Search

CASK, a MAGUK family scaffold protein, regulates gene expression as a transcription co-activator in neurons. However, the mechanism of CASK nucleus translocation and the regulatory function of CASK in myeloid cells remains unclear. Here, we investigated its role in H5N1-infected macrophages. We found that H5N1 triggers CASK nuclear translocation via PKR and SRC signaling. HCK, a SRC family kinase, enhances CASK phosphorylation at S395 via CDK5, facilitating CASK's nuclear entry. Knocking out CASK in myeloid cells specifically reduces interferon-alpha (IFNA) production by hindering the nuclear export of Ifna mRNA, while leaving its mRNA levels unchanged. Myeloid-specific CASK knockout (KO) mice display exacerbated lung inflammation, which correlates with reduced IFNA levels during H5N1 infection. Interactome studies show that H5N1 triggers associations between CASK and CCT4, STIP1, and TNK1. These associations recruit IRF7, POLR2C, TAF15, HNRNPs, and CRM1, enabling the CASK complex to bind to the Ifna promoter, bind co-transcriptionally to Ifna mRNA, and facilitate CRM1-dependent Ifna mRNA export. This underscores CASK's critical role in the antiviral response. [ABSTRACT FROM AUTHOR]

During intestinal and liver invasion by the protozoan parasite Entamoeba histolytica , extensive tissue destruction linked to large neutrophil infiltrates is observed. It has been proposed that microbicidal components of neutrophils are responsible for the damage, however, the mechanism by which they are released and act in the extracellular space remains unknown. In previous studies, we have shown that E. histolytica trophozoites induce NET formation, leading to the release of neutrophil granule content into extruded DNA. In this work, we evaluate the possible participation of NETs in the development of amoeba-associated pathology and analyze the contribution of anti-microbial components of the associated granules. E. histolytica- induced NETs were isolated and their effect on the viability and integrity of HCT 116 colonic and Hep G2 liver cultures were evaluated. The results showed that simple incubation of cell monolayers with purified NETs for 24 h resulted in cell detachment and death in a dose-dependent manner. The effect was thermolabile and correlated with the amount of DNA and protein present in NETs. Pretreatment of NETs with specific inhibitors of some microbicidal components suggested that serine proteases, are mostly responsible for the damage caused by NETs on HCT 116 cells, while the MPO activity was the most related to Hep G2 cells damage. Our study also points to a very important role of DNA as a scaffold for the activity of these proteins. We show evidence of the development of NETs in amoebic liver abscesses in hamsters as a preamble to evaluate their participation in tissue damage. In conclusion, these studies demonstrate that amoebic-induced NETs have potent cytotoxic effects on target cells and, therefore, may be responsible for the intense damage associated with tissue invasion by this parasite. [ABSTRACT FROM AUTHOR]

Introduction: The TrkB receptor is known for its role in regulating excitatory neuronal plasticity. However, accumulating evidence over the past decade has highlighted the involvement of TrkB in regulating inhibitory synapse stability and plasticity, particularly through regulation of the inhibitory scaffold protein gephyrin, although with contradicting results. Methods: In this study, we extended on these findings by overexpressing rat TrkB mutants deficient in either Shc-or PLCγ-dependent signaling, as well as a kinase-dead mutant, to dissect the contributions of specific TrkB-dependent signaling pathways to gephyrin clustering. Results: Our results demonstrate that TrkB signaling is required for gephyrin clustering on the perisomatic area of granule cells in the dentate gyrus in vivo. To further investigate, we expressed TrkB wild-type and mutants in hippocampal neurons in vitro. Discussion: Under basal conditions, TrkB-Shc signaling was important for the reduction of gephyrin cluster size, while TrkB-PLCγ signaling accounts for gephyrin clustering specifically at synaptic sites. Concomitant, impaired PLCγ signaling was associated with disinhibition of transduced neurons. Moreover, chemically induced inhibitory long-term potentiation (chem iLTP) depended on TrkB signaling and the activation of both Shc and PLCγ pathways. Conclusion: Our findings suggest a complex, pathway-specific regulation of TrkB-dependent gephyrin clustering, both under basal conditions and during chem iLTP. [ABSTRACT FROM AUTHOR]

Our knowledge of the assembly and dynamics of the cytokinetic contractile ring (CR) in animal cells remains incomplete. We have previously used super-resolution light microscopy and platinum replica electron microscopy to elucidate the ultrastructural organization of the CR in first division sea urchin embryos. To date, our studies indicate that the CR initiates as an equatorial band of clusters containing myosin II, actin, septin and anillin, which then congress over time into patches which coalesce into a linear array characteristic of mature CRs. In the present study, we applied super-resolution interferometric photoactivated localization microscopy to confirm the existence of septin filament-like structures in the developing CR, demonstrate the close associations between septin2, anillin, and myosin II in the CR, as well as to show that septin2 appears consistently submembranous, whereas anillin is more widely distributed in the early CR. We also provide evidence that the major actin cross-linking protein α-actinin only associates with the linearized, late-stage CR and not with the early CR clusters, providing further support to the idea that α-actinin associates with actomyosin structures under tension and can serve as a counterbalance. In addition, we show that inhibition of actomyosin contraction does not stop the assembly of the early CR clusters but does arrest the progression of these structures to the aligned arrays required for functional cytokinesis. Taken together our results reinforce and extend our model for a cluster to patch to linear structural progression of the CR in sea urchin embryos and highlight the evolutionary relationships with cytokinesis in fission yeast. [ABSTRACT FROM AUTHOR]

Understanding the interactions between fungal plant pathogens and host roots is crucial for developing effective disease management strategies. This study investigates the molecular mechanisms underpinning the chemotropic responses of the maize anthracnose fungus Colletotrichum graminicola to maize root exudates. Combining the generation of a deletion mutant with monitoring of disease symptom development and detailed analysis of chemotropic growth using a 3D-printed device, we identify the 7-transmembrane G-protein coupled receptor (GPCR) CgSte3 as a key player in sensing both plant-derived class III peroxidases and diterpenoids. Activation of CgSte3 initiates signaling through CgSo, a homolog to the Cell Wall Integrity Mitogen-Activated Protein Kinase (CWI MAPK) pathway scaffold protein identified in other filamentous fungi, facilitating the pathogen's growth towards plant defense molecules. The NADPH oxidase CgNox2 is crucial for peroxidase sensing but not for diterpenoid detection. These findings reveal that CgSte3 and CWI MAPK pathways are central to C. graminicola's ability to hijack plant defense signals, highlighting potential targets for controlling maize anthracnose. [ABSTRACT FROM AUTHOR]

Our knowledge of the assembly and dynamics of the cytokinetic contractile ring (CR) in animal cells remains incomplete. We have previously used superresolution light microscopy and platinum replica electron microscopy to elucidate the ultrastructural organization of the CR in first division sea urchin embryos. To date, our studies indicate that the CR initiates as an equatorial band of clusters containing myosin II, actin, septin and anillin, which then congress over time into patches which coalesce into a linear array characteristic of mature CRs. In the present study, we applied super-resolution interferometric photoactivated localization microscopy to confirm the existence of septin filament-like structures in the developing CR, demonstrate the close associations between septin2, anillin, and myosin II in the CR, as well as to show that septin2 appears consistently submembranous, whereas anillin is more widely distributed in the early CR. We also provide evidence that the major actin cross-linking protein a-actinin only associates with the linearized, late-stage CR and not with the early CR clusters, providing further support to the idea that a-actinin associates with actomyosin structures under tension and can serve as a counterbalance. In addition, we show that inhibition of actomyosin contraction does not stop the assembly of the early CR clusters but does arrest the progression of these structures to the aligned arrays required for functional cytokinesis. Taken together our results reinforce and extend our model for a cluster to patch to linear structural progression of the CR in sea urchin embryos and highlight the evolutionary relationships with cytokinesis in fission yeast. [ABSTRACT FROM AUTHOR]

The phospholipid bilayer nanodiscs (LNDs), as a rapidly-developing tool in recent years, provide a natural bio-memebrane environment to maintain the native conformation and functions of membrane proteins as well as a versatile delivery vehicle for a variety of hydrophobic and hydrophilic drugs. We have seen unprecedented advantages of phospholipid bilayer nanodiscs in membrane protein structure characterization, biochemical and physiological studies of membrane proteins, membrane environment studies, drug discovery & development, and drug delivery. Many previous reviews have been mainly focused on the advantages of nanodiscs in membrane protein researches, but few have touched upon the importance and potential application of nanodiscs in pharmaceutical industries. This review will provide general description of the structural characteristics, advantages, classification, and applications of phospholipid nanodiscs, with particular focus on nanodisc-enabled membrane protein drug discovery & development as well as drug delivery. [ABSTRACT FROM AUTHOR]

Nuclear envelope repair is a fundamental cellular response to stress, especially for cells experiencing frequent nuclear ruptures, such as cancer cells. Moreover, for chromosomally unstable cancer cells, characterized by the presence of micronuclei, the irreversible rupture of these structures constitutes a fundamental step toward cancer progression and therapy resistance. For these reasons, the study of nuclear envelope rupture and repair is of paramount importance. Nonetheless, due to the constraint imposed by the stochastic nature of rupture events, a precise characterization of the initial stage of nuclear repair remains elusive. In this study, we overcame this limitation by developing a new imaging pipeline that deterministically induces rupture while simultaneously imaging fluorescently tagged repair proteins. We provide a detailed step-by-step protocol to implement this method on any confocal microscope and applied it to study the major nuclear repair protein, barrier-to-autointegration factor (BAF). As a proof of principle, we demonstrated two different downstream analysis methods and showed how BAF is differentially recruited at sites of primary and micronuclear rupture. Additionally, we applied this method to study the recruitment at primary nuclei of the inner nuclear membrane protein LEM-domain 2 (LEMD2) and Charged Multivesicular Protein 7 (CHMP7), the scaffolding protein of the endosomal sorting complex required for transport III (ESCRT-III) membrane remodeling complex. The CHMP7-LEMD2 binding is the fundamental step allowing the recruitment of ESCRT-III, which represents the other major nuclear repair mechanism. This demonstrates the method's applicability for investigating protein dynamics at sites of nuclear and micronuclear envelope rupture and paves the way to more time-resolved studies of nuclear envelope repair. [ABSTRACT FROM AUTHOR]

The ability of skeletal muscles to contract is derived from the unique genes and proteins expressed within muscles, most notably myofilaments and elastic proteins. Here we investigated the role of the sallimus (sls) gene, which encodes a structural homologue of titin, in regulating development, structure, and function of Drosophila melanogaster. Knockdown of sls using RNA interference (RNAi) in all body-wall muscle fibers resulted in embryonic lethality. A screen for muscle-specific drivers revealed a Gal4 line that expresses in a single larval body wall muscle in each abdominal hemisegment. Disrupting sls expression in single muscle fibers did not impact egg or larval viability nor gross larval morphology but did significantly alter the morphology of individual muscle fibers. Ultrastructural analysis of individual muscles revealed significant changes in organization. Surprisingly, muscle-cell specific disruption of sls also severely impacted neuromuscular junction (NMJ) formation. The extent of motor-neuron (MN) innervation along disrupted muscles was significantly reduced along with the number of glutamatergic boutons, in MNIs and MN-Ib. Electrophysiological recordings revealed a 40% reduction in excitatory junctional potentials correlating with the extent of motor neuron loss. Analysis of active zone (AZ) composition revealed changes in presynaptic scaffolding protein (brp) abundance, but no changes in postsynaptic glutamate receptors. Ultrastructural changes in muscle and NMJ development at these single muscle fibers were sufficient to lead to observable changes in neuromuscular transduction and ultimately, locomotory behavior. Collectively, the data demonstrate that sls mediates critical aspects of muscle and NMJ development and function, illuminating greater roles for sls/titin. [ABSTRACT FROM AUTHOR]

Introduction: Lipopolysaccharide-responsive and beige-liκe anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PκA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. Aim: To compare and evaluate spleen-derived B cell responses to BCR crosslinκing in C57BL6 Lrba-/- and Lrba+/+ mice. Materials and methods: Spleen-derived B cells were obtained from 8 to 12-weeκ-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinκing was assessed by the F(ab')2 anti-m chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Results: Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinκing in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBa, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinκing in Lrba-/- B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. Discussion: These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is notoriously known for evolving strategies to modulate IFN signaling. Despite lots of efforts, the underlying mechanisms have remained incompletely understood. This study concerns the regulatory role of viral inner membrane protein p17. We found that the ASFV p17 shows a preferential interaction with cGAS-STING-IRF3 pathway, but not the RIG-IMAVS-NF-κB signaling, and can inhibit both poly(I:C)- and poly(A:T)-induced activation of IRF3, leading to attenuation of IFN-β induction. Mechanistically, p17 interacts with STING and IRF3 and recruits host scaffold protein PR65A, a subunit of cellular phosphatase PP2A, to down-regulate the level of p-IRF3. Also, p17 targets STING for partial degradation via induction of cellular apoptosis that consequently inhibits activation of both p-TBK1 and p-IRF3. Thus, our findings reveal novel regulatory mechanisms for p17 modulation of IFN signaling and shed light on the intricate interplay between ASFV proteins and host immunity. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid-beta (Aß) plaques and neurofibrillary Tau tangles in the brain. We previously identified a set of candidate AD microRNAs (miRNAs) in human cerebrospinal fluid (CSF) and used a target prediction pipeline to identify mRNAs and pathways that could potentially be regulated by the miRNAs. Of these pathways, clathrin mediated endocytosis (CME) was selected for further investigation. CME is altered in multiple brain cell types in AD and is implicated in early cellular phenotypes such as enlarged early endosomes and pathogenic processing of Aß. However, a comprehensive evaluation of major CME hub proteins in humans with AD across multiple brain regions is lacking. Thus, we used immunoblots to evaluate human postmortem AD and control (CTL) frontal cortex (FC; AD n = 22, CTL n = 23) and hippocampus (HP; AD n = 34, CTL n = 22) for changes in Intersectin 1 (ITSN1), Phosphatidylinositol Binding Clathrin Assembly Protein gene (PICALM), Clathrin Light Chain (CLT), FCH and Mu Domain Containing Endocytic Adaptor 1 (FCHO1), Adaptor Related Protein Complex 2 (AP2) Subunit Alpha 1 (AP2A1), and Dynamin 2 (DNM2). Of these, we found that in AD, ITSN1-long (ITSN1-L) was decreased in the FC of males and HP of females, while ITSN1-short was increased in the HP of both males and females. We further evaluated ITSN1-L levels in cortex (CTX) and HP of the 5xFAD mouse model of Aß pathology at different timepoints during aging and disease progression by immunoblot (n = 5-8 per group). At 3 months, female 5xFAD exhibited an increase of ITSN1-L in CTX but a decrease at 6 and 9 months. Additionally, immunofluorescent staining of 5xFAD primary HP neurons showed an increase of ITSN1-L in matured 5xFAD neurons at 21 and 28 days in vitro. Together, our studies show that in AD, isoforms of ITSN1 change in a brain region-and sex-dependent manner. Further, changes in ITSN1-L are transient with levels increasing during early Aß accumulation and decreasing during later progression. These findings suggest that ITSN1 expression, and consequently CME activity, may change depending on the stage of disease progression. [ABSTRACT FROM AUTHOR]

CARD-BCL10-MALT1 (CBM) signalosomes connect distal signaling of innate and adaptive immune receptors to proximal signaling pathways and immune activation. Four CARD scaffold proteins (CARD9, 10, 11, 14) can form seeds that nucleate the assembly of BCL10-MALT1 filaments in a cell- and stimulus-specific manner. MALT1 (also known as PCASP1) serves a dual function within the assembled CBM complexes. By recruiting TRAF6, MALT1 acts as a molecular scaffold that initiates IkB kinase (IKK)/NF-kB and c-Jun N-terminal kinase (JNK)/AP-1 signaling. In parallel, proximity-induced dimerization of the paracaspase domain activates the MALT1 protease which exerts its function by cleaving a set of specific substrates. While complete MALT1 ablation leads to immune deficiency, selective destruction of either scaffolding or protease function provokes autoimmune inflammation. Thus, balanced MALT1-TRAF6 recruitment and MALT1 substrate cleavage are critical to maintain immune homeostasis and to promote optimal immune activation. Further, MALT1 protease activity drives the survival of aggressive lymphomas and other nonhematologic solid cancers. However, little is known about the relevance of the cleavage of individual substrates for the pathophysiological functions of MALT1. Unbiased serendipity, screening and computational predictions have identified and validated ~20 substrates, indicating that MALT1 targets a quite distinct set of proteins. Known substrates are involved in CBM auto-regulation (MALT1, BCL10 and CARD10), regulation of signaling and adhesion (A20, CYLD, HOIL-1 and Tensin-3), or transcription (RelB) and mRNA stability/translation (Regnase-1, Roquin-1/2 and N4BP1), indicating that MALT1 often targets multiple proteins involved in similar cellular processes. Here, we will summarize what is known about the fate and functions of individual MALT1 substrates and how their cleavage contributes to the biological functions of the MALT1 protease. We will outline what is needed to better connect critical pathophysiological roles of the MALT1 protease with the cleavage of distinct substrates. [ABSTRACT FROM AUTHOR]

Introduction: Disrupted in schizophrenia-1 (DISC1) is a scaffolding protein whose mutated form has been linked to schizophrenia, bipolar affective disorders, and recurrent major depression. DISC1 regulates multiple signaling pathways involved in neurite outgrowth and cortical development and binds directly to glycogen synthase kinase-3β (GSK-3β). Since ketamine activates GSK-3β, we examined the impact of ketamine on DISC1 and GSK-3β expression. Methods: Postnatal day 7 rat pups were treated with ketamine with and without the non-specific GSK-3β antagonist, lithium. Cleaved-caspase-3, GSK-3β and DISC1 levels were measured by immunoblots and DISC1 co-localization in neurons by immunofluorescence. Binding of DISC1 to GSK-3β was determined by co-immunoprecipitation. Neurite outgrowth was determined by measuring dendrite and axon length in primary neuronal cell cultures treated with ketamine and lithium. Results: Ketamine decreased DISC1 in a dose and time-dependent manner. This corresponded to decreases in phosphorylated GSK-3β, which implicates increased GSK-3β activity. Lithium significantly attenuated ketamine-induced decrease in DISC1 levels. Ketamine decreased co-immunoprecipitation of DISC1 with GSK-3β and axonal length. Conclusion: These findings confirmed that acute administration of ketamine decreases in DISC1 levels and axonal growth. Lithium reversed this effect. This interaction provides a link between DISC1 and ketamine-induced neurodegeneration. [ABSTRACT FROM AUTHOR]

Introduction: Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However, its specific role in periodontium development remains less understood. This study aims to elucidate the expression pattern and function of PHB in periodontium development and its involvement in alveolar bone formation. Methods: Immunolocalization of PHB in the periodontium of postnatal (PN) mice were examined. Phb morpholino wasmicro-injected into the right-side mandible at PN5, corresponding to the positionwhere the alveolar bone process forms in relation to the lower firstmolar. The micro-injectionwith a scramble control (PF-127) and the left-side mandibles were used as control groups. Five days post-micro-injection, immunohistochemical analysis and micro-CT evaluation were conducted to assess bone mass andmorphological changes. Additionally, expression patterns of signaling molecules were examined following Phb downregulation using 24-h in vitro cultivation of developing dental mesenchyme at E14.5. Results: The immunostaining of PHB showed its localization in the periodontium at PN5, PN8, and PN10. The in vitro cultivation of dental mesenchyme resulted in alterations in Bmps, Runx2, and Wnt signalings after Phb knock-down. At 5 days post-micro-injection, Phb knocking down showed weak immunolocalizations of runt-related transcription factor (RUNX2) and osteocalcin (OCN). However, knocking down Phb led to histological alterations characterized by decreased bone mass and stronger localizations of Ki67 and PERIOSTIN in the periodontium compared 1 to control groups. The micro-CT evaluation showed decreased bone volume and increased PDL space in the Phb knock-down specimens, suggesting its regulatory role in bone formation. Discussion: The region-specific localization of PHB in the margin where alveolar bone forms suggests its involvement in alveolar bone formation and the differentiation of the periodontal ligament. Overall, our findings suggest that Phb plays amodulatory role in alveolar bone formation by harmoniously regulating boneforming-related signaling molecules during periodontium development. [ABSTRACT FROM AUTHOR]

Introduction: CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-kB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg). Method: In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-kB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-kB independent manner. Results and discuss: We found CARD11mutations causing hyper-activated NF-kB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-kB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-kB independent pathway for thymic Treg lineage commitment. [ABSTRACT FROM AUTHOR]

Deleterious variants of more than one hundred genes are associated with hearing loss including MYO3A, MYO6, MY07A and MYO15A and two conventional myosins MYH9 and MYH14. Variants of also manifest as Usher syndrome associated with dysfunction of the retina and vestibule as well as hearing loss. While the functions of MYH9 and MYH14 in the inner ear are debated, MYO3A, MYO6, MYO7A and MYO15A are expressed in inner ear hair cells along with class-I myosin MYO1C and are essential for developing and maintaining functional stereocilia on the apical surface of hair cells. Stereocilia are large, cylindrical, actin-rich protrusions functioning as biological mechanosensors to detect sound, acceleration and posture. The rigidity of stereocilia is sustained by highly crosslinked unidirectionally- oriented F-actin, which also provides a scaffold for various proteins including unconventional myosins and their cargo. Typical myosin molecules consist of an ATPase head motor domain to transmit forces to F-actin, a neck containing IQ-motifs that bind regulatory light chains and a tail region with motifs recognizing partners. Instead of long coiled-coil domains characterizing conventional myosins, the tails of unconventional myosins have various motifs to anchor or transport proteins and phospholipids along the F-actin core of a stereocilium. For these myosins, decades of studies have elucidated their biochemical properties, interacting partners in hair cells and variants associated with hearing loss. However, less is known about how myosins traffic in a stereocilium using their motor function, and how each variant correlates with a clinical condition including the severity and onset of hearing loss, mode of inheritance and presence of symptoms other than hearing loss. Here, we cover the domain structures and functions of myosins associated with hearing loss together with advances, open questions about trafficking ofmyosins in stereocilia and correlations between hundreds of variants in myosins annotated in ClinVar and the corresponding deafness phenotypes. [ABSTRACT FROM AUTHOR]

High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20-40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1). [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is the most common type of lung tumor; however, we lack effective early detection indicators and therapeutic targets. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is vital to initiate protein synthesis, acting as a scaffolding protein for the eukaryotic protein translation initiation factor complex, EIF4F, which regulates protein synthesis together with EIF4A, EIF4E, and other translation initiation factors. However, EIF4G1's function in NSCLC cancer is unclear. Herein, transcriptome sequencing showed that knockdown of EIF4G1 in H1299 NSCLC cells upregulated the expression of various inflammation-related factors. Inflammatory cytokines were also significantly overexpressed in NSCLC tumor tissues, among which CXCL8 (encoding C-X-C motif chemokine ligand 8) showed the most significant changes in both in the transcriptome sequencing data and tumor tissues. We revealed that EIF4G1 regulates the protein level of TNF receptor superfamily member 10a (TNFRSF10A) resulting in activation of the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NFB) pathways, which induces CXCL8 secretion, leading to targeted chemotaxis of immune cells. We verified that H1299 cells with EIF4G1 knockdown showed increased chemotaxis compared with the control group and promoted increased chemotaxis of macrophages. These data suggested that EIF4G1 is an important molecule in the inflammatory response of cancer tissues in NSCLC. [ABSTRACT FROM AUTHOR]

Background and aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibroinflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2. Methods: Anti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays. Results: Anti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDCinduced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx. Conclusions: A subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated. [ABSTRACT FROM AUTHOR]

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels. [ABSTRACT FROM AUTHOR]

Biologics developers are moving beyond antibodies for delivery of a wide range of therapeutic interventions. These non-antibody modalities are often based on ‘natural’ protein scaffolds that are modified to deliver bioactive sequences. Both human-derived and non-human-sourced scaffold proteins have been developed. New types of “non-antibody” scaffolds are still being discovered, as they offer attractive alternatives to monoclonals due to their smaller size, improved stability, and ease of synthesis. They are believed to have low immunogenic potential. However, while several human-sourced protein scaffolds have not been immunogenic in clinical studies, this may not predict their overall performance in other therapeutic applications. A preliminary evaluation of their potential for immunogenicity is warranted. Immunogenicity risk potential has been clearly linked to the presence of T “helper” epitopes in the sequence of biologic therapeutics. In addition, tolerogenic epitopes are present in some human proteins and may decrease their immunogenic potential. While the detailed sequences of many non-antibody scaffold therapeutic candidates remain unpublished, their backbone sequences are available for review and analysis. We assessed 12 example non-antibody scaffold backbone sequences using our epitope-mapping tools (EpiMatrix) for this perspective. Based on EpiMatrix scoring, their HLA DRB1-restricted T cell epitope content appears to be lower than the average protein, and sequences that may act as tolerogenic epitopes are present in selected human-derived scaffolds. Assessing the potential immunogenicity of scaffold proteins regarding self and non-self T cell epitopes may be of use for drug developers and clinicians, as these exciting new non-antibody molecules begin to emerge from the preclinical pipeline into clinical use. [ABSTRACT FROM AUTHOR]

Receptor Interacting Protein Kinase 1 (RIPK1) and caspase-8 (Casp8) jointly orchestrate apoptosis, a key mechanism for eliminating developing T cells which have autoreactive or improperly arranged T cell receptors. Mutations in the scaffolding domain of Ripk1 gene have been identified in humans with autoinflammatory diseases like Cleavage Resistant RIPK1 Induced Autoinflammatory (CRIA) and Inflammatory Bowel Disease. RIPK1 protein also contributes to conventional T cell differentiation and peripheral T cell homeostasis through its scaffolding domain in a cell death independent context. Ripk1 deficient mice do not survive beyond birth, so we have studied the function of this kinase in vivo against a backdrop Ripk3 and Casp8 deficiency which allows the mice to survive to adulthood. These studies reveal a key role for RIPK1 in mediating NK1.1 expression, including on thymic iNKT cells, which is a key requirement for thymic stage 2 to stage 3 transition as well as iNKT cell precursor development. These results are consistent with RIPK1 mediating responses to TcR engagement, which influence NK1.1 expression and iNKT cell thymic development. We also used in vivo and in vitro stimulation assays to confirm a role for both Casp8 and RIPK1 in mediating iNKT cytokine effector responses. Finally, we also noted expanded and hyperactivated iNKT follicular helper (iNKTFH) cells in both DKO (Casp8-, Ripk3- deficient) and TKO mice (Ripk1-, Casp8-, Ripk3- deficient). Thus, while RIPK1 and Casp8 jointly facilitate iNKT effector function, RIPK1 uniquely influenced thymic iNKT cell development most likely by regulating molecular responses to T cell receptor engagement. iNKT developmental and functional aberrances were not evident in mice expressing a kinase-dead version of RIPK1 (RIPK1kd), indicating that the scaffolding function of this protein exerts the critical regulation of iNKT cells. Our findings suggest that small molecule inhibitors of RIPK1 could be used to regulate iNKT cell development and effector function to alleviate autoinflammatory conditions in humans. [ABSTRACT FROM AUTHOR]

Introduction: Oligodendrocyte progenitor cells (OPCs) are vital for neuronal myelination and remyelination in the central nervous system. While the molecular mechanisms involved in OPCs' differentiation and maturation are not completely understood, GABA is known to positively influence these processes through the activation of GABAA receptors (GABAARs). The molecular identity of GABAARs expressed in human OPCs remains unknown, which restricts their specific pharmacological modulation to directly assess their role in oligodendrocytes' maturation and remyelination. Methods: In this study, we conducted a transcriptomic analysis to investigate the molecular stoichiometry of GABAARs in OPCs from the human brain. Using eight available transcriptomic datasets from the human brain cortex of control individuals, we analyzed the mRNA expression of all 19 known GABAARs subunit genes in OPCs, with variations observed across different ages. Results: Our analysis indicated that the most expressed subunits in OPCs are a1-3, ß1-3, α1-3, and e. Moreover, we determined that the combination of any a with ß2 and γ2 is likely to form heteropentameric GABAARs in OPCs. Importantly, we also found a strong correlation between GABAAR subunits and transcripts for postsynaptic scaffold proteins, suggesting the potential postsynaptic clustering of GABAARs in OPCs. Discussion: This study presents the first transcriptional-level identification of GABAAR subunits expressed in human OPCs, providing potential receptor combinations. Understanding the molecular composition of GABAARs in OPCs not only enhances our knowledge of the underlying mechanisms in oligodendrocyte maturation but also opens avenues for targeted pharmacological interventions aimed at modulating these receptors to promote remyelination in neurological disorders. [ABSTRACT FROM AUTHOR]

Postsynaptic neurotransmitter receptors and their associated scaffolding proteins assemble into discrete, nanometer-scale subsynaptic domains (SSDs) within the postsynaptic membrane at both excitatory and inhibitory synapses. Intriguingly, postsynaptic receptor SSDs are mirrored by closely apposed presynaptic active zones. These trans-synaptic molecular assemblies are thought to be important for efficient neurotransmission because they concentrate postsynaptic receptors near sites of presynaptic neurotransmitter release. While previous studies have characterized the role of synaptic activity in sculpting the number, size, and distribution of postsynaptic SSDs at established synapses, it remains unknown whether neurotransmitter signaling is required for their initial assembly during synapse development. Here, we evaluated synaptic nano-architecture under conditions where presynaptic neurotransmitter release was blocked prior to, and throughout synaptogenesis with tetanus neurotoxin (TeNT). In agreement with previous work, neurotransmitter release was not required for the formation of excitatory or inhibitory synapses. The overall size of the postsynaptic specialization at both excitatory and inhibitory synapses was reduced at chronically silenced synapses. However, both AMPARs and GABAARs still coalesced into SSDs, along with their respective scaffold proteins. Presynaptic active zone assemblies, defined by RIM1, were smaller and more numerous at silenced synapses, but maintained alignment with postsynaptic AMPAR SSDs. Thus, basic features of synaptic nano-architecture, including assembly of receptors and scaffolds into trans-synaptically aligned structures, are intrinsic properties that can be further regulated by subsequent activity-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Introduction: An altered neurodevelopmental trajectory associated with prenatal exposure to ?-9-tetrahydrocannabinol (THC) leads to aberrant cognitive processing through a perturbation in the effectors of hippocampal plasticity in the juvenile offspring. As adolescence presents a unique window of opportunity for "brain reprogramming", we aimed at assessing the role of the nonpsychoactive phytocannabinoid cannabidiol (CBD) as a rescue strategy to temper prenatal THC-induced harm. Methods: To this aim, Wistar rats prenatally exposed to THC (2 mg/kg s.c.) or vehicle (gestational days 5-20) were tested for specific indexes of spatial and configural memory in the reinforcement-motivated Can test and in the aversiondriven Barnes maze test during adolescence. Markers of hippocampal excitatory plasticity and endocannabinoid signaling--NMDAR subunits NR1 and 2A-, mGluR5-, and their respective scaffold proteins PSD95- and Homer 1-; CB1Rand the neuromodulatory protein HINT1 mRNA levels were evaluated. CBD (40 mg/kg i.p.) was administered to the adolescent offspring before the cognitive tasks. Results: The present results show that prenatal THC impairs hippocampal memory functions and the underlying synaptic plasticity; CBD is able to mitigate cognitive impairment in both reinforcement- and aversion-related tasks and the neuroadaptation of hippocampal excitatory synapses and CB1Rrelated signaling. Discussion: While this research shows CBD potential in dampening prenatal THCinduced consequences, we point out the urgency to curb cannabis use during pregnancy in order to avoid detrimental bio-behavioral outcomes in the offspring. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal (AM) fungi and rhizobia form two of the most important plant-microbe associations for the assimilation of phosphorus (P) and nitrogen (N). Symbiont-derived signals are able to coordinate the infection process by triggering multiple responses in the plant root, such as calcium influxes and oscillations, increased reactive oxygen species (ROS), cytoskeletal rearrangements and altered gene expression. An examination was made of the role of tetraspanins, which are transmembrane proteins that self-organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking, and ROS generation. In plant cells, tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, plasmodesmata and hormonal regulation. Some plant tetraspanins, such as Arabidopsis thaliana TETRASPANIN 8 and TETRASPANIN 9 (AtTET8 and AtTET9) are associated with exosomes during inter-kingdom communication. In this study, a homolog of AtTET8, PvTET8-1, in common bean (Phaseolus vulgaris L. var. Negro Jamapa) was examined in roots during interactions with Rhizobium tropici and Rhizophagus irregularis. The promoter of PvTET8-1 contained several cis-acting regulatory DNA elements potentially related to mutualistic interactions, and PvTET8-1 was transcriptionally activated during AM fungal and rhizobial associations. Silencing it decreased the size and number of nodules, nitrogen fixation, and mycorrhizal arbuscule formation, whereas overexpressing it increased the size and number of nodules, and mycorrhizal arbuscule formation but decreased nitrogen fixation. PvTET8-1 appears to be an important element in both of these mutualistic interactions, perhaps through its interaction with NADPH oxidase and the generation of ROS during the infection processes. [ABSTRACT FROM AUTHOR]

There are many special sites at the end of a synapse called active zones (AZs). Synaptic vesicles (SVs) fuse with presynaptic membranes at these sites, and this fusion is an important step in neurotransmitter release. The cytomatrix in the active zone (CAZ) is made up of proteins such as the regulating synaptic membrane exocytosis protein (RIM), RIM-binding proteins (RIM-BPs), ELKS/CAST, Bassoon/Piccolo, Liprin-α, and Munc13-1. RIM is a scaffold protein that interacts with CAZ proteins and presynaptic functional components to affect the docking, priming, and fusion of SVs. RIM is believed to play an important role in regulating the release of neurotransmitters (NTs). In addition, abnormal expression of RIM has been detected in many diseases, such as retinal diseases, Asperger’s syndrome (AS), and degenerative scoliosis. Therefore, we believe that studying the molecular structure of RIM and its role in neurotransmitter release will help to clarify the molecular mechanism of neurotransmitter release and identify targets for the diagnosis and treatment of the aforementioned diseases. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by a BCR-ABL fusion gene. Imatinib has significantly improved the treatment of CML as a first-generation tyrosine kinase inhibitor (TKIs). The T315I mutant form of BCRABL is the most common mutation that confers resistance to imatinib or the second-generation TKIs, resulting in poor clinical prognosis. In this work, we assessed the effect of a potent histone deacetylase (HDAC) inhibitor, I13, on the differentiation blockade in CML cells harboring T315I-mutated and wild-type BCR-ABL by MTT assay, flow cytometery, cell colony formation assay, mRNA Sequencing, Quantitative real-time PCR and Western blotting analysis. We found that I13 possessed highly potent activity against T315I-mutated BCR-ABL mutant-expressing cells and wild-type BCR-ABL-expressing cells. I13 induced cell differentiation and significantly suppressed the proliferation of these CML cells via the cell cycle G0/G1-phase accumulation. Moreover, it was revealed that I13 triggered the differentiation of BaF3-T315I cells, which was attributed to the block of the chronic myeloid leukemia signaling pathway via the depletion of BCRABL that was mediated by the inhibition of HDAC activity presented by the acetylation of histones H3 and H4. Taken together, I13 efficiently depleted BCR-ABL in CML cells expressing the BCR-ABL-T315I mutation, which blocked its function, serving as a scaffold protein that modulated the chronic myeloid leukemia signaling pathway mediating cell differentiation. The present findings demonstrate that I13 is a BCR-ABL modulator for the development of CML therapy that can override resistance caused by T315I-mutated BCR-ABL. [ABSTRACT FROM AUTHOR]

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B's inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites. [ABSTRACT FROM AUTHOR]

Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1–2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped selfgrooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments. [ABSTRACT FROM AUTHOR]

p62/Sequestosome-1 (SQSTM1) is a stress-inducible scaffold protein involved in multiple cellular processes, including apoptosis, inflammation, cell survival, and selective autophagy. SQSTM1 mutations are associated with a spectrum of multisystem proteinopathy, including Paget disease of the bone, amyotrophic lateral sclerosis, frontotemporal dementia, and distal myopathy with rimmed vacuoles (MRV). Herein, we report a new phenotype of SQSTM1-associated proteinopathy, a novel frameshift mutation in SQSTM1 causing proximal MRV. A 44-year-old Chinese patient presented with progressive limb-girdle weakness. She had asymmetric proximal limb weakness and myopathic features on electromyography. The magnetic resonance images showed fatty infiltration into muscles, predominantly in the thighs and medial gastrocnemius, sparing the tibialis anterior. Muscle histopathology revealed abnormal protein deposition, p62/SQSTM1-positive inclusions, and rimmed vacuoles. Next-generation sequencing showed a novel pathogenic SQSTM1 frameshift mutation, c.542_549delACAGCCGC (p. H181Lfs-66). We expanded the pathogenic genotype of SQSTM1 to include a new, related phenotype: proximal MRV. We suggest that SQSTM1 variations should be screened in cases of proximal MRV. [ABSTRACT FROM AUTHOR]

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, "spontaneous" transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the nonspecific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses. [ABSTRACT FROM AUTHOR]

TBK1 and IKKЄ are related, crucial kinases in antiviral immune signaling pathways downstream of cytosolic nucleic acid receptors such as cGAS and RIG-I-like receptors. Upon activation, they phosphorylate the transcription factors IRF3 and IRF7 and thereby initiate the expression of type I interferons and antiviral effectors. While point mutation-induced loss of TBK1 kinase activity results in clinical hyper-susceptibility to viral infections, a complete lack of TBK1 expression in humans is unexpectedly not associated with diminished antiviral responses. Here, we provide a mechanistic explanation for these so-far unexplained observations by showing that TBK1 controls the protein expression of its related kinase IKKЄ in human myeloid cells. Mechanistically, TBK1 constitutively diminishes the protein stability of IKKЄ independent of TBK1 kinase activity but dependent on its interaction with the scaffold protein TANK. In consequence, depletion of TBK1 protein but not mutation-induced kinase deficiency induces the upregulation of IKKЄ. Due to the functional redundancy of the kinases in cGAS-STING and RIG-I-like receptor signaling in human myeloid cells, enhanced IKKЄ expression can compensate for the loss of TBK1. We show that IKKЄ upregulation is crucial to ensure unmitigated type I interferon production in conditions of TBK1 deficiency: While the type I interferon response to Listeria monocytogenes infection is maintained upon TBK1 loss, it is strongly diminished in cells harboring a kinase-deficient TBK1 variant, in which IKKЄ is not upregulated. Many pathogens induce TBK1 degradation, suggesting that loss of TBK1-mediated destabilization of IKKЄ is a critical backup mechanism to prevent diminished interferon responses upon TBK1 depletion. [ABSTRACT FROM AUTHOR]

Introduction: Inflammation play important roles in the initiation and progression of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, clotting dysfunction, or even death associated with SARS-CoV-2 infection. However, the pathogenic mechanisms underlying SARS-CoV-2- induced hyperinflammation are still largely unknown. Methods: The animal model of septic shock and ALI was established after LPS intraperitoneal injection or intratracheal instillation. Bone marrow-derived macrophages (BMDMs) from WT and BPOZ-2 KO mouse strains were harvested from the femurs and tibias of mice. Immunohistology staining, ELISA assay, coimmunoprecipitation, and immunoblot analysis were used to detect the histopathological changes of lung tissues and the expression of inflammatory factors and protein interaction. Results and conclusions: We show a distinct mechanism by which the SARSCoV- 2 N (SARS-2-N) protein targets Bood POZ-containing gene type 2 (BPOZ-2), a scaffold protein for the E3 ubiquitin ligase Cullin 3 that we identified as a negative regulator of inflammatory responses, to promote NLRP3 inflammasome activation. We first demonstrated that BPOZ-2 knockout (BPOZ-2 KO) mice were more susceptible to lipopolysaccharide (LPS)-induced septic shock and ALI and showed increased serum IL-1b levels. In addition, BMDMs isolated from BPOZ-2 KO mice showed increased IL-1b production in response to NLRP3 stimuli. Mechanistically, BPOZ-2 interacted with NLRP3 and mediated its degradation by recruiting Cullin 3. In particular, the expression of BPOZ-2 was significantly reduced in lung tissues from mice infected with SARS-CoV-2 and in cells overexpressing SARS-2-N. Importantly, proinflammatory responses triggered by the SARS-2-N were significantly blocked by BPOZ-2 reintroduction. Thus, we concluded that BPOZ-2 is a negative regulator of the NLPR3 inflammasome that likely contributes to SARS-CoV-2-induced hyperinflammation. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) includes a group of multifactorial neurodevelopmental disorders characterized by impaired social communication, social interaction, and repetitive behaviors. Several studies have shown an association between cases of ASD and mutations in the genes of SH3 and multiple ankyrin repeat domain protein 3 (SHANK3). These genes encode many cell adhesion molecules, scaffold proteins, and proteins involved in synaptic transcription, protein synthesis, and degradation. They have a profound impact on all aspects of synaptic transmission and plasticity, including synapse formation and degeneration, suggesting that the pathogenesis of ASD may be partially attributable to synaptic dysfunction. In this review, we summarize the mechanism of synapses related to Shank3 in ASD. We also discuss the molecular, cellular, and functional studies of experimental models of ASD and current autism treatment methods targeting related proteins. [ABSTRACT FROM AUTHOR]

Macrophages (MФ), the primary cell of the innate immune system, serves as the first line of defense. During bacterial infection, Gram-negative (G-) bacteria release nanosized outer membrane vesicles (OMVs), facilitating the crosstalk between the microbe and the host. The underlying mechanisms by which OMVs induced proinflammatory (M1) activation are still unknown. Our study shows that OMVs caused M1 activation via modulating various toll-like receptor (TLR) expressions as they contain LPS, LTA, bacterial DNAs, and flagellins. Also, we found that caveolin-1 (cav-1), a 21-kDa scaffolding protein of caveolae and lipid rafts, plays a significant role in OMV-induced pro-inflammatory response in regulating various TLR signaling pathways. Specifically, cav-1 deletion increased the expression of OMV-induced TLRs, pro-inflammatory cytokine secretions (TNF-a and IL-1b), and the reactive oxygen species (ROS) production in MФs. Further, we examined the interaction between Cav-1 and TLR4 by immunoprecipitation, colocalization, and computational models, providing future direction to explore the role of cav-1 in OMV-induced other TLR signaling. Altogether, Cav-1 is a key regulator in OMVinduced multiple TLRs response. This study promotes future research to develop drugs by targeting the specific motif of cav-1 or TLRs against bacterial infection and macrophage-mediated inflammation. [ABSTRACT FROM AUTHOR]

Background: Shank3 is a scaffolding protein essential for the organization and function of the glutamatergic postsynapse. Monogenic mutations in SHANK3 gene are among the leading genetic causes of Autism Spectrum Disorders (ASD). The multiplicity of Shank3 isoforms seems to generate as much functional diversity and yet, there are no tools to study endogenous Shank3 proteins in an isoform-specific manner. Methods: In this study, we created a novel transgenic mouse line, the Shank3Venus/Venus knock in mouse, which allows to monitor the endogenous expression of the major Shank3 isoform in the brain, the full-length Shank3a isoform. Results: We show that the endogenous Venus-Shank3a protein is localized in spines and is mainly expressed in the striatum, hippocampus and cortex of the developing and adult brain. We show that Shank3Venus/+ and Shank3Venus/Venus mice have no behavioral deficiency. We further crossed Shank3Venus/Venus mice with Shank3ΔC/ΔC mice, a model of ASD, to track the Venus-tagged wild-type copy of Shank3a in physiological (Shank3Venus/+) and pathological (Shank3Venus/ΔC) conditions. We report a developmental delay in brain expression of the Venus-Shank3a isoform in Shank3Venus/ΔC mice, compared to Shank3Venus/+ control mice. Conclusion: Altogether, our results show that the Shank3Venus/Venus mouse line is a powerful tool to study endogenous Shank3a expression, in physiological conditions and in ASD. [ABSTRACT FROM AUTHOR]

Scaffold protein-mediated voltage-dependent ion channel clustering at unique membrane sites, such as nodes of Ranvier or the post-synaptic density plays an important role in determining action potential properties and information coding. Yet, the mechanism(s) by which scaffold protein-ion channel interactions lead to channel clustering and how cluster ion channel density is regulated are mostly unknown. This molecular-cellular gap in understanding channel clustering can be bridged in the case of the prototypical Shaker voltage-activated potassium channel (Kv), as the mechanism underlying the interaction of this channel with its PSD-95 scaffold protein partner is known. According to this mechanism, changes in the length of the intrinsically disordered channel C-terminal chain, brought about by alternative splicing to yield the short A and long B chain subunit variants, dictate affinity to PSD-95 and further controls cluster homo-tetrameric Kv channel density. These results raise the hypothesis that heteromeric subunit assembly serves as a means to regulate Kv channel clustering. Since both clustering variants are expressed in similar fly tissues, it is reasonable to assume that hetero-tetrameric channels carrying different numbers of high- (A) and low-affinity (B) subunits could assemble, thereby giving rise to distinct cluster Kv channel densities. Here, we tested this hypothesis using high-resolution microscopy, combined with quantitative clustering analysis. Our results reveal that the A and B clustering variants can indeed assemble to form heteromeric channels and that controlling the number of the high-affinity A subunits within the hetero-oligomer modulates cluster Kv channel density. The implications of these findings for electrical signaling are discussed.

Suppression of human cytomegalovirus (HCMV) major immediate early gene (IE) expression from the viral major immediate early promoter (MIEP) is known to be crucial for the establishment and maintenance of HCMV latency in myeloid progenitor cells and their undifferentiated derivatives. This suppression of the MIEP during latent infection is known to result from epigenetic histone modification imparting a repressive chromatin structure around the MIEP in undifferentiated myeloid cells. In contrast, reactivation, resulting from, e.g., myeloid cell differentiation, is associated with activatory chromatin marks around the MIEP. Recently, recruitment of the transcriptional repressor SETDB1, via KAP1, to latent HCMV genomes was shown to be involved in latency-associated MIEP suppression in CD34+ progenitor cells. KAP1 is also known to associate with Chromodomain-helicase-DNA-binding protein 3 (CHD3) as part of the NuRD complex which can aid transcriptional silencing. We now show that the cellular protein Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1), a known interactor of CHD3, is significantly upregulated during HCMV latency and that this protein is required for MIEP suppression during latent infection of myeloid cells. We further show that SERBP1 mediates CHD3 association with the MIEP as well as KAP1 association with viral genomic DNA. We suggest that SERBP1 functions as a scaffold protein to recruit transcriptional repressors to the latent viral genome and to mediate transcriptional silencing of the MIEP during latent carriage. [ABSTRACT FROM AUTHOR]

Although a large part of the genome is transcribed, only 1.9% has a proteincoding potential; most of the transcripts are non-coding RNAs such as snRNAs, tRNAs, and rRNAs that participate in mRNA processing and translation. In addition, there are small RNAs with a regulatory role, such as siRNAs, miRNAs, and piRNAs. Finally, the long non-coding RNAs (lncRNAs) are transcripts of more than 200 bp that can positively and negatively regulate gene expression (both in cis and trans), serve as a scaffold for protein recruitment, and control nuclear architecture, among other functions. An essential process regulated by lncRNAs is genome stability. LncRNAs regulate genes associated with DNA repair and chromosome segregation; they are also directly involved in the maintenance of telomeres and have recently been associated with the activity of the centromeres. In cancer, many alterations in lncRNAs have been found to promote genomic instability, which is a hallmark of cancer and is associated with resistance to chemotherapy. In this review, we analyze the most recent findings of lncRNA alterations in cancer, their relevance in genomic instability, and their impact on the resistance of tumor cells to anticancer therapy. [ABSTRACT FROM AUTHOR]

The Mercator projection map of the world provides a useful, but distorted, view of the relative scale of countries. Current cellular models suffer from a similar distortion. Here, we undertook an in-depth structural analysis of the molecular dimensions in the cell's computational machinery, the MeshCODE, that is assembled from a meshwork of binary switches in the scaffolding proteins talin and vinculin. Talin contains a series of force-dependent binary switches and each domain switching state introduces quantised step-changes in talin length on a micrometre scale. The average dendritic spine is 1 mm in diameter so this analysis identifies a plausible Gearbox-like mechanism for dynamic regulation of synaptic function, whereby the positioning of enzymes and substrates relative to each other, mechanically-encoded by the MeshCODE switch patterns, might control synaptic transmission. Based on biophysical rules and experimentally derived distances, this analysis yields a novel perspective on biological digital information. [ABSTRACT FROM AUTHOR]

Background: Plasma neurofilament light chain (pNFL) represents one of the scaffolding proteins of central nervous system axonal injury. The aim of this study was to evaluate pNFL as a predictive biomarker for early neurological deterioration (END) in medically managed patients with large vessel occlusion (LVO) and mild presentation (NIHSS < 6). Methods: This retrospective study was developed from a prospectively collected stroke database, which was conducted at a large academic comprehensive stroke center in western China. Patients who first presented with acute ischemic stroke (AIS) within 24 h of symptom onset were continuously included. Stroke severity was analyzed at admission using the NIHSS score. The pNFL drawn on admission was analyzed with a novel ultrasensitive single-molecule array. Results: Thirty-nine consecutive patients were included in the analysis, and 19 (48.72%) patients experienced END. Patients who experienced END had significantly higher pNFL levels (mean, 65.20 vs. 48.28 pg/mL; P < 0.001) and larger infarct volume (mean, 15.46 vs. 9.56mL; P < 0.001). pNFL was valuable for the prediction of END (OR, 1.170; 95% CI, 1.049-1.306; P = 0.005), even after adjusted for age and sex (OR, 1.178; 95% CI, 1.038-1.323; P = 0.006), blood sampling time, baseline NIHSS, TOAST classification, and infarct volume (OR, 1.168; 95% CI, 1.034-1.320; P = 0.012). The area under the ROC curve was 85.0% (95% CI, 0.731-0.970; P < 0.001). The sensitivity was 73.7%, and the specificity was 80%. Conclusion: END in minor stroke with LVO was distinguishable from those without END following the determination of pNFL in the blood samples within 24 h of onset. The pNFL is a promising biomarker of END in minor stroke with LVO. [ABSTRACT FROM AUTHOR]

pHLA complexes represent the largest class of cell surface markers on cancer cells, making them attractive for targeted cancer therapies. Adoptive cell therapies expressing TCRs that recognize tumor specific pHLAs take advantage of the unique selectivity and avidity of TCR: pHLA interactions. More recently, additional protein binding domains binding to pHLAs, known as TCR mimics (TCRm), were developed for tumor targeting of high potency therapeutic modalities, including bispecifics, ADCs, CAR T and -NK cells. TCRm compounds take advantage of the exquisite tumor specificity of certain pHLA targets, including cell lineage commitment markers and cancer testis antigens (CTAs). To achieve meaningful anti-tumor responses, it is critical that TCRm compounds integrate both, high target binding affinities and a high degree of target specificity. In this review, we describe the most advanced approaches to achieve both criteria, including affinity- and specificity engineering of TCRs, antibodies and alternative protein scaffolds. We also discuss the status of current TCRm based therapeutics developed in the clinic, key challenges, and emerging trends to improve treatment options for cancer patients treated with TCRm based therapeutics in Oncology. [ABSTRACT FROM AUTHOR]

Shank3 is an abundant excitatory postsynaptic scaffolding protein implicated in various neurodevelopmental disorders, including autism spectrum disorder (ASD), Phelan-McDermid syndrome, intellectual disability, and schizophrenia. Shank3-mutant mice show various molecular, synaptic, and behavioral deficits, but little is known about how transcriptomic phenotypes vary across different ages, brain regions, and gene dosages. Here, we report transcriptomic patterns in the forebrains of juvenile and adult homozygous Shank3-mutant mice that lack exons 14-16 and also the prefrontal, hippocampal, and striatal transcriptomes in adult heterozygous and homozygous Shank3-mutant mice. The juvenile and adult mutant transcriptomes show patterns opposite from and similar to those observed in ASD (termed reverse-ASD and ASD-like patterns), respectively. The juvenile transcriptomic changes accompany synaptic upregulations and ribosomal and mitochondrial downregulations, whereas the adult transcriptome show opposite changes. The prefrontal, hippocampal, and striatal transcriptomes show differential changes in ASD-related gene expressions and biological functions associated with synapse, ribosome, mitochondria, and spliceosome. These patterns also differ across heterozygous and homozygous Shank3- mutant mice. These results suggest age, brain region, and gene dosagedifferential transcriptomic changes in Shank3-mutant mice. [ABSTRACT FROM AUTHOR]

Purposes: Dynein axonemal assembly factor 5 (DNAAF5) is the transcription factor of regulating the cytoskeleton and hydrodynamic protein complex assembly, however, it was not well elucidated in the malignant progression of hepatocellular carcinoma (HCC). Methods: We investigated the role of DNAAF5 in hepatocellular carcinoma by using multiple groups of clinical tissues combined with data from the TCGA database. Then we overexpressed DNAAF5 in hepatocellular carcinoma tumor tissues, which correlates with poor patient survival outcomes. Furthermore, we constructed stable cell lines of HCC cells to confirm the cancer-promoting effects of DNAAF5 in hepatocellular carcinoma. To explore the mechanisms of DNAAF5, transcriptome sequencing combined with mass spectrometry was also performed, which showed that DNAAF5 affects its downstream signaling pathway by interacting with PFKL and that DNAAF5 regulates PFKL protein stability by recruiting the deubiquitination protein, USP39. To corroborate these findings, the same series of tissue microarrays were used to confirm correlations between DNAAF5 and PFKL expressions. In animal experiments, DNAAF5 also promoted the proliferation of HCC cells. Results: We found that DNAAF5 expressions were markedly higher in HCC tissues, compared to the adjacent normal tissues. Increased levels of DNAAF5 were associated with significantly worse prognostic outcomes for HCC patients. Cell function experiments showed that HCC cells of overexpressing DNAAF5 exhibited faster proliferation rates, stronger clone formation abilities and higher drug resistance rates. However, tumor cell proliferation rates and colony formation were significantly decreased after DNAAF5 knockout, accompanied by an increase in sensitivity to sorafenib. In addition, the results of our study showed that DNAAF5 accelerates PFKL protein deubiquitination by recruiting USP39 in HCC cells. Furthermore, The overexpression of DNAAF5 could promote HCC cell proliferation in vivo and in vitro, whereas USP39 knockdown inhibited this effect. Overall, DNAAF5 serves as a scaffold protein to recruit USP39 to form a ternary complex by directly binding the PFKL protein, thereby improving the stability of the latter, which promotes the malignant process of hepatocellular carcinoma. Conclusions: These findings revealed DNAAF5 was negatively correlated with the prognosis of patients with hepatocellular carcinoma. It underlying mechanism showed that DNAAF5 directly binds PFKL and recruits the deubiquitinated protein (USP39) to improve the stability of the PFKL protein, thus enhancing abnormal glycolysis in HCC cells. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells perform immune surveillance functions in tumors. The antitumor effects of NK cells are closely related to tumor occurrence and development. However, the molecular factors that determine NK cell antitumor activity remain to be characterized. In the present study, we identified a novel long noncoding RNA (lncRNA), NK cell activity-associated lncRNA 1 (NCAL1), and investigated its function in NK cells. NCAL1 was primarily located in NK cell nuclei, where it functioned by activating Gab2, a scaffold protein with an essential role in immune cells. Gab2 positively regulated the killing activity of NK cells. Mechanistically, NCAL1 upregulated Gab2 epigenetically by binding to the Gab2 promoter, which decreased methylation, recruited the transcription factor Sp1, and increased H3K4me3 and H3K27ac levels in the Gab2 promoter. Furthermore, NCAL1 enhanced the cytotoxicity of NK cells toward tumor cells through the Gab2-PI3K-AKT pathway. Thus, NCAL1 potentiates NK cell cytotoxicity and is a promising therapeutic target to improve NK cell therapy. [ABSTRACT FROM AUTHOR]

Tumor-associated tertiary lymphoid structures are ectopic lymphoid aggregates that have considerable morphological, cellular, and molecular similarity to secondary lymphoid organs, particularly lymph nodes. Tumor vessels expressing peripheral node addressin (PNAd) are hallmark features of these structures. Previous work from our laboratory demonstrated that PNAd is displayed on intratumoral vasculature of murine tumors, and its expression is controlled by the engagement of lymphotoxin-a3, secreted by effector CD8 T cells, with tumor necrosis factor receptors (TNFR) on tumor endothelial cells (TEC). The goals of the present work were: 1) to identify differences in expression of genes encoding the scaffolding proteins and glycosyl transferases associated with PNAd biosynthesis in TEC and lymph node blood endothelial cells (LN BEC); and 2) to determine which of these PNAd associated components are regulated by TNFR signaling. We found that the same genes encoding scaffolding proteins and glycosyl transferases were upregulated in PNAd+ LN BEC and PNAd+ TEC relative to their PNAdneg counterparts. The lower level of PNAd expression on TEC vs LN BEC was associated with relatively lower expression of these genes, particularly the carbohydrate sulfotransferase Chst4. Loss of PNAd on TEC in the absence of TNFR signaling was associated with lack of upregulation of these same genes. A small subset of PNAd+ TEC remaining in the absence of TNFR signaling showed normal upregulation of a subset of these genes, but reduced upregulation of genes encoding the scaffolding proteins podocalyxin and nepmucin, and carbohydrate sulfotransferase Chst2. Lastly, we found that checkpoint immunotherapy augmented both the fraction of TEC expressing PNAd and their surface level of this ligand. This work points to strong similarities in the regulation of PNAd expression on TEC by TNFR signaling and on LN BEC by lymphotoxin-b receptor signaling, and provides a platform for the development of novel strategies that manipulate PNAd expression on tumor vasculature as an element of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Left atrial appendage (LAA) thrombus detachment resulting in intracranial embolism is a major complication of atrial fibrillation (AF). Endocardial endothelial cell (EEC) injury leads to thrombosis, whereas autophagy protects against EEC dysfunction. However, the role and underlying mechanisms of autophagy in EECs during AF have not been elucidated. In this study, we isolated EECs from AF model mice and observed reduced autophagic flux and intracellular calcium concentrations in EECs from AF mice. In addition, we detected an increased expression of the mechanosensitive protein PLXND1 in the cytomembranes of EECs. PLXND1 served as a scaffold protein to bind with ORAI1 and further decreased ORAI1-mediated calcium influx. The decrease in the calcium influx-mediated phosphorylation of CAMK2 is associated with the inhibition of autophagy, which results in EEC dysfunction in AF. Our study demonstrated that the change in PLXND1 expression contributes to intracellular calcium dyshomeostasis, which inhibits autophagy flux and results in EEC dysfunction in AF. This study provides a potential intervention target for EEC dysfunction to prevent and treat intracardiac thrombosis in AF and its complications. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are a special type of endogenous RNAs with extensive roles in multiple human diseases. They are formed by back-splicing of partial sequences of the parental precursor mRNAs. Unlike linear RNAs, their covalently closed loop structure without a 5' cap and a 3' polyadenylated tail confers on them high stability and they are difficult to be digested by RNase R. Increasing evidence has proved that aberrant expressions of many circRNAs are detected and that circRNAs exert essential biological functions in disease development and progression via acting as a molecular sponge of microRNA, interacting with proteins as decoys or scaffolds, or self-encoding small peptides. Circular RNA zinc finger protein 609 (circ-ZNF609) originates from exon2 of ZNF609, which is located at chromosome 15q22.31, and it has recently been proved that it can translate into a protein. Being aberrantly upregulated in various diseases, it could promote malignant progression of human tumors, as well as tumor cell proliferation, migration, and invasion. Here in this review, we concluded the biological functions and potential mechanisms of circ-ZNF609 in multiple diseases, which could be further explored as a targetable molecule in future accurate diagnosis and prognosis. [ABSTRACT FROM AUTHOR]