Repeat Domain Research Articles

Chaperone-mediated autophagy contributes to chromosomal stability by controlling TTC28 degradation.

While macroautophagy (autophagy) contributes to maintaining chromosomal stability via multiple pathways, including regulating chromatin ubiquitination and cytoplasmic DNA fragment degradation, the impacts of microautophagy and chaperone-mediated autophagy (CMA) on maintaining chromosomal stability are not known. The TTC28 (tetratricopeptide repeat domain 28) gene is frequently mutated and downregulated in human cancers. The molecular mass of the TTC28 protein is 271 kDa, which makes its functional study very difficult. Recently, we reported that TTC28 plays a key role in maintaining chromosomal stability, probably through regulating mitosis and cytokinesis, and that TTC28 downregulation may contribute to the high chromosomal instability (CIN) of cancer cells, according to the results of serial experiments and bioinformatics analyses. Notably, our findings demonstrate that TTC28 is a substrate of CMA and microautophagy and that these pathways also play a role in maintaining chromosomal stability in a TTC28-dependent manner. These findings demonstrate that CMA- and microautophagy-mediated degradation is a master regulator of the ability of TTC28 to maintain genome stability.

Methods for detecting, building, and improving tryptophan mannosylation in glycoprotein structures.

Tryptophan mannosylation, the covalent addition of an α-ᴅ-mannose sugar to a tryptophan side chain, is a post-translational modification (PTM) that can affect protein stability, folding, and interactions. Compared to other forms of protein glycosylation, it is relatively uncommon but is affected by conformational anomalies and modeling errors similar to those seen in N- and O-glycans in the Protein Data Bank (PDB). In this work, we report methods for detecting, building, and improving mannose structures linked to tryptophans. These methods have been used to mine X-ray crystallographic and cryo-electron microscopy maps in the PDB looking for unmodeled mannosylation, resulting in a number of cases where the modification can be placed in the map with high confidence. Additionally, we address most conformational issues affecting this modification. Finally, the development of a structural template to recognize thrombospondin repeats (TSR) domains where tryptophan mannosylation occurs will allow for the mannosylation of candidate-predicted models, for example, those predicted with AlphaFold.

Functional classification of tauopathy strains reveals the role of protofilament core residues.

Distinct tau amyloid assemblies underlie diverse tauopathies but defy rapid classification. Cell and animal experiments indicate tau functions as a prion, as different strains propagated in cells cause unique, transmissible neuropathology after inoculation. Strain amplification requires compatibility of the monomer and amyloid template. We used cryo-electron microscopy to study one cell-based yellow fluorescent protein (YFP)-tagged strain, resolving its amyloid nature. We then used sequential alanine (Ala) substitution (scan) within tau repeat domain (RD) to measure incorporation to preexisting tau RD-YFP aggregates. This robustly discriminated strains, defining sequences critical for monomer incorporation. We then created 3R/4R or 4R wild-type RD (amino acids 246 to 408) biosensors. Ala scan of recombinant tau seeds with the Alzheimer's disease (AD) fold matched that of AD homogenate. We scanned 22 brain lysates comprising four tauopathies. This clustered cases by neuropathological syndrome, revealed the role of amino acids in protofilament folds, and allowed strain discrimination based on amino acid requirements for prion replication.

The Role of WDR77 in Cancer: More Than a PRMT5 Interactor.

WD repeat domain 77 protein (WDR77), a WD-40 domain-containing protein, is a crucial regulator of cellular pathways in cancer progression. While much of the past research on WDR77 has focused on its interaction with PRMT5 in histone methylation, WDR77's regulatory functions extend beyond this pathway, influencing diverse mechanisms such as mRNA translation, chromatin assembly, cell cycle regulation, and apoptosis. WDR77 is a key regulator of cell cycle progression, regulating the transition from the G1 phase. WDR77 regulates many signaling pathways such as TGFβ where its role in these cellular pathways underscores its broad oncogenic potential. WDR77 also assists and promotes certain transcription factors such as E2F. Furthermore, in certain cancers, WDR77 enhances steroid hormone receptor activity, uniquely linking it to hormone-driven malignancies. WDR77 often translocates between the nucleus and the cytoplasm, with its location dictating its role in the cell. WDR77 has the ability to adapt its function depending on its location which emphasizes its dynamic role in both promoting and inhibiting tumor growth, depending on cellular context. This dual function makes WDR77 an attractive therapeutic target, as disrupting its interactions with critical signaling pathways or modulating its translocation could yield novel strategies for cancer treatment. Given WDR77's role in oncogenic pathways independent of PRMT5, further exploration of WDR77 and its non-PRMT5-related activities may reveal additional therapeutic opportunities in an array of cancers.

Recombinant Antibodies Inhibit Enzymatic Activity of the E3 Ubiquitin Ligase CHIP via Multiple Mechanisms.

Carboxyl-terminus of Hsp70-Interacting Protein (CHIP) is an E3 ubiquitin ligase that marks misfolded substrates for degradation. Hyper-activation of CHIP has been implicated in multiple diseases, including cystic fibrosis and cancer, suggesting that it may be a potential drug target. However, there are few tools available for exploring this possibility. Moreover, the best ways of inhibiting CHIP's function are not obvious, as this complex protein is composed of a tetratricopeptide repeat (TPR) domain, a U-box domain, and a coiled-coil domain that mediates homodimerization. To probe the structure and function of CHIP, we report an antibody panning campaign that yielded six recombinant Fabs with affinity for CHIP. Interestingly, these antibodies varied in their binding site(s) and impact on CHIP function, such as inhibiting TPR interactions, autoubiquitination, and/or substrate ubiquitination. Of particular interest, antibody 2F1 nearly eliminated substrate binding (IC50 = 2.7 μM) and limited ubiquitination and autoubiquitination. Cryo-electron microscopy of the 2F1:CHIP complex revealed a 2:1 binding mode (Fab:CHIP dimer), with 2F1 bound to the U-box domain and simultaneously displacing the TPR domain. Together, these studies provide insight into ways of inhibiting CHIP's activity and provide a series of new probes for exploring the function of this important E3 ubiquitin ligase.

Apaf-1 is an evolutionarily conserved DNA sensor that switches the cell fate between apoptosis and inflammation

Apoptotic protease activating factor 1 (Apaf-1) was traditionally defined as a scaffold protein in mammalian cells for assembling a caspase activation platform known as the ‘apoptosome’ after its binding to cytochrome c. Although Apaf-1 structurally resembles animal NOD-like receptor (NLR) and plant resistance (R) proteins, whether it is directly involved in innate immunity is still largely unknown. Here, we found that Apaf-1-like molecules from lancelets, fruit flies, mice, and humans have conserved DNA sensing functionality. Mechanistically, mammalian Apaf-1 recruits receptor-interacting protein 2 (RIP2, also known as RIPK2) via its WD40 repeat domain and promotes RIP2 oligomerization to initiate NF-κB-driven inflammation upon cytoplasmic DNA recognition. Furthermore, DNA binding of Apaf-1 determines cell fate by switching the cellular processes between intrinsic stimuli-activated apoptosis and inflammation. These findings suggest that Apaf-1 is an evolutionarily conserved DNA sensor and may serve as a cell fate checkpoint, which determines whether cells initiate inflammation or undergo apoptosis by distinct ligand binding.

WDR62 mediates MAPK/ERK pathway to stimulate DNA damage repair and attenuate cisplatin sensitivity in lung adenocarcinoma.

Chemotherapy resistance has long stood in the way of therapeutic advancement for lung cancer patients, the malignant tumor with the highest incidence and fatality rate in the world. Patients with lung adenocarcinoma (LUAD) now have a dismal prognosis due to the development of cisplatin (DDP) resistance, forcing them to use more costly second-line therapies. Therefore, overcoming resistance and enhancing patient outcomes can be achieved by comprehending the regulatory mechanisms of DDP resistance in LUAD. WD repeat domain 62 (WDR62) expression in LUAD tissues and in DDP-resistant or sensitive LUAD patients was analyzed bioinformatically, and a K-M plot was utilized to assess survival status. Real-time quantitative PCR was employed for WDR62 expression detection, cell-counting kit-8 assay for half maximal inhibitory concentration determination, flow cytometry for cell apoptosis detection, immunofluorescence for γ-H2AX expression analysis, and western blot for nonhomologous end joining repair and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway-related protein expression analysis. Poor prognosis was linked to WDR62, which was overexpressed in LUAD tissues and cells. Compared to sensitive cells, DDP-resistant cells had increased WDR62 expression. WDR62 knockdown may enhance DDP-induced cell apoptosis while reducing cell proliferation and DNA damage repair. Functional investigations verified that overexpressed WDR62's encouraging impact on DNA damage repair in A549/DDP cells could be reversed by MAPK inhibitors, increasing the cells' susceptibility to DDP. LUAD cells became less sensitive to DDP when WDR62 activated the MAPK/ERK pathway, which promoted DNA damage repair, indicating that DDP resistance might be reversed by treating LUAD with inhibitors of the MAPK pathway.

GEMIN5 and neurodevelopmental diseases: from functional insights to disease perception.

GEMIN5 is a predominantly cytoplasmic multifunctional protein, known to be involved in recognizing snRNAs through its WD40 repeats domain placed at the N-terminus. A dimerization domain in the middle region acts as a hub for protein-protein interaction, while a non-canonical RNA-binding site is placed towards the C-terminus. The singular organization of structural domains present in GEMIN5 enables this protein to perform multiple functions through its ability to interact with distinct partners, both RNAs and proteins. This protein exerts a different role in translation regulation depending on its physiological state, such that while GEMIN5 down-regulates global RNA translation, the C-terminal half of the protein promotes translation of its mRNA. Additionally, GEMIN5 is responsible for the preferential partitioning of mRNAs into polysomes. Besides selective translation, GEMIN5 forms part of distinct ribonucleoprotein complexes, reflecting the dynamic organization of macromolecular complexes in response to internal and external signals. In accordance with its contribution to fundamental cellular processes, recent reports described clinical loss of function mutants suggesting that GEMIN5 deficiency is detrimental to cell growth and survival. Remarkably, patients carrying GEMIN5 biallelic variants suffer from neurodevelopmental delay, hypotonia, and cerebellar ataxia. Molecular analyses of individual variants, which are defective in protein dimerization, display decreased levels of ribosome association, reinforcing the involvement of the protein in translation regulation. Importantly, the number of clinical variants and the phenotypic spectrum associated with GEMIN5 disorders is increasing as the knowledge of the protein functions and the pathways linked to its activity augments. Here we discuss relevant advances concerning the functional and structural features of GEMIN5 and its separate domains in RNA-binding, protein interactome, and translation regulation, and how these data can help to understand the involvement of protein malfunction in clinical variants found in patients developing neurodevelopmental disorders.

Inhibition of RIPK1-driven necroptosis ameliorates inflammatory hyperalgesia caused by lipopolysaccharide: involvement of TLR-, NLRP3-, and caspase-11-mediated signaling pathways.

Increasing evidence suggests that inhibition of receptor-interacting serine/threonine-protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain-like pseudokinase (MLKL) necrosome has protective effects in vivo models of painful conditions seen in humans associated with inflammation and demyelination in the central nervous system. However, the contribution of RIPK1-driven necroptosis to inflammatory pain remains unknown. Therefore, this study aims to determine the effect of necrostatin (Nec) -1s, a selective RIPK1 inhibitor, on lipopolysaccharide (LPS)-induced inflammatory pain and related underlying mechanisms. In the saline-, LPS-, and/or Nec-1s-injected male mice, thermal hyperalgesia was evaluated by hot plate test. Alterations in the expression of proteins involved in the RIPK1, toll-like receptor (TLR) 4, myeloid differentiation factor (MyD) 88/toll-interleukin (IL)-1 receptor domain-containing adapter-inducing interferon-β (TRIF)/nuclear factor (NF)-kB, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing (NLRP) 3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1, and caspase-11/gasdermin D (GSDMD) signaling pathways, as well as proteins related to demyelination and remyelination in the brain and spinal cord were determined by the immunoblotting method. The LPS-induced alleviation of thermal hyperalgesia was prevented by necrostatin-1s. Necrostatin-1s reversed (1) increased activity of RIPK1, RIPK3, MLKL, and NF-kB p65, (2) enhanced expression of TLR4, MyD88, TRIF, NF-kB p65, HMGB1, NLRP3, ASC, caspase-1 p20, IL-1β, caspase-11 p20, p30-GSDMD, and semaphorin 3A, and (3) diminished myelin PLP expression induced by LPS. These findings suggest that the use of RIPK1 inhibitors could be a therapeutic approach in the management of inflammatory pain associated with necroptosis, pyroptosis, and demyelination.

Identification of narciclasine as a novel NRF2 inhibitor

Cancer genome sequencing studies have identified somatic mutations in the KEAP1/NRF2 pathway. In an effort to identify novel NRF2 small molecule inhibitor(s), we have screened a natural compound library comprising 1,330 chemicals in A549-ARE-GFP-luciferase cells and identified that narciclasine significantly inhibits NRF2-dependent luciferase activity. Narciclasine suppressed the expression of NRF2 and NRF2 target genes, caused significant oxidative stress, and sensitized cisplatin-mediated apoptosis in A549 cells. In addition, we have observed that WD Repeat Domain 43 (WDR43) serves as a direct target of narciclasine for the inhibition of NRF2 as narciclasine binds to recombinant WDR43 in vitro and silencing WDR43 attenuated the inhibition of NRF2 by narciclasine in A549 cells. Finally, we observed that administration of narciclasine significantly decreased the growth of A549 xenografts. Together, our results demonstrate that the inhibition of NRF2 by narciclasine is mediated by WDR43 and future studies are necessary to elucidate the exact mechanism how WDR43 mediates the inhibition of NRF2 by narciclasine.

Intraspecific variation and functional study of VERL polymorphism in Pacific abalone (Haliotis discus hannai Ino) and giant abalone (H. gigantea Gmelin).

Sperm and eggs have interacting proteins on their surfaces that affect their compatibility during fertilization. These gamete recognition proteins (GRPs) are typically polymorphic within species, resulting in variations in gamete affinity. This study presents the full-length sequence of VERL, an egg vitelline envelope receptor for sperm lysin, in Pacific abalone (Haliotis discus hannai Ino) and giant abalone (Haliotis gigantea Gmelin). The full-length VERL cDNA sequence in Pacific abalone (11,373 bp) encodes 3790 amino acids, most of which are organized into 21 tandem repeats. In giant abalone, the VERL spans 9405 bp and encodes 3134 amino acids, including 17 tandem repeats. By resolving the nucleotide polymorphisms from resequencing data into haplotypes, high allelic diversity was found in VERL domain repeats 1-2 in both species. Further analysis of intraspecific variation in this region revealed that the alleles of VERL repeats 1-2 in Pacific abalone clustered into two clades, yet no significant divergence was observed among the three Pacific abalone geographic populations. In contrast, alleles in giant abalone were subdivided into two distinct branches, separated by a maximum of 12 amino acid substitutions. Notably, this study provides the first evidence of assortative mating between male and female gametes based on VERL genotypes in giant abalone. Overall, this study revealed the amino acid polymorphism of VERL in two abalone species and its functional effects on fertilization potential. These findings provide a foundation for future research on gamete recognition mechanisms and support selective breeding strategies to enhance abalone reproductive efficiency.

The N17 domain of huntingtin as a multifaceted player in Huntington's disease.

Huntington's disease (HD) is primarily caused by the aberrant aggregation of the N-terminal exon 1 fragment of mutant huntingtin protein (mHttex1) with expanded polyglutamine (polyQ) repeats in neurons. The first 17 amino acids of the N-terminus of Httex1 (N17 domain) immediately preceding the polyQ repeat domain are evolutionarily conserved across vertebrates and play multifaceted roles in the pathogenesis of HD. Due to its amphipathic helical properties, the N17 domain, both alone and when membrane-associated, promotes mHttEx1 aggregation. Diverse post-translational modifications (PTMs) in the N17 domain alter the aggregation state, thus modulating the cellular toxicity of mHttex1. Furthermore, the N17 domain serves as a nuclear export signal (NES) and mediates the cytoplasmic localization of mHttex1. This review summarizes the four main roles of the N17 domain in regulating HD pathology and discusses potential therapeutic approaches targeting this N17 domain to mitigate HD progression.

Upregulation of WDR4 mediated by RBFOX2 promotes laryngeal cancer progression through the WDR4/m7G/lncRNA ZFAS1/RBFOX2 axis.

High levels of the N7 methylguanosine (m7G) methyltransferase WD repeat domain 4 (WDR4) are associated with the progression of multiple tumors, including head and neck squamous cell carcinoma. Laryngeal cancer (LC) is the second most common malignant tumor of the head and neck. However, the role of WDR4 in LC remains unclear. Here, we found that WDR4 expression was significantly upregulated in LC tissues and cells. Silencing WDR4 inhibited proliferation, invasion, and epithelial-mesenchymal transition (EMT, manifested by an increase in E-cadherin protein levels and a decrease in N-cadherin and Vimentin protein levels) in TU177 and M4E cells. Furthermore, the levels of m7G and ZFAS1 were significantly upregulated in LC tissues and cells. Mechanistic studies revealed that WDR4 upregulated the levels of ZFAS1 and RBFOX2 proteins by promoting the stability of ZFAS1 in an m7G-dependent manner, and RBFOX2 promoted WDR4 expression by binding to WDR4 mRNA. Overexpression of WDR4 increased m7G and ZFAS1 levels, whereas overexpression of WDR4 with m7G catalytic site mutation had no effect on m7G and ZFAS1 levels in TU177 and M4E cells. Silencing ZFAS1 or RBFOX2 counteracted the promoting effect of WDR4 overexpression on the malignant proliferation of TU177 and M4E cells. TU177 cells transfected with sh-WDR4 lentiviral vectors were intraperitoneally injected into nude mice to construct xenograft tumor models. Knockdown of WDR4 significantly inhibited LC tumor growth in vivo. In conclusion, RBFOX2-mediated upregulation of WDR4 promoted LC progression through the WDR4/m7G/ZFAS1/RBFOX2 axis.

Exploration of ANKRD27 as an immune-related prognostic factor in pan-cancer and hepatocellular carcinoma.

Ankyrin repeat domain 27 (ANKRD27) has been found to be associated with certain cancers. However, its clinical potential in pan-cancer remains unclear. Public datasets (TCGA and GTEx) were applied to analyze ANKRD27 expression in multiple cancer types and its correlations with immune scores, immune checkpoint genes, and immune modulatory genes. We also examined ANKRD27 expression in hepatocellular carcinoma (HCC) patients using TCGA and GSE14520 datasets. The upregulation of ANKRD27 was verified via qRT-PCR in vitro. Based on TCGA-HCC, external, and GSE14520 cohorts, the associations between ANKRD27 expression and survival outcome were explored via the Kaplan-Meier survival curve. The effects of ANKRD27 reduction on HCC cell growth, movement, and invasion were evaluated by CCK-8, Wound healing, and Transwell assays. ANKRD27 exhibited aberrant expression in multiple cancers and was correlated with immune traits, including immune infiltration, immune checkpoint genes, and immune modulatory genes. Elevated expression of ANKRD27 was found in TCGA-HCC and GSE14520 cohorts and was confirmed in HCC cell lines. HCC patients with high ANKRD27 expression had poorer prognosis. In vitro, reducing ANKRD27 decreased the capability of proliferation, migration, and invasion in HCC cells. High ANKRD27 expression was associated with sensitivity to certain drugs. ANKRD27 displays abnormal levels of expression in different cancer types and is linked to immune status in cancer. Furthermore, ANKRD27 may serve as a prognostic predictor for HCC.

Passaging Human Tauopathy Patient Samples in Cells Generates Heterogeneous Fibrils with a Subpopulation Adopting Disease Folds.

The discovery by cryo-electron microscopy (cryo-EM) that the neu-ropathological hallmarks of different tauopathies, including Alzheimer's disease, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP), are caused by unique misfolded conformations of the protein tau is among the most profound developments in neurodegenerative disease research. To capitalize on these discoveries for therapeutic development, one must achieve in vitro replication of tau fibrils that adopt the representative tauopathy disease folds, which represents a grand challenge for the field. A widely used approach has been seeded propagation using pathological tau fibrils derived from post-mortem patient samples in biosensor cells that expresses a fragment of the tau protein known as K18, or Tau4RD, containing the microtubule-binding repeat domain of tau as the substrate. The new insights from cryo-EM raised the question of whether the Tau4RD fragment is capable of adopting characteristic tau folds found in CBD and PSP patient fibrils, and whether cell-passaged and amplified tau fibrils can be used as seeds to achieve cell-free assembly of recombinant 4R tau into fibrils without the addition of cofactors. Using Double Electron Electron Resonance (DEER) spectroscopy, we discovered that cell-passaged pathological seeds generate heterogeneous fibrils that are, however, distinct between the CBD and PSP lysate-seeded fibrils, and vastly different from heparin-induced tau fibril structures. Moreover, the lysate-seeded fibrils contain a characteristic sub-population that resembles the disease fold corresponding to the respective starting patient sample. These findings indicate that templated propagation using CBD and PSP patient-derived fibrils is possible with a tau fragment that does not contain the entire pathological fibril core, but also that additional mechanisms must be tuned to converge on a homogeneous fibril population.

ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 Inversely Regulates Programmed Death-Ligand 1 Through Negative Feedback of Phosphorylated Epithelial Growth Factor Receptor and Activation of Nuclear Factor-Kappa B in Non-Small Cell Lung Cancer.

Signaling pathways centered on the G-protein ADP-ribosylation factor 6 (Arf6) and its downstream effector ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 (AMAP1) drive cancer invasion, metastasis, and therapy resistance. The Arf6-AMAP1 pathway has been reported to promote receptor recycling leading to programmed cell death-ligand 1 (PD-L1) overexpression in pancreatic ductal carcinoma. Moreover, AMAP1 regulates of nuclear factor-kappa B (NF-κB), which is an important molecule in inflammation and immune activation, including tumor immune interaction through PD-L1 regulation. In this study, we investigated the function of AMAP1 on PD-L1 expression using lung cancer cells. We used two non-small cell lung cancer cell lines. Protein expression was evaluated by Western blotting. AMAP1 and NF-kB expression were reduced by conventional siRNA methods, and osimertinib was used as an epithelial growth factor receptor (EGFR) inhibitor. Multiple analysis of receptor tyrosine kinases (RTKs) was conducted using a semi-comprehensive RTKs assay. We found that AMAP1 inversely regulated PD-L1 expression. Based on these results, we examined the activation levels of RTKs associated with both AMAP1 and PD-L1. Following a semi-comprehensive phosphorylated RTK assay, we observed the upregulation of phosphorylated EGFR (pEGFR) led by the downregulation of AMAP1. The inhibition of pEGFR by osimertinib downregulates PD-L1 expression. We investigated the relationships between AMAP1, NF-κB, and PD-L1 expression. AMAP1 knockdown upregulated the expression of both NF-κB and PD-L1. Subsequently, NF-κB knockdown downregulated PD-L1 levels, while double knockdown of AMAP1 and NF-κB, restored PD-L1 expression. AMAP1 may inversely regulate PD-L1 through negative feedback of pEGFR and activation of NF-κB in NSCLC cell lines.

Revealing the dance of NLRP3: spatiotemporal patterns in inflammasome activation.

The nucleotide-binding domain, leucine-rich repeat, and pyrin domain containing-protein 3 (NLRP3) inflammasome is a multiprotein complex that plays a critical role in the innate immune response to both infections and sterile stressors. Dysregulated NLRP3 activation has been implicated in a variety of autoimmune and inflammatory diseases, including cryopyrin-associated periodic fever syndromes, diabetes, atherosclerosis, Alzheimer's disease, inflammatory bowel disease, and cancer. Consequently, fine-tuning NLRP3 activity holds significant therapeutic potential. Studies have implicated several organelles, including mitochondria, lysosomes, the endoplasmic reticulum (ER), the Golgi apparatus, endosomes, and the centrosome, in NLRP3 localization and inflammasome assembly. However, reports of conflict and many factors regulating interactions between NLRP3 and subcellular organelles remain unknown. This review synthesizes the current understanding of NLRP3 spatiotemporal dynamics, focusing on recent literature that elucidates the roles of subcellular localization and organelle stress in NLRP3 signaling and its crosstalk with other innate immune pathways converging at these organelles.

Transcriptome-wide identification and characterization of Toll pathway genes in Riptortus pedestris (Hemiptera: Alydidae)

The Toll pathway was first identified in Drosophila and plays an essential role in defense against infection by various pathogens. To date, various noncoding RNAs (ncRNAs) have been demonstrated to maintain immune homeostasis by regulating several target genes in the insect Toll pathway. However, the characterization and function of Toll pathway genes involved in the response to environmental changes at the posttranscriptional level associated with gut bacterial changes in Riptortus pedestris, which is a significant pest of soybeans, remain unclear. In this study, we identified and classified six Toll genes into three subtypes with typical Toll domain arrangements, including a Toll/interleukin receptor (TIR) domain, a transmembrane domain, and multiple leucine-rich repeat (LRR) domains; in addition, only one positive selection site was found in hemipteran sPP-Tolls, and a total of five downstream members in the Toll signaling pathway were selected and characterized. The expression patterns revealed that all these genes were widely expressed at all developmental stages of R. pedestris, and they presented variable expression levels among the different feeding treatments in the R. pedestris gut. Our comprehensive prediction analysis revealed that there are sixty miRNA‒mRNA interaction pairs, including fifty-six miRNA and six Toll pathway genes (P‒Toll1, sP‒Toll, Myd88, Pelle, Tube, and Cactus), and a ceRNA network comprising two lncRNA‒miRNA‒Toll pairs was constructed in response to environmental changes. Finally, the expression of some above genes and ncRNAs from the ceRNA network exhibited positive or negative association with the most changes in gut bacterial genera via Pearson correlation analysis. These findings provide valuable insights into how the Toll pathway of R. pedestris is involved in environmental adaptation at the posttranscriptional level and identifies new avenues for developing more effective methods for pest control through integration with gut bacteria.

Shank3 Overexpression Leads to Cardiac Dysfunction in Mice by Disrupting Calcium Homeostasis in Cardiomyocytes.

SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms. Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca²⁺ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca²⁺ homeostasis was assessed by analyzing cytosolic Ca²⁺ transients and sarcoplasmic reticulum Ca²⁺ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometry-based identification was employed to identify proteins in the cardiac Shank3 interactome. Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes. The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca²⁺ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca²⁺ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes. This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca²⁺ homeostasis and contraction, with a notable reduction in troponin I.

PhSLB1 Regulates Branch Development in Petunia

F-box proteins play crucial roles in various developmental stages in plants, such as shoot branching. As an important ornamental and model plant, the branch development of petunia has always been a hot research topic. In our study, the homologous gene of SMALL LEAF AND BUSHY1 (SLB1) was isolated from Petunia ×hybrida cv. Mitchell Diploid. PhSLB1 contained a C-terminal WD40 repeat domain and an N-terminal F-box domain. An analysis of expression indicated that PhSLB exhibited the highest expression in roots, whereas expression levels were lowest in stems and leaves. Subcellular localization assays demonstrated that PhSLB1 was localized on the nucleus. Furthermore, RNA interference (RNAi) of PhSLB1 in petunia significantly increased the branch number. Quantitative real-time polymerase chain reaction showed that PhSLB1 not only regulated cytokinin and strigolactone pathway but also affected the expression of some key branching-related genes, such as PhBRC1, PhKNOX, PhH2A, and PhL27. Our research establishes a theoretical groundwork for revealing the mechanism by which SLB1 regulates branch development.