Essential Protein Research Articles

Genome-wide conditional degron libraries for functional genomics.

Functional genomics with libraries of knockout alleles is limited to non-essential genes and convoluted by the potential accumulation of suppressor mutations in knockout backgrounds, which can lead to erroneous functional annotations. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for inducible degradation of AID-tagged proteins in the budding yeast Saccharomyces cerevisiae. First, we determined that N-terminal tagging is at least twice as likely to inadvertently impair protein function across the proteome. We thus constructed two libraries with over 5,600 essential and non-essential proteins fused at the C-terminus with an AID tag and an optional fluorescent protein. Approximately 90% of AID-tagged proteins were degraded in the presence of the auxin analog 5-Ph-IAA, with initial protein abundance and tag accessibility as limiting factors. Genome-wide screens for DNA damage response factors revealed a role for the glucose signaling factor GSF2 in resistance to hydroxyurea, highlighting how the AID libraries extend the yeast genetics toolbox.

Fission yeast essential nuclear pore protein Nup211 regulates the expression of genes involved in cytokinesis.

Nuclear pore proteins control nucleocytoplasmic transport; however, certain nucleoporins play regulatory roles in activities such as transcription and chromatin organization. The fission yeast basket nucleoporin Nup211 is implicated in mRNA export and is essential for cell viability. Nup211 preferentially associates with heterochromatin, however, it is unclear whether it plays a role in regulating transcription. To better understand its functions, we constructed a nup211 "shut-off" strain and observed that Nup211 depletion led to severe defects in cell cycle progression, including septation and cytokinesis. Using RNA-Seq and RT-qPCR, we revealed that loss of Nup211 significantly altered the mRNA levels of a set of genes crucial for cell division. Using domain analysis and CRISPR/cas9 technology, we determined that the first 655 residues of Nup211 are sufficient for viability. This truncated protein was detected at the nuclear periphery. Furthermore, exogenous expression of this domain in nup211 shut-off cells effectively restored both cell morphology and transcript abundance for some selected genes. Our findings unveil a novel role for Nup211 in regulating gene expression.

Assessment of inhibitory potentiality of natural compounds against worrisome rice blast fungus

Rice blast, a severe fungal disease, is a substantial threat to global food security, particularly in rice-oriented areas. The Magnaporthe oryzae fungus is increasingly resistant and fast developing in nature. However, chemical fungicides are not only detrimental to the environment but eventually also lose their efficiency. To Tackle this issue, we used an in silico based strategy to identify plant metabolites as bio-fungicides to combat rice blast. Therefore, we screened a total of 56 antifungal natural compounds for the ability to inhibit fungal development through the targeted inhibition of essential proteins in the rice blast pathogen. Molecular docking analysis identified curcumin, myricetin, sterigmatocystin, and versicolorin B as promising candidates with superior binding affinities compared to conventional fungicides like strobilurin, azoxystrobin, and tricyclazole. Notably, myricetin showed the docking score for the SD protein of −233.20, whereas versicolorin B demonstrated the highest binding affinity for the SD protein of −234.23. Among the control fungicides, azoxystrobin displayed the lowest docking score of −177.53. The docked complexes were found to be stable based on molecular dynamics simulations results; the binding free energies of SD-Versicolorin B (−156.018 ± 24.881 kJ/mol) and SD-Myricetin (−137.526 ± 19.977 kJ/mol) complexes were also found to be favorable. Taking everything considered, these naturally occurring substances showed strong fungicidal effects against rice blast causative agent while remaining non-toxic, providing encouraging substitutes for traditional fungicides. In conclusion, these non-toxic natural compounds exhibited strong fungicidal action against rice blast, suggesting promising alternatives to conventional fungicides.

Cloning genes of p49, p72, pe199l, pe248r and cd2vinto <i>Pichia Pastoris</i> gs115

African swine fever (ASF), caused by the African swine fever virus (ASFV), poses a significant threat to pig populations worldwide. ASFV is a double-stranded DNA virus. In recent years, 54 structural proteins and more than 100 proteins have been involved in viral infection found in macrophages of diseased pigs. Among these, p49 and p72 are essential capsid proteins crucial for forming the viral capsid. Additionally, pE199L and pE248R, located in the inner viral membrane, are critical for membrane fusion, a necessary step for viral entry into host cells. Another important player is CD2v, a type I transmembrane protein involved in the infection process. In this research, to develop subunit vaccines against ASFV, we focused on cloning the genes encoding these five proteins—p49, p72, pE199L, pE248R, and CD2v—into the pPIC9K plasmid for expression in the Pichia pastoris GS115 yeast strain. The viral genomic DNA was extracted from blood samples of infected pigs, and the genes encoding the five proteins were successfully amplified using Phusion PCR. The PCR products of each gene were then digested with EcoRI and NotI restriction enzymes and ligated into the pPIC9K plasmid. After that, we transformed the recombinant plasmids into Escherichia coli DH5α for amplification and purification. The plasmids were subsequently linearized with SalI and introduced into P. pastoris GS115 through electroporation. The selection of appropriate media and PCR analysis of the genomic DNA confirmed the successful generation of five recombinant P. pastoris GS115 strains. This work paves the way for the development of a recombinant protein vaccine against ASF by using the Pichia pastoris GS115 in the future.

Proteomics analysis in rats reveals convergent mechanisms between major depressive disorder and dietary zinc deficiency

Abstract Background Preclinical and clinical studies have shown that dietary zinc deficiency can lead to symptoms similar to those observed in major depressive disorder (MDD). However, the underlying molecular mechanisms remain unclear. To investigate these mechanisms, we examined proteomic changes in the prefrontal cortex (PFC) and hippocampus (HP) of rats, two critical brain regions implicated in the pathophysiology of depression. Methods Rats were fed diets either adequate in zinc (ZnA, 50 mg Zn/kg) or deficient in zinc (ZnD, <3 mg/kg) for four weeks. High-throughput proteomic analysis was used to detect changes in protein expression, supplemented by enzyme activity assay for mitochondrial complexes I and IV, examining their functional impacts. Results ZnD led to significant alterations in protein expression related to zinc transport and mitochondrial function. Proteomic analysis revealed changes in zinc transporter family members such as Slc30a1 (6.64 log2FC), Slc30a3 (-2.32 log2FC), Slc30a4 (2.87 log2FC), Slc30a5 (5.90 log2FC), Slc30a6 (1.50 log2FC), and Slc30a7 (2.17 log2FC) in the PFC, and Slc30a3 (-1.02 log2FC), Slc30a5 (-1.04 log2FC), and Slc30a7 (1.08 log2FC) in the HP of rats subjected to ZnD. Furthermore, ZnD significantly affected essential mitochondrial activity proteins, including Atp5pb (3.25 log2FC), Cox2 (2.28 log2FC), Atp5me (2.04 log2FC), Cyc1 (2.30 log2FC), Cox4i1 (1.23 log2FC), Cox7c (1.63 log2FC), and Cisd1 (1.55 log2FC), with a pronounced decrease in complex I activity in the PFC. Conclusions Our study demonstrates that ZnD leads to significant proteomic changes in the PFC and HP of rats. Specifically, ZnD alters the expression of zinc transporter proteins and proteins critical for mitochondrial function. The significant decrease in complex I activity in the PFC further underscores the impact of ZnD on mitochondrial function. These results highlight the molecular mechanisms by which ZnD can influence brain function and contribute to symptoms similar to those observed in depression.

AttentionEP: Predicting Essential Proteins via Fusion of Multiscale Features by Attention Mechanisms

Identifying essential proteins is of utmost importance in the field of biomedical research due to their essential functions in cellular activities and their involvement in mechanisms related to diseases. In this research, a novel approach called AttentionEP for predicting essential proteins (EP) is introduced by attention mechanisms. This method leverages both cross-attention and self-attention frameworks, focusing on enhancing prediction accuracy through the integration of features across diverse scales. Spatial characteristics of proteins are obtained from the protein-protein interaction (PPI) network by employing Graph Convolutional Networks (GCN) and Graph Attention Networks (GAT). Following this, Bidirectional Long Short-Term Memory networks (BiLSTM) are employed to derive temporal features from gene expression datasets. Furthermore, spatial characteristics are derived by integrating data on subcellular localization with the application of Deep Neural Networks (DNN). In order to effectively integrate features across multiple scales, initial steps involve the application of self-attention techniques to derive essential insights from each unique data set. Following this, mechanisms involving self-attention and cross-attention are employed to enhance the interaction between diverse information sources. To identify essential proteins, a classifier based on the ResNet architecture is developed. The findings from the experiments indicate that the method introduced here shows superior performance in identifying essential proteins, recording an Area Under the Curve (AUC) value of 0.9433. This approach shows a considerable advantage over established techniques. The findings of this study provide a significant advancement in the comprehension of critical proteins, revealing promising potential for applications in the development of therapeutics and addressing various diseases.

Neuroprotective Effects of Chaperonin Containing TCP1 Subunit 2 (CCT2) on Motor Neurons Following Oxidative or Ischemic Stress.

Chaperonin containing TCP1 (CCT) is an essential protein that controls proteostasis following spinal cord damage. In particular, CCT2 plays an important role in neuronal death in various neurological disorders; however, few studies have investigated the effects of CCT2 on ischemic damage in the spinal cord. In the present study, we synthesized a cell-permeable Tat-CCT2 fusion protein and observed its effects on H2O2-induced oxidative damage in NSC34 motoneuron-like cells and in the spinal cord after ischemic injury. Tat-CCT2, but not its control protein CCTs, was delivered into NSC34 cells in a concentration- and incubation time-dependent manner, and a clear cytosolic location of the delivered protein was observed. In addition, the delivered protein gradually degraded, and nearly control levels were observed 24h after Tat-CCT2 treatment. Tat-CCT2 treatment significantly ameliorated 200 µM H2O2-induced neuronal damage in NSC34 cells at 8.0 µM protein treatment. Additionally, Tat-CCT2 significantly ameliorated H2O2-induced reactive oxygen species formation and DNA fragmentation. In the rabbit spinal cord, Tat-CCT2 was efficiently delivered into the spinal cord 4h after 0.125mg/kg protein treatment. In addition, treatment with Tat-CCT2 significantly improved the neurological scores based on the Tarlov criteria 24 and 72h after ischemia/reperfusion. Moreover, the number of surviving neurons in the ventral horn of the spinal cord was significantly increased in the Tat-CCT2-treated group 3 and 7 days after ischemia compared to vehicle-treated group. Treatment with Tat-CCT2 alleviated the ischemia-induced oxidative stress and ferroptosis-related factor (malondialdehyde, 8-iso-prostaglandin F2α, and high mobility group box 1) and pro-inflammatory cytokine (interleukin-1β, interleukin-6, and tumor necrosis factor-α) releases in the ventral horn of the spinal cord 8 and 24h after ischemia/reperfusion. In addition, Tat-CCT2 treatment significantly ameliorated ischemia-induced microglial activation in the ventral horn of spinal cord 24h after reperfusion. These results suggest that Tat-CCT2 mitigates ischemia-induced neuronal damage in the spinal cord.

Climate change and poultry production: Enhancing resilience and sustainability through climate-smart practices, a review

Chicken production plays a vital role in global food systems, providing essential protein to billions of people, particularly in developing nations. However, climate change poses significant challenges to the poultry industry, including temperature stress, water scarcity, feed supply disruptions, disease prevalence, and economic impacts. This review examines the intricate effects of climate change on chicken production, highlighting primary challenges and exploring potential solutions. Proactive measures such as improved housing, water management, feed innovation, disease control, and genetic selection are crucial for enhancing the resilience of poultry farming systems. Collaborative efforts among industry stakeholders, researchers, and policymakers are essential to effectively implement these strategies and safeguard food security, economic stability, and environmental sustainability. By prioritizing adaptation and mitigation efforts, stakeholders can mitigate the adverse effects of climate change on chicken production, ensuring continued productivity and profitability in a changing climate. It is imperative that decisive action is taken to address these challenges, fostering innovation, sustainability, and resilience in chicken production systems worldwide.

Machine learning-aided identification of natural compounds targeting Marburg virus VP40 with potential antiviral activity

Marburg virus infection poses a significant threat to humans due to its high fatality rate. The application of in-silico drug design to target the essential protein target of the virus has been proven to be a fundamental technique to inhibit viral growth. Here, VP40 (a matrix protein) was used as an essential protein target of Marburg, and 2569 natural compounds were screened using the molecular docking and neural network-based DeepPurpose architecture. The top 138 compounds that exhibited a binding score of −8 kcal/mol in molecular docking were used in the best DeepPurpose model to predict the IC50. The best model in DeepPurpose was composed of Morgan and CNN-based encoding for protein and ligand, respectively. The top three compounds, NPL130 (CHEMBL2087156), NPL313 (CHEMBL76073), and NPL371 (CHEMBL54440), were selected from the machine learning model, and molecular dynamics simulation was performed for their best complex along with the control compound complex, Nilotinib (CHEMBL255863). Protein showed less than 0.3 nm of deviation in the complex formed with control and NPL130 compounds. Control had shown the best average MM/GBSA binding energy of −36.97 kcal/mol with the lowest standard deviation of 1.86. A stable complex was indicated by the negative binding free energies of −20 to −25 kcal/mol for all three hits. The free energy landscape showed that, along with the control compound, NPL130 had the biggest conformational space with the lowest free energy. Overall, this study proposed NPL130 as a potential hit compound to target Marburg viral growth.

High-Sensitivity Cardiac Troponin Assays in Acute Heart Failure, Moving Beyond Myocardial Infarction.

Cardiac troponins are essential structural proteins found in the contractile apparatus of cardiac myocytes. During myocardial damage, such as in myocardial infarction (MI), these troponins are released into the bloodstream. As a result, they play a central role in the diagnosis of MI, serving as sensitive and specific markers for cardiac injury. Earlier assays that were used for measuring troponin levels were considered as a dichotomous test, categorizing patients as being positive or negative for MI. The recent introduction of high-sensitivity cardiac troponin assays has revolutionized cardiac troponin detection. These assays can detect troponin levels that are 100 times lower than what traditional methods can detect. Hence it is now considered a quantitative measure of cardiac myocyte injury not only in the setting of MI but also in subjects without MI such as heart failure, in whom it can be regarded as a marker for myocardial stress. This review aims to establish the relationship between high-sensitivity cardiac troponin levels and the prognosis of patients suffering from acute heart failure. Additionally, this seeks to identify other applications where the release of troponin from the cardiomyocyte can provide prognostic information. This information can be vital in determining the appropriate treatment options for patients, ultimately improving their quality of life and positively impacting health economics.

BCL2 regulates antibacterial autophagy in the intestinal epithelium

Autophagy is a key innate immune defense mechanism in intestinal epithelial cells. Bacterial invasion of epithelial cells activates antibacterial autophagy through a process that requires the innate immune adaptor protein MYD88, yet how MYD88 signaling connects to the autophagy machinery is unknown. Here, we show that the mouse intestinal pathogen Salmonella enterica Serovar Typhimurium (Salmonella Typhimurium) triggers MYD88 signaling that regulates binding of the anti-autophagy factor B cell lymphoma 2 (BCL2) to the essential autophagy protein Beclin1 (BECN1) in small intestinal enterocytes, a key epithelial cell lineage. Salmonella infection activated the kinase c-Jun N-terminal protein kinase 1 (JNK1) downstream of MYD88. JNK1 induced enterocyte BCL2 phosphorylation, promoting dissociation of the inhibitory BCL2-BECN1 complex and releasing BECN1 to initiate autophagy. Mice with BCL2 phosphorylation site mutations that prevent BCL2-BECN1 dissociation showed increased Salmonella invasion of enterocytes and dissemination to extraintestinal sites. These findings reveal that BCL2 links MYD88 signaling to enterocyte autophagy initiation, providing mechanistic insight into how invading bacteria trigger autophagy in the intestinal epithelium.

Comprehensive prediction and analysis of human protein essentiality based on a pretrained large language model.

Human essential proteins (HEPs) are indispensable for individual viability and development. However, experimental methods to identify HEPs are often costly, time consuming and labor intensive. In addition, existing computational methods predict HEPs only at the cell line level, but HEPs vary across living human, cell line and animal models. Here we develop a sequence-based deep learning model, Protein Importance Calculator (PIC), by fine-tuning a pretrained protein language model. PIC not only substantially outperforms existing methods for predicting HEPs but also provides comprehensive prediction results across three levels: human, cell line and mouse. Furthermore, we define the protein essential score, derived from PIC, to quantify human protein essentiality and validate its effectiveness by a series of biological analyses. We also demonstrate the biomedical value of the protein essential score by identifying potential prognostic biomarkers for breast cancer and quantifying the essentiality of 617,462 human microproteins.

PKP2 Gene Mutation Causing Left Ventricular Non-compaction with Atrial Septal Defect in a Neonate: A Case Report

Arrhythmogenic ventricular cardiomyopathy (AVC) is an inherited cardiomyopathy that predisposes to ventricular arrhythmias (VA), leading to sudden cardiac death in young patients. Familial arrhythmogenic right ventricular dysplasia-9 (ARVD9) is caused by heterozygous mutations in the PKP2 gene (602861), which encodes plakophilin-2, an essential armadillo repeats protein of the cardiac desmosome, on chromosome 12p11. We are reporting a 15-day-old neonate (after obtaining proper written consent from the parents) with a history of two sibling deaths in early childhood who presented with VA with left ventricular non-compaction with atrial septal defect later diagnosed as AVC on the genetic study, the first of its kind, who survived on conservative management and did well on follow-up.

Combined Therapeutic Strategies Based on the Inhibition of Non-Oncogene Addiction to Improve Tumor Response in EGFR- and KRAS-Mutant Non-Small-Cell Lung Cancer.

Oncogene-driven NSCLC is usually treated with targeted therapies using tyrosine kinase inhibitors (TKIs) to inhibit oncogene downstream signaling pathways, affecting tumor survival and proliferation. EGFR- and KRAS-mutant NSCLCs are the most represented subtypes, and they are treated in clinical practice with oncogene-targeting drugs in the first and second line, respectively. Unfortunately, the development of oncogene-independent resistant clones limits TKI efficacy. Here, we used non-oncogene addiction (NOA) as an innovative therapeutic strategy to target other essential proteins that support changes in tumor phenotype. Specifically, we tested, for the first time, a combination of inhibitors, namely ATR, involved in DNA damage response, and pyruvate dehydrogenase kinases (PDKs), involved in energy metabolism. Sensitive PC9 and the corresponding EGFR-TKI-resistant PC9/OR, EGFR-mutant H1975, and KRAS-mutant A549 NSCLC cells, were treated with TKIs (osimertinib and selumetinib, respectively). In parallel, cells were exposed to two combination regimens: one using the TKI with an ATR inhibitor and the other one combining the two selected NOA inhibitors (ATR inhibitor, M4344; and PDK inhibitor, DCA). The effect of these two combined approaches, compared to TKI alone, produced similar results in terms of cell proliferation, cell death, and migration. Thus, depending on tumor biology, selecting between the proposed therapeutic strategies will be different, to maximize tumor response. The major translational relevance of this study is to exploit new targets for the development of innovative and improved therapeutic strategies with NOA drugs, over combinations including target genes within the oncogene pathway, to overcome resistance to TKI therapies in patients with NSCLC who are oncogene-addicted.

A simple MiMIC based approach for tagging endogenous genes to visualise live transcription in Drosophila.

Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem loops into endogenous genes requires laborious CRISPR genome editing. Here we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging and live imaging transcription of endogenous genes and the long non-coding RNA, roX1, in the embryo. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues.

Protective Effects of L-Cysteine Against Cisplatin-Induced Oxidative Stress-Mediated Reproductive Damage

Cisplatin (CIS) is a widely used chemotherapeutic agent, but its side effects, such as oxidative stress, inflammation, and apoptosis, often lead to male reproductive damage. Oxidative stress, primarily caused by the excessive generation of reactive oxygen species (ROS), plays a critical role in disrupting testicular homeostasis, resulting in spermatogenic impairment and tissue injury. L-cysteine (CYS), a semi-essential amino acid with potent antioxidant and anti-inflammatory properties, may offer protection against CIS-induced oxidative damage. This study aimed to assess the protective potential of CYS against CIS-induced male reproductive toxicity using in vivo and in vitro models. In vitro, treatment of TM3 (Leydig) and TM4 (Sertoli) cells with CIS led to increased ROS levels, reduced cell viability, and elevated apoptosis and inflammation, all of which were significantly ameliorated by subsequent CYS exposure. In vivo, CIS-treated male rats displayed heightened oxidative stress, impaired spermatogenesis, and histopathological damage in reproductive organs. However, CYS administration for 21 days significantly reduced oxidative stress, improved sperm viability, and protected testicular tissues from damage. These findings suggest that CYS has a protective effect against CIS-induced oxidative stress and male reproductive damage, making it a promising therapeutic agent for mitigating CIS-induced reproductive toxicity.

Attitudes and Perception of Nursing Mothers towards Exclusive Breastfeeding in the Federal Capital Territory (FCT)

Breastfeeding is one of the oldest practices known to mankind because the breastmilk contains the essential fats, proteins, carbohydrates, and immunological factors needed for infants to thrive. This study assessed the attitude and perception of nursing mothers towards exclusive breastfeeding. An evaluation was conducted to assess the attitude and perception using a qualitative research method which involved 401 nursing mothers in the Federal Capital Territory (FCT) who were selected randomly to answer questionnaires regarding their knowledge and practice of exclusive breastfeeding. The data was analysed using the chi-square test. The result was presented using frequency tables and charts, the chi-square was used to test for hypotheses between variables. 344 of 401 respondents (85.7%) have a good understanding of exclusive breastfeeding. 280 of 401 respondents (69.8%) practice exclusive breastfeeding, 167 of 401 respondents (41.64%) were informed about exclusive breastfeeding from healthcare practitioners and 121 of 401 respondents (30.17%) heard about it from family members. From this study, we derived that the level of education, marital status, income level and religious beliefs have no significant effect on exclusive breastfeeding. This study highlighted that majority of the respondents have knowledge of exclusive breastfeeding and practice it. It is necessary to provide lactation services especially to new mothers during the antenatal and postnatal care, fathers should be made aware of the benefits of exclusive breastfeeding and the importance of supporting the nursing mothers by feeding them healthy meals which would help nourish both the mother and infant. Exclusive breastfeeding is recommended for the first six months of a child’s life.

Combatting cellular immortality in cancers by targeting the shelterin protein complex

Shelterin proteins (TERF1, TERF2, TPP1, TINF2, POT1) protect telomeres, prevent unwarranted repair activation, and regulate telomerase activity. Alterations in these proteins can lead to cancer progression. This study uses an in-silico approach to examine shelterin in tumour samples across various cancers, employing mutation plots, phylogenetic trees, and sequence alignments. Network pharmacology identified TERF1 as an essential shelterin protein and transcription factors RUNX1, CTCF, and KDM2B as potential biomarkers due to their interactions with miRNAs and shelterin proteins. We performed MCODE analysis to identify subnetworks of ncRNAs interacting with the shelterin proteins. Shelterin expression predicted patient survival in 24 cancer types, with TERF1, TERF2, TINF2, and POT1 significantly expressed in testicular, AML, prostate, breast and renal cancers, respectively, and TPP1 in AML and skin cancer. Spearman and Pearson's analyses showed significant correlations of TERF1 across cancers, with near-significant correlations for all five proteins in different cancer datasets like breast cancer, kidney renal papillary and lung squamous cell carcinoma, skin cutaneous melanoma, etc.,. Shelterin expression correlated with patient survival in breast, renal, lung, skin, uterine, and gastric cancers. Insights into TPP1-associated glycans highlighted glycosylated sites contributing to tumorigenesis. This study provides molecular signatures for further functional and therapeutic research on shelterin, highlighting its potential as a target for anti-cancer therapies and promising prospects for cancer prognosis and prediction.

Regulation of MiR-206 in denervated and dystrophic muscles and its effect on AChR clustering.

Muscle-specific microRNA miR-206 has recently emerged as a potential regulator of genes involved in the formation and regeneration of the neuromuscular junction (NMJ). This study investigated miR-206-3p (miR-206) expression in synaptic and non-synaptic regions of denervated and in alpha-dystrobrevin (Dtnb) knockout mice, as well as its impact on the formation and/or maintenance of agrin-induced acetylcholine receptor (AChR) clusters. In denervated, Dtnb-deficient, and crushed muscles, miR-206 expression significantly increased compared to innervated muscles. While miR-206 expression is slightly elevated in the synaptic regions of innervated muscles, it dramatically rises in non-synaptic areas of denervated muscles. miR-206 targets transcripts of essential NMJ proteins such as Dtnb, alpha-syntrophin (Snta1), and rapsyn, but not AchRα subunit or Lrp4 in innervated muscles. However, in denervated muscles, AChRα transcripts, which increase significantly, become a target of miR-206. Co-expression of miR-206 with rapsyn, Dtnb, and Snta1 in C2C12 myoblasts significantly reduced their protein levels, and overexpression of miR-206 in myotubes disrupted agrin-induced AChR clustering. These results indicate that miR-206 fine-tunes NMJ signaling proteins by regulating transcripts of various proteins with different localizations under normal and pathological conditions.

Re-localization of a repeat-containing fungal effector by apoplastic protein Chitinase-like 1 blocks its toxicity

A fungal effector that is toxic to plant cells was identified in Verticillium dahliae. The effector contains a non-canonical Common in several Fungal Extracellular Membrane proteins (CFEM) domain, a tandem repeat region consisting of four 14-amino acid repeats rich in proline, and a C-terminal region, thus is designated V. dahliae tetrapeptide repeat protein (VdTRP). The membrane targeting of VdTRP is vital for its cell toxicity. CFEM mediates the membrane targeting and the tandem repeat region exerts the toxic function upon cell membrane. The chitinase-like 1 (CTL1), an essential apoplastic protein of cotton, can redirect VdTRP from cell membrane to apoplast. Transgenic cotton overexpressing CTL1 greatly enhances cotton resistance to V. dahliae without affecting cotton growth and development, implicating its potential application in breeding cotton with high wilt resistance. Our data demonstrates that genetic manipulation of effector target constitutes potential strategy for improving crop resistance to fungal pathogens.