Tandem Domains Research Articles

Copines, a Family of Calcium Sensor Proteins and Their Role in Brain Function.

The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share a unique architecture and contain tandem C2 domains and a Von Willebrand factor type A (VWA) domain. C2 domains in Copines bind calcium, phospholipids, and other proteins and mediate the transient association of these proteins with biological membranes at elevated calcium levels. The VWA domain also binds calcium and is involved in protein-protein interactions. Here, we provide a comprehensive review of the sequences, structures, expression, targeting, and function of the entire family of known Copine proteins (Copine 1-9 in mammals) with a particular emphasis on their functional roles in the mammalian brain. Neuronal Copines are implicated in a wide array of processes from cell differentiation to synaptic transmission and plasticity and are also linked to several pathological conditions from cancers to brain diseases. This review provides the most up-to-date insights into the structure and function of Copines, with an emphasis on their role in brain function.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Molecular mechanism of specific HLA-A mRNA recognition by the RNA-binding-protein hMEX3B to promote tumor immune escape

Immunotherapy, including immune checkpoint inhibitors and adoptive cell transfer, has obtained great progress, but their efficiencies vary among patients due to the genetic and epigenetic differences. Human MEX3B (hMEX3B) protein is an RNA-binding protein that contains two KH domains at the N-terminus and a RING domain at its C-terminus, which has the activity of E3 ubiquitin ligase and is essential for RNA degradation. Current evidence suggests that hMEX3B is involved in many important biological processes, including tumor immune evasion and HLA-A regulation, but the sequence of substrate RNA recognized by hMEX3B and the functional molecular mechanisms are unclear. Here, we first screened the optimized hMEX3B binding sequence on the HLA-A mRNA and reported that the two tandem KH domains can bind with their substrate one hundred times more than the individual KH domains. We systematically investigated the binding characteristics between the two KH domains and their RNA substrates by nuclear magnetic resonance (NMR). Based on this information and the small-angle X-ray scattering (SAXS) data, we used molecular dynamics simulations to obtain structural models of KH domains in complex with their corresponding RNAs. By analyzing the models, we noticed that on the KH domains’ variable loops, there were two pairs of threonines and arginines that can disrupt the recognition of the RNA completely, and this influence had also been verified both in vitro and in vivo. Finally, we presented a functional model of the hMEX3B protein, which indicated that hMEX3B regulated the degradation of its substrate mRNAs in many biological processes. Taken together, our research illustrated how the hMEX3B protein played a key role in translation inhibition during the immune response to tumor cells and provided an idea and a lead for the study of the molecular mechanism and function of other MEX3 family proteins.

A Revised Molecular Model of Ovarian Cancer Biomarker CA125 (MUC16) Enabled by Long-read Sequencing.

Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.

Identification of Fusarium wilt resistance gene SiRLK1 in Sesamum indicum L.

Sesame Fusarium wilt (SFW), caused by Fusarium oxysporum f. sp. sesami (Fos), is one of the most devastating diseases affecting sesame cultivation. Deciphering the genetic control of SFW resistance is pivotal for effective disease management in sesame. An inheritance study on a cross between the highly resistant variety Yuzhi 11 and the highly susceptible accession Sp1 using a Fos pathogenicity group 1 isolate indicated that resistance was conferred by a single dominant allele. The target locus was located in a 1.24 Mb interval on chromosome 3 using a combination of cross-population association mapping and bulked segregant analysis. Fine genetic mapping further narrowed the interval between 21,350 and 21,401 kb. The locus Sindi_0812400 was identified as the SFW resistance gene and officially designated SiRLK1. This gene encodes a specific malectin/receptor-like protein kinase with three putative tandem kinase domains and is considered a kinase fusion protein. Sequence analysis revealed that a high proportion (49.44%) of variants within the locus was located within the kinase domain III, and several of which were evidently associated with the diversity in SFW response, indicating the critical role of kinase domain III in expression of disease resistance. These findings provide valuable information for further functional analysis of SFW resistance genes and marker-assisted resistance breeding in sesame.

Simulations of a PKA RIα homodimer reveal cAMP-coupled conformational dynamics of each protomer and the dimer interface with functional implications.

Protein kinase A (PKA) is a ubiquitous cAMP-dependent enzyme in mammalian tissues. The inactive PKA holoenzyme disassociates into a homodimer of regulatory (R) subunits and two active catalytic (C) subunits upon cAMP binding to two tandem domains (termed CBD-A and CBD-B) in R subunits. The release of cAMP facilitates reassociation of R and C subunits, resetting PKA to its basal state. The cAMP-mediated structural changes in the activation-termination cycle remain partially understood. The multimeric states of PKA complicate the issue and are particularly less studied. Therefore, we computationally investigated the conformational dynamics of the PKA RIα homodimer in different cAMP-bound states. The absence of cAMP in two CBDs differently affects the conformational dynamics of protomers. Moreover, such disparate responses are extended to the dimer interface constituted by the N-terminal helical sub-domains termed N3A motifs. The removal of cAMP from CBD-A induces large-scale structural changes of individual R subunits towards the holoenzyme state, consistent with previous simulations of a single R subunit. Meanwhile it keeps the structural heterogeneity of the N3A-N3A' dimer interface observed in the fully bound state. By contrast, the removal of cAMP from CBD-B does not affect individual R subunits but alters the conformational space of the N3A-N3A' dimer interface. The cAMP-coupled structural changes of each protomer and conserved conformational space of the N3A-N3A' dimer interface are essential for the transition between the fully cAMP-bound R2 homodimer and the R2C2 holoenzyme as suggested by their crystal structures. Our work provides structural insights into the regulatory mechanism of cAMP in PKA signaling.

Ectopic expression of TTP gene from human in poplar promotes xylem differentiation and confers plant drought tolerance.

The CCCH zinc finger proteins play critical roles in a wide variety of growth, development, and stress responses. Currently, limited reports are available about the roles of animal CCCH proteins in plants. In this study, we report the identification of human TTP (hTTP) with functional similarity to PdC3H17 in a hybrid poplar. hTTP and PdC3H17 shared highly similar tandem CCCH zinc-finger RNA-binding domains. The fragments excluding the CCCH domain of both hTTP and PdC3H17 possessed transcriptional activation activities in yeast cells. Compared to the controls, ectopic expression of hTTP in poplar caused dwarfism, and resulted in significant increases in stem xylem vessel number and photosynthetic and ROS-scavenging abilities, thereby enhancing plant tolerance to drought stress. Our results suggest that hTTP may perform a function in poplar through the PdC3H17-mediated system, and provide an example for the application of animal genes in plants.

Exon shuffling and alternative splicing of ROCO genes in brown algae enables a diverse repertoire of candidate immune receptors.

The ROCO family is a family of GTPases characterized by a central ROC-COR tandem domain. Interest in the structure and function of ROCO proteins has increased with the identification of their important roles in human disease. Nevertheless, the functions of most ROCO proteins are still unknown. In the present study, we characterized the structure, evolution, and expression of ROCOs in four species of brown algae. Brown algae have a larger number of ROCO proteins than other organisms reported to date. Phylogenetic analyses showed that ROCOs have an ancient origin, likely originated in prokaryotes. ROCOs in brown algae clustered into four groups and showed no strong relationship with red algae or green algae. Brown algal ROCOs retain the ancestral LRR-ROC-COR domain arrangement, which is found in prokaryotes, plants and some basal metazoans. Remarkably, individual LRR motifs in ROCO genes are each encoded by separate exons and exhibit intense exon shuffling and diversifying selection. Furthermore, the tandem LRR exons exhibit alternative splicing to generate multiple transcripts. Both exon shuffling and alternative splicing of LRR repeats may be important mechanisms for generating diverse ligand-binding specificities as immune receptors. Besides their potential immune role, expression analysis shows that many ROCO genes are responsive to other stress conditions, suggesting they could participate in multiple signal pathways, not limited to the immune response. Our results substantially enhance our understanding of the structure and function of this mysterious gene family.

Features of the rare pathogen Meyerozyma guilliermondii strain SO and comprehensive in silico analyses of its adherence-contributing virulence factor agglutinin-like sequences

Meyerozyma guilliermondii is a rare yeast pathogen contributing to the deadly invasive candidiasis. M. guilliermondii strain SO, as a promising protein expression host, showed 99% proteome similarity with the clinically isolated ATCC 6260 (type strain) in a recent comparative genomic analysis. However, their in vitro virulence features and in vivo pathogenicity were uncharacterized. This study aimed to characterize the in vitro and in vivo pathogenicity of M. guilliermondii strain SO and analyze its Als proteins (MgAls) via comprehensive bioinformatics approaches. M. guilliermondii strain SO showed lower and higher sensitivity towards β-mercaptoethanol and lithium, respectively than the avirulent S. cerevisiae but exhibited the same tolerance towards cell wall-perturbing Congo Red with C. albicans. With 7.5× higher biofilm mass, M. guilliermondii strain SO also demonstrated 75% higher mortality rate in the zebrafish embryos with a thicker biofilm layer on the chorion compared to the avirulent S. cerevisiae. Being one of the most important Candida adhesins, sequence and structural analyses of four statistically identified MgAls showed that MgAls1056 was predicted to exhibit the most conserved amyloid-forming regions, tandem repeat domain and peptide binding cavity (PBC) compared to C. albicans Als3. Favoured from the predicted largest ligand binding site and druggable pockets, it showed the highest affinity towards hepta-threonine. Non-PBC druggable pockets in the most potent virulence contributing MgAls1056 provide new insights into developing antifungal drugs targeting non-albicans Candida spp. Virtual screening of available synthetic or natural bioactive compounds and MgAls1056 deletion from the fungal genome should be further performed and validated experimentally. Communicated by Ramaswamy H. Sarma

Harmonin homology domain-mediated interaction of RTEL1 helicase with RPA and DNA provides insights into its recruitment to DNA repair sites.

The regulator of telomere elongation helicase 1 (RTEL1) playsrolesin telomere DNA maintenance, DNA repair, and genome stability by dismantling D-loops and unwinding G-quadruplex structures. RTEL1 comprises a helicase domain, two tandem harmonin homology domains 1&2 (HHD1 and HHD2), and a Zn2+-binding RING domain. In vitro D-loop disassembly by RTEL1 is enhanced in the presence of replication protein A (RPA). However, the mechanism of RTEL1 recruitment at non-telomeric D-loops remainsunknown. In this study, we have unravelled a direct physical interaction between RTEL1 and RPA. Under DNA damage conditions, we showed that RTEL1 and RPA colocalise in the cell. Coimmunoprecipitation showed that RTEL1 and RPA interact, and the deletion of HHDs of RTEL1 significantly reduced this interaction. NMR chemical shift perturbations (CSPs) showed that RPA uses its 32C domain to interact with the HHD2 of RTEL1.Interestingly, HHD2 also interacted with DNA in thein vitro experiments. HHD2 structure was determined using X-ray crystallography, and NMR CSPs mapping revealed that both RPA32C and DNAcompetitivelybind toHHD2 on an overlapping surface. These results establish novel roles of accessory HHDs in RTEL1's functionsand provide mechanistic insights into the RPA-mediated recruitment of RTEL1to DNA repairsites.

A study of the synthetic route and resistance of gilteritinib

Gilteritinib is used to treat acute myeloid leukemia (AML) carrying FMS-like tyrosine kinase 3 (FLT3) mutations. As it is a new drug, there are still many aspects that need improvement. This article will be written according to the following three points: Analysis of its mechanism of action with the receptor; Analysis of the three main synthetic routes of gilteritinib and reasonable suggestions are given; Causes and solutions to the emergence of drug resistance. The following 4 points have been found that:(1) FLT3-internal tandem duplication (ITD) mutation and FLT3-tyrosine kinase domain (TKD) mutation, leukemic cells' ability to proliferate, differentiate, and survive aberrant changes due to both kinds of mutations. (2) Gilteritinib combines with FLT3's ATP pocket, and the compound shaped from FLT3 to gilteritinib was synthesized by the juxtamembrane (JM) domain. (3) Synthesis route 3 in this paper is most suitable for industrial mass production. (4) Resistance to gilteritinib is divided into primary resistance and secondary resistance, research in the future should focus on finding new therapeutic targets.

Efforts toward discovering inhibitors of the SYK SH2–FCER1γ interaction as potential Alzheimer’s disease chemical probes and therapeutics

AbstractBackgroundSpleen associated tyrosine kinase (SYK) plays a potential role in several neurodegenerative conditions. To date, a few pharmacological inhibitors have been developed, targeting the ATP‐binding kinase domain, and some of these inhibitors have been reported to show beneficial effects on the pathophysiology of Alzheimer’s (PMC: 9179326).In 2015, the Accelerating Medicines Partnership: Alzheimer’s Disease (AMP‐AD) Knowledge Portal analyzed and identified SYK and the FC gamma receptor (FCεR1γ) as targets for Alzheimer’s Disease (AD), through multidimensional human “omic” (genomic, epigenomic, RNAseq, and proteomic) data (PMID: 26853544). Thus, the interaction between FCER1G p‐ITAM and SYK tandem SH2 domain was selected for further target validation and small molecule hit discovery efforts through the portfolio of novel targets to treat the Alzheimer’s disease via the Target Enablement to Accelerate Therapy Development for Alzheimer’s Disease (TREAT‐AD) program were initiated. As part of the TREAT‐AD effort, we aim to provide experimental validation of a SYK–FCεR1γ interaction inhibitor for AD and begin drug discovery campaigns to identify novel inhibitors of this interaction, with the goal of developing an in vivo chemical probe.MethodUsing purified SYK tandem‐SH2 (tSH2) domain protein, a time‐resolved fluorescence energy transfer (TR‐FRET) based assay platform was developed with a fluoroprobe‐labeled phospho‐ITAM peptide of FCεR1γ. This assay was miniaturized for ultra‐high‐throughput screening (uHTS) of 100K compounds in parallel with a DNA encoded library (DEL) screening approach (Ref: X‐Chem). The hits from these screening methods were evaluated in secondary assays in an attempt to validate the initial hits.ResultWe have previous crystallography data of the SYK tSH2 domains bound to phospho‐ITAM peptides and have established a TR‐FRET based assay to discover and characterize inhibitors of the interaction between the FCεR1γ peptide and SYK tSH2 domains. Several thio‐uric acids hit compounds have been identified from uHTS. Most of them demonstrate potency in the TR‐FRET based assay of <10 µM. Although, orthogonal experimental validation is still under process.ConclusionThrough the TREAT‐AD program, we have explored the SYK–FCεR1γ interaction and demonstrated novel screening strategies, which can be further exploited to generate novel inhibitors of this interaction.

Investigating two pore potassium channel THIK‐1 expression in Alzheimer’s disease

AbstractBackgroundThe neuroinflammatory response, particularly the activation of the NLRP3 pathway, has been suggested as one of the key mechanisms of the pathological process in neurodegenerative diseases. Tandem pore domain halothane‐inhibited K+ channel 1 (THIK‐1) provides an upstream regulation of this neuroinflammatory response by modulating intracellular K+. While in vitro and in vivo model systems provide strong support for these mechanisms, unequivocal evidence from neurodegenerative disorders has been lacking.MethodThis research project investigated gene expression of markers in the THIK‐1/NLRP3 pathway by studying post‐mortem brain tissue of animal model representing Alzheimer’s disease (AD). In addition, THIK‐1 gene and protein expression in human post‐mortem brains of AD and Parkinson’s disease (PD) was investigated. Furthermore, DNA methylation of THIK‐1 gene in human post‐mortem brains of AD was analysed to explore a potential underlying mechanism of the changes of THIK‐1 gene expression.ResultA rat model of AD pathogenesis by intrahippocampal Aβ oligomer injection showed substantial up regulation of THIK‐1, glial activation markers, NLRP3 inflammasome components and IL‐1β. Changes in cortical gene expression for THIK‐1 support an inflammatory glial cell activation in both advanced AD and PD with dementia, not observed in the substantia nigra of the latter group. The increase in THIK‐1 expression was also supported by down‐regulation in DNA methylation of its corresponding gene in AD.ConclusionThe association between THIK‐1 expression and pathology changes observed in this study indicate a THIK‐1‐induced activation of this glial subtype in AD and PD. Therefore, specific blocks of the microglial THIK‐1 K+ channels at the early stage of AD and PD provides a potential therapeutic target.

The MC160 protein of the molluscum contagiosum virus dampens cGAS/STING-induced interferon-β activation

Molluscum contagiosum virus (MCV) is a poxvirus that causes benign, persistent skin lesions. MCV encodes a variety of immune evasion molecules to dampen host immune responses. Two of these proteins are the MC159 and MC160 proteins. Both MC159 and MC160 contain two tandem death effector domains and share homology to the cellular FLIPs, FADD, and procaspase-8. MC159 and MC160 dampen several innate immune responses such as NF-κB activation and mitochondrial antiviral signaling (MAVS)-mediated induction of type 1 interferon (IFN). The type 1 IFN response is also activated by the cytosolic DNA sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Both cGAS and STING play a vital role in sensing a poxvirus infection. In this study, we demonstrate that there are nuanced differences between both MC160 and MC159 in terms of how the viral proteins modulate the cGAS/STING and MAVS pathways. Specifically, MC160 expression, but not MC159 expression, dampens cGAS/STING-mediated induction of IFN in HEK 293 T cells. Further, MC160 expression prevented the K63-ubiquitination of both STING and TBK1, a kinase downstream of cGAS/STING. Ectopic expression of the MC160 protein, but not the MC159 protein, resulted in a measurable decrease in the TBK1 protein levels as detected via immunoblotting. Finally, using a panel of MC160 truncation mutants, we report that the MC160 protein requires both DEDs to inhibit cGAS/STING-induced activation of IFN-β. Our model indicates MC160 likely alters the TBK1 signaling complex to decrease IFN-β activation at the molecular intersection of the cGAS/STING and MAVS signaling pathways.

DUF916 and DUF3324 in the WxL protein cluster bind to WxL and link bacterial and host surfaces.

Bacterial WxL proteins contain peptidoglycan-binding WxL domains, which have a dual Trp-x-Leu motif and are involved in virulence. It was recently shown that WxL proteins occur in gene clusters, containing typically a small WxL protein (which in the mature protein consists only of a WxL domain), a large WxL protein (which contains a C-terminal WxL domain with N-terminal host-binding domains), and a conserved protein annotated as a Domain of Unknown Function (DUF). Here we analyse this DUF and show that it contains two tandem domains - DUF916 and DUF3324 - which both have an IgG-like fold and together form a single functional unit, connected to a C-terminal transmembrane helix. DUF3324 is a stable domain, while DUF916 is less stable and is likely to require a stabilizing interaction with WxL. The protein is suggested to have an important role to bind and stabilize WxL on the peptidoglycan surface, via the DUF916 domain, and to bind to host cells via the DUF3324 domain. AlphaFold2 predicts that a β-hairpin strand from DUF916 inserts into WxL adjacent to its N-terminus. We therefore propose to rename the DUF916-DUF3324 pair as WxL Interacting Protein (WxLIP), with DUF916, DUF3324 and the transmembrane helix forming the first, second and third domains of WxLIP, which we characterize as peptidoglycan binding domain (PGBD), host binding domain (HBD) and transmembrane helix (TMH) respectively. This article is protected by copyright. All rights reserved.

TC2N Promotes Cell Proliferation and Metastasis in Hepatocellular Carcinoma by Targeting the Wnt/β-Catenin Signaling Pathway

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancer worldwide, has an exceedingly poor prognosis. Tandem C2 domain nuclear protein (TC2N) has been implicated in tumorigenesis and serves as an oncogene or tumor suppressor in different types of cancer. Here, we explore the possible regulatory activities and molecular mechanisms of TC2N in HCC progression. However, TC2N expression was significantly upregulated in HCC tissues and hepatoma cell lines, and this upregulation was positively correlated with tumor progression in HCC patients. The ectopic overexpression of TC2N accelerated the proliferation, migration, and invasion of HCC cells, whereas its knockdown showed the opposite effects. Bioinformatics analysis showed that TC2N participates in the regulation of the Wnt/β-catenin signaling pathway. Mechanistically, TC2N activated the Wnt/β-catenin signaling pathway by regulating the expression levels of β-catenin and its downstream targets CyclinD1, MMP7, c-Myc, c-Jun, AXIN2, and glutamine synthase. Furthermore, the deletion of β-catenin effectively neutralized the regulation of TC2N in HCC proliferation and metastasis. Overall, this study showed that TC2N promotes HCC proliferation and metastasis by activating the Wnt/β-catenin signaling pathway, indicating that TC2N might be a potential molecular target for the treatment of HCC.

Elevated Expression of Two Pore Potassium Channel THIK-1 in Alzheimer's Disease: An Inflammatory Mechanism.

Tandem pore domain halothane-inhibited K+ channel 1 (THIK-1, coded by KCNK13) provides an upstream regulation of the activation of the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, which has been suggested as one of the key mechanisms of the pathological process in neurodegeneration mainly from in vitro and in vivo model systems studies. However, unequivocal evidence from neurodegenerative disorders has been lacking. To investigate the involvement of the THIK-1/NLRP3 pathway in the pathological process of Alzheimer's disease (AD) and Parkinson's disease (PD). This study investigated gene expression of markers in the THIK-1/NLRP3 pathway in an animal model representing AD as well as in human postmortem brains of AD and PD by quantitative real-time PCR. THIK-1 protein expression was determined using automated capillary electrophoresis immunoblotting. Furthermore, DNA methylation of KCNK13 was analysed in AD cohort by pyrosequencing. A substantial upregulation of KCNK13, glial activation markers, NLRP3 inflammasome components, and IL1B was observed in the animal study. Increased expression of KCNK13 support an inflammatory glial cell activation in both advanced AD and PD. The increase in KCNK13 expression was also supported by downregulation in DNA methylation of KCNK13 in AD. The association between THIK-1 K+ channels expression and pathology changes indicates a THIK-1-induced activation of this glial subtype in AD and PD. Therefore, specific blocks of the microglial THIK-1 K+ channels at the early stage of AD and PD may be beneficial for the patients.

Discovery of a 53BP1 Small Molecule Antagonist Using a Focused DNA-Encoded Library Screen.

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 μM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 μM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission.

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease Calpain-7 (CAPN7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem microtubule-interacting and trafficking (MIT) domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for (1) CAPN7 recruitment to midbodies, (2) efficient abscission, and (3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.

Grass carp (Ctenopharyngodon idella) Mex3B positively regulates innate immunity by promoting the K63-linked ubiquitination of TLR3

As a member of Mex3 (muscle excess protein-3) family, Mex3B (Mex-3 RNA binding family member B) is crucial in cell proliferation and migration in mammals. In this study, an ortholog of mammalian Mex3B (denominated CiMex3B, MT276802.1) was cloned and identified in grass carp (Ctenopharyngodon idella). CiMex3B is 1578 bp in length and encodes a polypeptide of 525 amino acids. Consistent with its mammalian counterpart, CiMex3B also contains one C-terminal RING domain and two N-terminal conserved tandem KH domains. CiMex3B up-regulates the expressions of IFN1, ISG15, MX2, as well as the expressions of inflammatory cytokines such as IL6, IL8 and TNFα in response to poly(I:C). A screening test for identifying potential targets indicated that CiMex3B is associated with TLR3 and TRIF. CiMex3B co-localizes with TLR3 in the late endosome, mitochondria and endoplasmic reticulum after poly(I:C) stimulation, whereas they are rarely discovered in the lysosomes. CiMex3B serves as a positive regulator in the phosphorylation of IRF3 and induces IFN1 expression. In addition, two truncation mutants of CiMex3B (1–220 and 221–525) were constructed to better understand the molecular mechanism of CiMex3B-mediated ubiquitination of TLR3. In line with wild-type protein, CiMex3B mutant (1–220) was found mainly in the cytoplasm; however, CiMex3B mutant (221–525) resided in the cytoplasm and the nucleus as well, and it was further confirmed that CiMex3B mutant (221–525) still interacts with TLR3. We also observed that CiMex3B promotes the K63-linked ubiquitination of TLR3, while neither of the truncation mutants (1–220 or 221–525) retains this activity. To sum up, this study revealed that CiMex3B potentiates the K63-linked ubiquitination of TLR3, and then elicits the IRF3-mediated antiviral innate immune responses.