinal inflammation in mice by inhibiting MAPK/NF-kB (p65) axis.

Ulcerative colitis (UC) is a chronic problem of the intestine and relapsing in nature. Biochanin A is a nature-derived isoflavonoid and has numerous bioactivities. However, its role against UC and intestinal inflammation remains obscure. We aimed to comprehensively explore the pharmacological effect of biochanin A in alleviating colitis and to evaluate the potential mechanisms. Initially, we explored the anti-inflammatory action of biochanin A (15, 30, and 60 μM) by employing lipopolysaccharide (LPS)-activated RAW 264.7 cells. In RAW 264.7 cells under LPS stimulation, biochanin A inhibited the elevation of reactive oxygen species (ROS) (p < 0.0001), interleukin (IL)-1β(p < 0.0001), IL-18 (p < 0.01), and tumor necrosis factor (TNF)-α (p < 0.01) release, nitrite production (p < 0.0001), and the expression of inducible nitric oxide synthase (iNOS)and cyclooxygenase-2 (COX-2)proteins. Next, we studied the effectiveness of biochanin A (20 and 40 mg/kg) in mouse colitis induced with dextran sulfate sodium (DSS)by assessing colon length, disease activity index (DAI)scoring, and performing colonoscopy and histological analysis. The pro-inflammatory cytokines were estimated using ELISA. Western blot studies were performed to assess underlying mechanisms. In mice, biochanin A treatment alleviated DAI score (p < 0.0001), restored colon length (p < 0.05) and morphology, and re-established colon histopathology. Biochanin A affects the phosphorylation of proteins associated with NF-κB (p65) and mitogen-activated protein kinase (MAPK)axis and regulates colonic inflammation by reducing the expression of inflammatory cytokines and myeloperoxidase (MPO)activity. Altogether, our findings support the idea that the anticolitis potential of biochanin A is allied with anti-inflammatory activity by inhibiting the MAPK/NF-κB(p65) axis. Hence, biochanin A may be an alternative option to alleviate the risk of colitis.

Phosphorylation of DNA-binding domains of CLOCK–BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

HSP70-mediated mitochondrial dynamics and autophagy represent a novel vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancersanalyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mousemodels of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Fisetin induces G2/M phase arrest and caspase-mediated cleavage of p21Cip1 and p27Kip1 leading to apoptosis and tumor growth inhibition in HNSCC.

The anticancer potential and associated mechanisms of flavonoid fisetin are yet to be fully investigated on human head and neck squamous cell carcinoma (HNSCC). In the present study, fisetin (25-75 µM for 24-48 h) dose-dependently inhibited growth and induced death in HNSCC Cal33 and UM-SCC-22B cells, without showing any death in normal cells. Fisetin (25-50 µM) induced G2/M phase arrest via decrease in Cdc25C, CDK1, cyclin B1 expression, and an increase in p53(S15). A concentration-dependent increase in fisetin-induced DNA damage and apoptosis in HNSCC cells was authenticated by comet assay, gamma-H2A.X(S139) phosphorylation, and marked cleavage of PARP protein. Interestingly, fisetin-induced cell death occurred independently of p53 and reactive oxygen species production. The activation of JNK and inhibition of PI3K/Akt, ERK1/2, EGFR, and STAT-3 signaling were identified. Further, fisetin-induced apoptosis was mediated, in part, via p21Cip1 and p27Kip1 cleavage by caspase, which was reversed by z-VAD-FMK, a pan-caspase inhibitor. Subsequently, fisetin was also found to induce autophagy; nevertheless, autophagy attenuation exaggerated apoptosis. Oral fisetin (50 mg/kg body weight) treatment to establish Cal33 xenograft in mice for 19 days showed 73% inhibition in tumor volume (p < 0.01) along with a decrease in Ki67-positive cells and an increase in cleaved caspase-3 level in tumors. Consistent with the effect of 50 µM fisetin in vitro, the protein levels of p21Cip1 and P27Kip1 were also decreased by fisetin in tumors. Together, these findings showed strong anticancer efficacy of fisetin against HNSCC with downregulation of EGFR-Akt/ERK1/2-STAT-3 pathway and activation of JNK/c-Jun, caspases and caspase-mediated cleavage of p21Cip1 and p27Kip1.

The role of SRPK1-mediated phosphorylation of SR proteins in the chromatin configuration transition of mouse germinal vesicle oocytes.

Meiotic resumption in mammalian oocytes involves nucleus and organelle structural changes, notably chromatin configuration transitioning from non-surrounding nucleolus (NSN) to surrounding nucleolus (SN) in germinal vesicle (GV) oocytes. Our study found that nuclear speckles, a subnuclear structure mainly composed of serine-arginine (SR) proteins, changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregation pattern in SN oocytes. We further discovered that SRPK1, an enzyme phosphorylating SR proteins, co-localized with NS at SN stage and NSN oocytes failed to convert into SN oocytes after inhibiting the activity of SRPK1. Furthermore, the typical structure of chromatin ring around the nucleolus in SN oocytes collapsed after inhibitor treatment. To explore the underlying mechanism, phosphorylated SR proteins were confirmed to be associated with chromatin by salt extraction experiment, and in situ DNase I assay showed that the accessibility of chromatin enhanced in SN oocytes with SRPK1 inhibited, accompanied by decreased repressive modification on histone and abnormal recurrence of transcriptional signal. In conclusion, our results indicated that SRPK1-regulated phosphorylation on SR proteins was involved in the NSN to SN transition and played an important role in maintaining the condensation nucleus of SN oocytes via interacting with chromatin.

Effects of Different Na+ Concentrations on cAMP-Dependent Protein Kinase Activity in Postmortem Meat

cAMP-dependent protein kinase (PKA) activity regulates protein phosphorylation, with Na+ playing a crucial role in PKA activity. The aim of this study was to investigate the effects of different Na+ concentrations on PKA activity and protein phosphorylation level in postmortem muscle. The study consisted of two experiments: (1) NaCl of 0, 20, 100, 200 and 400 mM was added to a muscle homogenate incubation model to analyze the effect of Na+ concentration on PKA activity, and (2) the same concentrations were added to pure PKA in vitro incubation models at 4 °C to verify the effect of Na+ on PKA activity. The PKA activity of the muscle homogenate model increased with storage time in groups with different Na+ concentrations. High concentrations of Na+ inhibited sarcoplasmic protein phosphorylation. The PKA activity at 24 h of storage and the sarcoplasmic protein phosphorylation level at 12 h of storage in the group with 200 mM Na+ was lower than that of the other groups. After 1 h incubation, the PKA activity of samples in the 200 mM Na+ group was inhibited and lower than that in the other Na+ groups in the in vitro incubation model. These results suggest that the Na+ concentration at 200 mM could better inhibit PKA activity. This study provided valuable insights for enhancing curing efficiency and improving meat quality.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Bioinformatic Analysis of Topoisomerase IIα Reveals Interdomain Interdependencies and Critical C-Terminal Domain Residues

DNA Topoisomerase IIα (Top2A) is a nuclear enzyme that is a cancer drug target, and there is interest in identifying novel sites on the enzyme to inhibit cancer cells more selectively and to reduce off-target toxicity. The C-terminal domain (CTD) is one potential target, but it is an intrinsically disordered domain, which prevents structural analysis. Therefore, we set out to analyze the sequence of Top2A from 105 species using bioinformatic analysis, including the PSICalc algorithm, Shannon entropy analysis, and other approaches. Our results demonstrate that large (10th-order) interdependent clusters are found including non-proximal positions across the major domains of Top2A. Further, CTD-specific clusters of the third, fourth, and fifth order, including positions that had been previously analyzed via mutation and biochemical assays, were identified. Some of these clusters coincided with positions that, when mutated, either increased or decreased relaxation activity. Finally, sites of low Shannon entropy (i.e., low variation in amino acids at a given site) were identified and mapped as key positions in the CTD. Included in the low-entropy sites are phosphorylation sites and charged positions. Together, these results help to build a clearer picture of the critical positions in the CTD and provide potential sites/regions for further analysis.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Inhibition of mTORC2 promotes natriuresis in Dahl salt-sensitive rats via the decrease of NCC and ENaC activity.

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin 1 (mTORC1), partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (NS) or 4.0% NaCl (HS) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide (inhibitors of ENaC, NKCC2, or NCC, respectively) either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K+ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.

Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407.

Cofactors interacting with PPARγ can regulate adipogenesis and adipocyte metabolism by modulating the transcriptional activity and selectivity of PPARγ signaling. ZFP407 was previously demonstrated to regulate PPARγ target genes such as GLUT4, and its overexpression improved glucose homeostasis in mice. Here, using a series of molecular assays, including protein-interaction studies, mutagenesis, and ChIP-seq, ZFP407 was found to interact with the PPARγ/RXRα protein complex in the nucleus of adipocytes. Consistent with this observation, ZFP407 ChIP-seq peaks significantly overlapped with PPARγ ChIP-seq peaks, with more than half of ZFP407 peaks overlapping with PPARγ peaks. Transcription factor binding motifs enriched in these overlapping sites included CTCF, RARα/RXRγ, TP73, and ELK1, which regulate cellular development and function within adipocytes. Site-directed mutagenesis of frequent PPARγ phosphorylation or SUMOylation sites did not prevent its regulation by ZFP407, while mutagenesis of ZFP407 domains potentially necessary for RXR and PPARγ binding abrogated any impact of ZFP407 on PPARγ activity. These data suggest that ZFP407 controls the activity of PPARγ, but does so independently of post-translational modifications, likely by direct binding, establishing ZFP407 as a newly identified PPARγ cofactor. In addition, ZFP407 ChIP-seq analyses identified regions that did not overlap with PPARγ peaks. These non-overlapping peaks were significantly enriched for the transcription factor binding motifs of TBX19, PAX8, HSF4, and ZKSCAN3, which may contribute to the PPARγ-independent functions of ZFP407 in adipocytes and other cell types.

Genome-wide characterization and expression profiling of E2F/DP gene family members in response to abiotic stress in tomato (Solanum lycopersicum L.)

BackgroundE2F/DP (Eukaryotic 2 transcription factor/dimerization partner) family proteins play an essential function in the cell cycle development of higher organisms. E2F/DP family genes have been reported only in a few plant species. However, comprehensive genome-wide characterization analysis of the E2F/DP gene family of Solanum lycopersicum has not been reported so far.ResultsThis study identified eight nonredundant SlE2F/DP genes that were classified into seven groups in the phylogenetic analysis. All eight genes had a single E2F-TDP domain and few genes had additional domains. Two segmental duplication gene pairs were observed within tomato, in addition to cis-regulatory elements, miRNA target sites and phosphorylation sites which play an important role in plant development and stress response in tomato. To explore the three-dimensional (3D) models and gene ontology (GO) annotations of SlE2F/DP proteins, we pointed to their putative transporter activity and their interaction with several putative ligands. The localization of SlE2F/DP-GFP fused proteins in the nucleus and endoplasmic reticulum suggested that they may act in other biological functions. Expression studies revealed the differential expression pattern of most of the SlE2F/DP genes in various organs. Moreover, the expression of E2F/DP genes against abiotic stress, particularly SlE2F/DP2 and/or SlE2F/DP7, was upregulated in response to heat, salt, cold and ABA treatment. Furthermore, the co-expression analysis of SlE2F/DP genes with multiple metabolic pathways was co-expressed with defence genes, transcription factors and so on, suggested their crucial role in various biological processes.ConclusionsOverall, our findings provide a way to understand the structure and function of SlE2F/DP genes; it might be helpful to improve fruit development and tolerance against abiotic stress through marker-assisted selection or transgenic approaches.

Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling.

AMPylation is a posttranslational modification that generally modifies amino acid side chains of proteins with adenosine monophosphate (AMP)1,2. Here we report that with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity toward the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family effectors and deubiquitinases DupA/B in an E1/E2-independent ubiquitination process3-7. The product of LnaB is further hydrolyzed by an ADP-ribosyl hydrolase, MavL, to be Ub, thereby preventing accumulation of PRR42-Ub and ADPRR42-Ub and protecting the canonical ubiquitination in host cells. LnaB represents a large family of AMPylases adopting a common structural fold, which is distinct from those of the previously known AMPylases, in bacterial pathogens of more than 20 species. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity toward phosphorylated residues and produces unique ADPylation modification in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family kinases8,9, which dampens the host downstream phosphorylation signaling. Structural studies revealed the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study presents an unprecedented regulation and molecular mechanism in bacterial pathogenesis and protein phosphorylation.

Molecular cloning of TPM3 gene in qinchuan cattle and its effect on myoblast proliferation and differentiation

Tropomyosin 3 (TPM3) plays a significant role as a regulatory protein in muscle contraction, affecting the growth and development of skeletal muscles. Despite its importance, limited research has been conducted to investigate the influence of TPM3 on bovine skeletal muscle development. Therefore, this study revealed the role of TPM3 in bovine myoblast growth and development. This research involved conducting a thorough examination of the Qinchuan cattle TPM3 gene using bioinformatics tools to examine its sequence and structural characteristics. Furthermore, TPM3 expression was evaluated in various bovine tissues and cells using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the coding region of TPM3 spans 855 bp, with the 161st base being the T base, encoding a protein with 284 amino acids and 19 phosphorylation sites. This protein demonstrated high conservation across species while displaying a predominant α-helix secondary structure despite being an unstable acidic protein. Notably, a noticeable increase in TPM3 expression was observed in the longissimus dorsi muscle and myocardium of calves and adult cattle. Expression patterns varied during different stages of myoblast differentiation. Functional studies that involved interference with TPM3 in Qinchuan cattle myoblasts revealed a very significantly decrease in S-phase cell numbers and EdU-positive staining (P < 0.01), and disrupted myotube morphology. Moreover, interference with TPM3 resulted in significantly (P < 0.05) or highly significantly (P < 0.01) decreased mRNA and protein levels of key proliferation and differentiation markers, indicating its role in the modulation of myoblast behavior. These findings suggest that TPM3 plays an essential role in bovine skeletal muscle growth by influencing myoblast proliferation and differentiation. This study provides a foundation for further exploration into the mechanisms underlying TPM3-mediated regulation of bovine muscle development and provides valuable insights that could guide future research directions as well as potential applications for livestock breeding and addressing muscle-related disorders.

Neuroprotective effects and mechanisms of action of artemisinin in retinal ganglion cells in a mouse model of traumatic optic neuropathy

IntroductionTraumatic optic neuropathy is known to be a critical condition that can cause blindness; however, the specific mechanism underlying optic nerve injury is unclear. Recent studies have reported that artemisinin, considered vital in malaria treatment, can also be used to treat neurodegenerative diseases; however, its precise role and mechanism of action remain unknown. Therefore, in this study, we aimed to investigate the impact and probable mechanism of action of artemisinin in retinal ganglion cells (RGCs) in a mouse model of traumatic optic neuropathy induced by optic nerve crush (ONC). MethodsONC was induced in the left eye of mice by short-term clamping of the optic nerve; oral artemisinin was administered daily. The neuroprotective effect of the drug was assessed using Tuj-1 staining in RGCs. In addition, the inflammatory response and the expression levels of phosphorylated tau protein and tau oligomers were observed using RT-qPCR, TUNEL assay, and fluorescence staining to investigate the underlying mechanisms. ResultsArtemisinin increased the survival rate of RGCs 14 days after ONC. Artemisinin significantly reduced the levels of inflammatory factors such as CXCL10, CXCR3, and IL-1β in the retina and decreased the apoptosis of RGCs. Moreover, downregulation of the phosphorylation of tau proteins and the expression of tau oligomers were observed after artemisinin treatment. ConclusionOur results suggest that artemisinin can increase the survival rate of RGCs after ONC and reduce their apoptosis. This effect may be achieved by inhibiting the inflammatory response it triggers and downregulating tau protein phosphorylation and tau oligomer expression. These findings suggest the potential application of artemisinin as a therapeutic agent for neuropathy.