Repeat Domain Research Articles

The VDAC1 oligomerization regulated by ATP5B leads to the NLRP3 inflammasome activation in the liver cells under PFOS exposure

As a persistent organic pollutant, perfluorooctane sulfonate (PFOS) has a serious detrimental impact on human health. It has been suggested that PFOS is associated with liver inflammation. However, the underlying mechanisms are still unclear. Here, PFOS was found to elevate the oligomerization tendency of voltage-dependent anion channel 1 (VDAC1) in the mice liver and human normal liver cells L-02. Inhibition of VDAC1 oligomerization alleviated PFOS-induced nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome activation. Cytoplasmic membrane VDAC1 translocated to mitochondria was also observed in response to PFOS. Therefore, the oligomerization of VDAC1 occurred mainly in the mitochondria. VDAC1 was found to interact with the ATP synthase beta subunit (ATP5B) under PFOS treatment. Knockdown of ATP5B or immobilization of ATP5B to the cytoplasmic membrane alleviated the increased VDAC1 oligomerization and NLRP3 inflammasome activation. Therefore, our results suggested that PFOS induced NLRP3 inflammasome activation through VDAC1 oligomerization, a process dependent on ATP5B to transfer VDAC1 from the plasma membrane to the mitochondria. The findings offer novel perspectives on the activation of the NLRP3 inflammasome, the regulatory mode on VDAC1 oligomerization, and the mechanism of PFOS toxicity.

Oligomerization regulates the interaction of Gemin5 with members of the SMN complex and the translation machinery

RNA-binding proteins are multifunctional molecules impacting on multiple steps of gene regulation. Gemin5 was initially identified as a member of the survival of motor neurons (SMN) complex. The protein is organized in structural and functional domains, including a WD40 repeats domain at the N-terminal region, a tetratricopeptide repeat (TPR) dimerization module at the central region, and a non-canonical RNA-binding site at the C-terminal end. The TPR module allows the recruitment of the endogenous Gemin5 protein in living cells and the assembly of a dimer in vitro. However, the biological relevance of Gemin5 oligomerization is not known. Here we interrogated the Gemin5 interactome focusing on oligomerization-dependent or independent regions. We show that the interactors associated with oligomerization-proficient domains were primarily annotated to ribosome, splicing, translation regulation, SMN complex, and RNA stability. The presence of distinct Gemin5 protein regions in polysomes highlighted differences in translation regulation based on their oligomerization capacity. Furthermore, the association with native ribosomes and negative regulation of translation was strictly dependent on both the WD40 repeats domain and the TPR dimerization moiety, while binding with the majority of the interacting proteins, including SMN, Gemin2, and Gemin4, was determined by the dimerization module. The loss of oligomerization did not perturb the predominant cytoplasmic localization of Gemin5, reinforcing the cytoplasmic functions of this essential protein. Our work highlights a distinctive role of the Gemin5 domains for its functions in the interaction with members of the SMN complex, ribosome association, and RBP interactome.

Improved Expression of Aggregation-Prone Tau Proteins Using a Spidroin-Derived Solubility Tag

Tauopathies, a group of neurodegenerative disorders, are characterized by the abnormal aggregation of microtubule-associated Tau proteins in neurons and glial cells. The process of Tau proteins transitioning from soluble, intrinsically disordered monomers to disease-associated aggregates is still unclear. Investigating these molecular mechanisms requires the reconstitution of such processes in cellular and in vitro models using recombinant proteins at high purity and yield. However, the production of phase-separating or aggregation-prone recombinant proteins like Tau’s hydrophobic-rich domains or disease mutation-carrying variants on a large scale is highly challenging due to their limited solubility. To overcome this challenge, we have developed an improved strategy for expressing and purifying recombinant Tau proteins using the major ampullate spidroin-derived solubility tag (MaSp-NT*). This approach involves using NT* as a fusion tag to enhance the solubility and stability of expressed proteins by forming micelle-like particles within the cytosol of E. coli cells. We found that fusion with the NT* tag significantly increased the solubility and yield of highly hydrophobic and/or aggregation-prone Tau constructs. Our purification method for NT* fusion proteins yielded up to twenty-fold higher amounts than proteins purified using our novel tandem-tag (6xHis-SUMO-Tau-Heparin) purification system. This enhanced expression and yield were demonstrated with full-length Tau (hT40/Tau441), its particularly aggregation-prone repeat domain (Tau-MTBR), and Frontotemporal dementia (FTD)-associated mutant (Tau-P301L). These advancements offer promising avenues for the production of large quantities of Tau proteins suitable for in vitro experimental techniques such as nuclear magnetic resonance (NMR) spectroscopy without the need for a boiling step, bringing us closer to effective treatments for tauopathies.

Structure of the E3 ligase CRL2-ZYG11B with substrates reveals the molecular basis for N-degron recognition and ubiquitination.

ZYG11B is a substrate specificity factor for Cullin-RING ubiquitin ligase (CRL2) involved in many biological processes, including Gly/N-degron pathways. Yet how the binding of ZYG11B with CRL2 is coupled to substrate recognition and ubiquitination is unknown. We present the Cryo-EM structures of the CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-peptide from the inflammasome-forming pathogen sensor NLRP1. The structures indicate ZYG11B folds into a Leucine-Rich Repeat followed by two armadillo repeat domains that promote assembly with CRL2 and recognition of NLRP1 Gly/N-degron. ZYG11B promotes activation of the NLRP1 inflammasome through recognition and subsequent ubiquitination of the NLRP1 Gly/N-degron revealed by viral protease cleavage. Our structural and functional data indicate that blocking ZYG11B recognition of the NLRP1 Gly/N-degron inhibits NLRP1 inflammasome activation by a viral protease. Overall, we show how the CRL2-ZYG11B E3 ligase complex recognizes Gly/N-degron substrates, including those that are involved in viral protease-mediated activation of the NLRP1 inflammasome.

Analysis of endogenous NOTCH1 from POFUT1 S162L patient fibroblasts reveals the importance of the O-fucose modification on EGF12 in human development.

NOTCH1 is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of NOTCH1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on NOTCH1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous NOTCH1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. NOTCH1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of NOTCH1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 is known to have significant effects on NOTCH1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.

Evolution of paralogous multicomponent systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria

Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a)missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.

The initiation of mitochondrial DNA replication.

Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.

Development of an anti-tauopathy mucosal vaccine specifically targeting pathologic conformers

Alzheimer’s disease (AD) and related tauopathies are associated with pathological tau protein aggregation, which plays an important role in neurofibrillary degeneration and dementia. Targeted immunotherapy to eliminate pathological tau aggregates is known to improve cognitive deficits in AD animal models. The tau repeat domain (TauRD) plays a pivotal role in tau-microtubule interactions and is critically involved in the aggregation of hyperphosphorylated tau proteins. Because TauRD forms the structural core of tau aggregates, the development of immunotherapies that selectively target TauRD-induced pathological aggregates holds great promise for the modulation of tauopathies. In this study, we generated recombinant TauRD polypeptide that form neurofibrillary tangle-like structures and evaluated TauRD-specific immune responses following intranasal immunization in combination with the mucosal adjuvant FlaB. In BALB/C mice, repeated immunizations at one-week intervals induced robust TauRD-specific antibody responses in a TLR5-dependent manner. Notably, the resulting antiserum recognized only the aggregated form of TauRD, while ignoring monomeric TauRD. The antiserum effectively inhibited TauRD filament formation and promoted the phagocytic degradation of TauRD aggregate fragments by microglia. The antiserum also specifically recognized pathological tau conformers in the human AD brain. Based on these results, we engineered a built-in flagellin-adjuvanted TauRD (FlaB-TauRD) vaccine and tested its efficacy in a P301S transgenic mouse model. Mucosal immunization with FlaB-TauRD improved quality of life, as indicated by the amelioration of memory deficits, and alleviated tauopathy progression. Notably, the survival of the vaccinated mice was dramatically extended. In conclusion, we developed a mucosal vaccine that exclusively targets pathological tau conformers and prevents disease progression.

Stable ubiquitin conjugation for biological interrogation of ubiquitinated tau repeat domain

Protein semisynthesis approaches are key for gaining insights into the effects of post-translational modifications (PTMs) on the structure and function of modified proteins. Among PTMs, ubiquitination involves the conjugation of a small protein modifier to a substrate amino acid residue and is unique in controlling a variety of cellular processes. Interest has grown in understanding the role of ubiquitination in neurodegenerative conditions, including tauopathies. The latter are characterized by the accumulation of the intrinsically disordered protein tau in the form of neurofibrillary tangles in the brains of patients. The presence of ubiquitinated tau in the pathological aggregates suggests that ubiquitination might play a role in the formation of abnormal protein deposits. In this study, we developed a new strategy, based on dehydroalanine chemistry, to install wild type ubiquitin on a tau repeat domain construct with site-specificity. We optimized a three-step reaction which yielded a good amount of highly pure tau repeat domain ubiquitinated in position 353. The structural features of the conjugate were examined by circular dichroism and NMR spectroscopy. The ubiquitinated tau was challenged in a number of assays: fibrils formation under aggregating conditions in vitro, chemical stability upon exposure to a variety of biological media including cell extracts, and internalization into astrocytes. The results demonstrated the wide applicability of the new semisynthetic strategy for the investigation of ubiquitinated substrates in vitro or in cell, and in particular for studying if ubiquitination has a role in the molecular mechanisms that underlie the aberrant transition of tau into pathological aggregates.

MUC21: a new target for tumor treatment.

MUC21, also known as Epiglycanin, is a high-molecular-weight glycoprotein with transmembrane mucin properties. It consists of a tandem repeat domain, a stem domain, a transmembrane domain and a cytoplasmic tail. MUC21 is expressed is observed in normal tissues in organs like the thymus, testes, lungs, and large intestine. Research has shown that MUC21 is expressed in esophageal squamous cell carcinoma, lung adenocarcinoma, glioblastoma, thyroid cancer, melanoma, and various other malignant tumors in distinctive manner. Additionally, tumor invasion, metastasis, and poor prognosis are linked to it. Some researchers believe that MUC21 has the potential to become a new target in cancer treatment. This review aims to deliver a comprehensive overview of the glycosylation, function, and research progress of MUC21 in multiple types of cancer and infectious diseases.

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotianabenthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

Pathogenic FLNA variants affecting the hinge region of filamin A are associated with male survival.

Pathogenic variants in FLNA cause a diversity of X-linked developmental disorders associated with either preserved or diminished levels of filamin A protein and are conceptualized dichotomously as relating to underlying gain- or loss-of-function pathogenic mechanisms. Hemizygosity for germline deletions or truncating variants in FLNA is generally considered to result in embryonic lethality. Structurally, filamin A is composed of an N-terminal actin-binding region, followed by 24 immunoglobulin-like repeat units. The repeat domains are separated into distinct segments by two regions of low-complexity known as hinge-1 and hinge-2. Hinge-1 is proposed to confer flexibility to the otherwise rigid protein and is a target for cleavage by calpain with the resultant filamin fragments mediating crucial cellular signaling processes. Here, three families with pathogenic variants in FLNA that impair the function of hinge-1 in males are described, leading to distinct clinical phenotypes. One large in-frame deletion that includes the hinge leads to frontometaphyseal dysplasia in affected males and females, while two germline truncating variants located within the exon encoding hinge 1 result in phenotypes in males that are explained by exon skipping and under-expression of a transcript that deletes hinge-1 from the resultant protein. These three variants affecting hinge-1 indicate that this domain does not mediate cellular functions that, when deficientresult in embryonic lethality in males and that germline truncating variants in this region of FLNA can result in viable phenotypes in males.

Biochemical properties and application of a multi-domain β-1,3-1,4-glucanase from Fibrobacter sp.

A novel glycoside hydrolase (GH) family 16 multi-domain β-1,3-1,4-glucanase (FsGlc16A) from Fibrobacter sp. UWP2 was identified, heterogeneously expressed, and its enzymatic properties, protein structure and application potential were characterized. Enzymological characterization showed that FsGlc16A performed the optimal catalytic activity at pH 4.5 and 50 °C with a specific activity of 3263 U/mg. FsGlc16A exhibited the substrate specificity towards oat β-glucan, barley β-glucan and lichenan, and in addition, it hydrolyzed oat β-glucan and lichenan into different β-glucooligosaccharides with polymerization degrees of 3–4, which further illustrated that it belonged to the endo-type β-1,3-1,4-glucanase. FsGlc16A was classified in subfamily25 of GH16. A ‘PXSSSS’ repeats domain was identified at the C-terminus of FsGlc16A, which was distinct from the typical GH family 16 β-1,3-1,4-glucanases. Removing the ‘PXSSSS’ repeats domain affected the binding of the substrate to FsGlc16A and reduced the enzyme activity. FsGlc16A displayed good potential for the applications, which hydrolyzed oat bran into β-glucooligosaccharides, and reduced filtration time (18.89 %) and viscosity (3.64 %) in the saccharification process. This study investigated the enzymatic properties and domain function of FsGlc16A, providing new ideas and insights into the study of β-1,3-1,4-glucanase.

The draft genome of Nitzschia closterium f. minutissima and transcriptome analysis reveals novel insights into diatom biosilicification

BackgroundNitzschia closterium f. minutissima is a commonly available diatom that plays important roles in marine aquaculture. It was originally classified as Nitzschia (Bacillariaceae, Bacillariophyta) but is currently regarded as a heterotypic synonym of Phaeodactylum tricornutum. The aim of this study was to obtain the draft genome of the marine microalga N. closterium f. minutissima to understand its phylogenetic placement and evolutionary specialization. Given that the ornate hierarchical silicified cell walls (frustules) of diatoms have immense applications in nanotechnology for biomedical fields, biosensors and optoelectric devices, transcriptomic data were generated by using reference genome-based read mapping to identify significantly differentially expressed genes and elucidate the molecular processes involved in diatom biosilicification.ResultsIn this study, we generated 13.81 Gb of pass reads from the PromethION sequencer. The draft genome of N. closterium f. minutissima has a total length of 29.28 Mb, and contains 28 contigs with an N50 value of 1.23 Mb. The GC content was 48.55%, and approximately 18.36% of the genome assembly contained repeat sequences. Gene annotation revealed 9,132 protein-coding genes. The results of comparative genomic analysis showed that N. closterium f. minutissima was clustered as a sister lineage of Phaeodactylum tricornutum and the divergence time between them was estimated to be approximately 17.2 million years ago (Mya). CAFF analysis demonstrated that 220 gene families that significantly changed were unique to N. closterium f. minutissima and that 154 were specific to P. tricornutum, moreover, only 26 gene families overlapped between these two species. A total of 818 DEGs in response to silicon were identified in N. closterium f. minutissima through RNA sequencing, these genes are involved in various molecular processes such as transcription regulator activity. Several genes encoding proteins, including silicon transporters, heat shock factors, methyltransferases, ankyrin repeat domains, cGMP-mediated signaling pathways-related proteins, cytoskeleton-associated proteins, polyamines, glycoproteins and saturated fatty acids may contribute to the formation of frustules in N. closterium f. minutissima.ConclusionsHere, we described a draft genome of N. closterium f. minutissima and compared it with those of eight other diatoms, which provided new insight into its evolutionary features. Transcriptome analysis to identify DEGs in response to silicon will help to elucidate the underlying molecular mechanism of diatom biosilicification in N. closterium f. minutissima.

The unconventional resistance protein PTR recognizes the Magnaporthe oryzae effector AVR-Pita in an allele-specific manner.

Blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating rice diseases. Disease resistance genes such as Pi-ta or Pi-ta2 are critical in protecting rice production from blast. Published work reports that Pi-ta codes for a nucleotide-binding and leucine-rich repeat domain protein (NLR) that recognizes the fungal protease-like effector AVR-Pita by direct binding. However, this model was challenged by the recent discovery that Pi-ta2 resistance, which also relies on AVR-Pita detection, is conferred by the unconventional resistance gene Ptr, which codes for a membrane protein with a cytoplasmic armadillo repeat domain. Here, using NLR Pi-ta and Ptr RNAi knockdown and CRISPR/Cas9 knockout mutant rice lines, we found that AVR-Pita recognition relies solely on Ptr and that the NLR Pi-ta has no role in it, indicating that it is not the Pi-ta resistance gene. Different alleles of Ptr confer different recognition specificities. The A allele of Ptr (PtrA) detects all natural sequence variants of the effector and confers Pi-ta2 resistance, while the B allele of Ptr (PtrB) recognizes a restricted set of AVR-Pita alleles and, thereby, confers Pi-ta resistance. Analysis of the natural diversity in AVR-Pita and of mutant and transgenic strains identified one specific polymorphism in the effector sequence that controls escape from PtrB-mediated resistance. Taken together, our work establishes that the M. oryzae effector AVR-Pita is detected in an allele-specific manner by the unconventional rice resistance protein Ptr and that the NLR Pi-ta has no function in Pi-ta resistance and the recognition of AVR-Pita.

Pick's Disease Presenting as Tremulous Parkinsonism with Limited Levodopa Response-A Rare Cause of Corticobasal Syndrome.

Corticobasal syndrome is a clinical diagnosis and common pathological causes are corticobasal degeneration, progressive supranuclear palsy and Alzheimer's disease. We would like to highlight a rare but important differential of corticobasal syndrome. A 78-year-old female had a 4-year history of predominantly right-hand rest tremor, worsening of handwriting but no change in cognition. The clinical examination showed right upper limb postural and kinetic tremor, mild wrist rigidity and reduced amplitude of right-sided finger tapping. She was initially diagnosed as idiopathic Parkinson's disease. Five years after onset of symptoms, she demonstrated bilateral myoclonic jerks and right upper limb dystonic posturing. She could not copy movements with the right hand. The magnetic resonance imaging (MRI) revealed disproportionate atrophy in the parietal lobes bilaterally. The clinical diagnosis was changed to probable corticobasal syndrome. She passed away 11 years from onset of symptoms at the age of 85 years. She underwent a post-mortem. The anterior and posterior frontal cortex, anterior cingulate, temporal neocortex, hippocampus and amygdaloid complex demonstrated considerable tau-related pathology consisting of a dense background of neuropil threads, and rounded, paranuclear neuronal inclusions consistent with Pick bodies. The immunostaining for three microtubule binding domain repeats (3R) tau performed on sections from the frontal and temporal lobes, basal ganglia and midbrain highlighted several inclusions whilst no 4R tau was observed. She was finally diagnosed with Pick's disease. Pick's disease can rarely present with clinical features of corticobasal syndrome.

Exploration of the binding determinants of protein phosphatase 5 (PP5) reveals a chaperone-independent activation mechanism

The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C-termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves auto-inhibition in PP5’s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5’s enzymatic activity. The enhanced affinity for PP5’s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry (paDSF) assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C-termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (ELP1/IKBKAP) as a putative partner and, indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5’s TPR domain in vitro. Consistent with this idea, mutation of ELP1’s terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5’s TPR domain and expand the scope of PP5’s functions to include chaperone-independent complexes.

TLR21 is involved in the NF-κB and IFN-β pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Chang'an decoction alleviates endoplasmic reticulum stress by regulating mitofusin 2 to improve colitis.

To evaluate the protective effects of Chang'an decoction (, CAD) on colitis, and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum (ER) stress induced by mitofusin 2 (MFN2). The composition of CAD was identified by liquid chromatography-mass spectrometry technology. A mice model of dextran sulfate sodium (DSS) induced colitis was established and therapeutic effects of CAD were determined by detecting body weight, disease activity index, colon length and histopathological changes. Then, the expression levels of MFN2, ER stress markers and Nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) relevant proteins were detected by polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence staining. Subsequently, knockdown and overexpression cell model were constructed to further investigate the underlying mechanism of MFN2 mediating ER stress and energy metabolism by PCR, Western blot, electron microscopy and reactive oxygen species (ROS) staining. Finally, inflammatory indicator and tight junction proteins were measured by PCR and immunofluorescence staining to evaluate the protective effects of CAD. Results showed that the indispensable regulatory role of MFN2 in mediating ER stress and mitochondrial damage was involved in the protective effects of CAD on colitis in mice fed with DSS. Network pharmacology analysis also revealed CAD may play a protective effect on colitis by affecting mitochondrial function. In addition, our data also suggested a causative role for MFN2 in the development of inflammatory responses and energy metabolic alterations by constructing a knockdown and overexpression cell model whereby alter proper ER-mitochondria interaction in Caco-2 cells. Furthermore, relative expression analyses of ER stress markers and NLRP3 inflammasome showed the onset of ER stress and activation of NLRP3 inflammasome, which is consistent with the above findings. In contrast, intervention of CAD could improve the mucosal barrier integrity and colonic inflammatory response effectively through inhibiting ER stress response mediated by MFN2. CAD could alleviate ER stress by regulating MFN2 to exert therapeutic effects on DSS-induced colitis, which might provide an effective natural therapeutic approach for the treatment of ulcerative colitis.

Titanium particle-induced inflammasome in human gingival epithelial cells

Background/purposePeri-implantitis remains a substantial challenge. This study investigated the effect of titanium particles on human oral epithelial cells, focusing on the nucleotide-binding domain and leucine-rich repeat protein (NLRP) 3 inflammasome. Materials and methodsThe Ca9-22 human gingival epithelial cell line was subjected to incubation with titanium particles. To evaluate cell viability, the MTT assay was employed. Total RNA was extracted, and messenger RNA (mRNA) expressions of COX2, TGF-β1, NLRP1, NLPR3, CASP1, and AIM2 were analyzed. The concentration of interleukin (IL)1β in cell supernatants was quantified through enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) were visualized using an ROS assay Kit. ResultsCa9-22 cells treated with titanium particles showed >75% cell viability across all concentrations tested, with consistent results. mRNA expressions of inflammation-related genes (COX2 and TGF-β1) significantly increased in a dose-dependent manner. The mRNA expression of NLRP3 and CASP1, as well as the secretion of IL1β, increased after 6-h incubation with titanium particles. Moreover, the ROS assay results showed increased production of ROS after treatment with titanium particles, whereas NLRP3 expression and IL1β secretion reduced after treatment with N-acetyl-l-cysteine (ROS scavenger). ConclusionOur findings indicate that titanium particles possess a distinct ability to trigger the NLRP3 inflammasome, partly by producing ROS.