Proximity Ligation Research Articles

CD74 is a functional MIF receptor on activated CD4+ T cells

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

Importin subunit beta-1 mediates ERK5 nuclear translocation, and its inhibition synergizes with ERK5 kinase inhibitors in reducing cancer cell proliferation.

The mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 5 (ERK5) is emerging as a promising target in cancer. Indeed, alterations of the MEK5/ERK5 pathway are present in many types of cancer, including melanoma. One of the key events in MAPK signalling is MAPK nuclear translocation and its subsequent regulation of gene expression. Likewise, the effects of ERK5 in supporting cancer cell proliferation have been linked to its nuclear localization. Despite many processes regulating ERK5 nuclear translocation having been determined, the nuclear transporters involved have not yet been identified. Here, we investigated the role of importin subunit alpha (α importin) and importin subunit beta-1 (importin β1) in ERK5 nuclear shuttling to identify additional targets for cancer treatment. Either importin β1 knockdown or the α/β1 importin inhibitor ivermectin reduced the nuclear amount of overexpressed and endogenous ERK5 in HEK293T and A375 melanoma cells, respectively. These results were confirmed in single-molecule microscopy in HeLa cells. Moreover, immunofluorescence analysis showed that ivermectin impairs epidermal growth factor(EGF)-induced ERK5 nuclear shuttling in HeLa cells. Both co-immunoprecipitation experiments and proximity ligation assay provided evidence that ERK5 and importin β1 interact and that this interaction is further induced by EGF administration and prevented by ivermectin treatment. The combination of ivermectin and the ERK5 inhibitor AX15836 synergistically reduced cell viability and colony formation ability in A375 and HeLa cells and was more effective than single treatments in preventing the growth of A375 and HeLa spheroids. The increased reduction of cell viability upon the same combination was also observed in patient-derived metastatic melanoma cells. The combination of ivermectin and ERK5 inhibitors other than AX15836 provided similar effects on cell viability. The identification of importin β1 as the nuclear transporter of ERK5 may be exploited for additional ERK5-inhibiting strategies for cancer therapy.

Kinin B1 receptor and TLR4 interaction in inflammatory response.

We aimed to broaden our understanding of a potential interaction between B1R and TLR4, considering earlier studies suggesting that lipopolysaccharide (LPS) may trigger B1R stimulation. We assessed the impact of DBK and LPS on the membrane potential of thoracic aortas from C57BL/6, B1R, or TLR4 knockout mice. Additionally, we examined the staining patterns of these receptors in the thoracic aortas of C57BL/6 and in endothelial cells (HBMEC). DBK does not affect the resting membrane potential of aortic rings in C57BL/6 mice, but it hyperpolarizes preparations in B1KO and TLR4KO mice. The hyperpolarization mechanism in B1KO mice involves B2R, and the TLR4KO response is independent of cytoplasmic calcium influx but relies on potassium channels. Conversely, LPS hyperpolarizes thoracic aorta rings in both C57BL/6 and B1KO mice, with the response unaffected by a B1R antagonist. Interestingly, the absence of B1R alters the LPS response to potassium channels. These activities are independent of nitric oxide synthase (NOS). While exposure to DBK and LPS does not alter B1R and TLR4 mRNA expression, treatment with these agonists increases B1R staining in endothelial cells of thoracic aortic rings and modifies the staining pattern of B1R and TLR4 in endothelial cells. Proximity ligation assay suggests a interaction between the receptors. Our findings provide additional support for a putative connection between B1R and TLR4 signaling. Given the involvement of these receptors and their agonists in inflammation, it suggests that drugs and therapies targeting their effects could be promising therapeutic avenues worth exploring.

Targeting senescence induced by age or chemotherapy with a polyphenol-rich natural extract improves longevity and healthspan in mice

Accumulating senescent cells within tissues contribute to the progression of aging and age-related diseases. Botanical extracts, rich in phytoconstituents, present a useful resource for discovering therapies that could target senescence and thus improve healthspan. Here, we show that daily oral administration of a standardized extract of Salvia haenkei (Haenkenium (HK)) extended lifespan and healthspan of naturally aged mice. HK treatment inhibited age-induced inflammation, fibrosis and senescence markers across several tissues, as well as increased muscle strength and fur thickness compared with age-matched controls. We also found that HK treatment reduced acutely induced senescence by the chemotherapeutic agent doxorubicin, using p16LUC reporter mice. We profiled the constituent components of HK by mass spectrometry, and identified luteolin—the most concentrated flavonoid in HK—as a senomorphic compound. Mechanistically, by performing surface plasmon resonance and in situ proximity ligation assay, we found that luteolin disrupted the p16–CDK6 interaction. This work demonstrates that administration of HK promotes longevity in mice, possibly by modulating cellular senescence and by disrupting the p16–CDK6 interaction.

Selective Assembly of TRPC Channels in the Rat Retina during Photoreceptor Degeneration.

Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex, even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that an increase in TRPC1/5 heteromers may contribute to the slowing of the degeneration of the inner retina during the outer retinal degeneration.

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis

BackgroundEnterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive.MethodsA siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein’s GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A’s host interacting partners during infection.ResultsSmall GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A’s involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A’s top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection.ConclusionsThis study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

Harnessing host enhancers of SARS-CoV-2 entry as novel targets for antiviral therapy

The WHO declared the official end of the SARS-CoV-2 caused public health emergency on May 5th, 2023, after two years in which the virus infected approximately 750 Mio individuals causing estimated up to 7 Mio deaths. Likely, the virus will continue to evolve in the human population as a seasonal respiratory pathogen. To now prevent severe infection outcomes in vulnerable individuals, effective antivirals are urgently needed to complement the protection provided by vaccines. SARS-CoV-2 enters its host cell via ACE2 mediated membrane fusion, either at the plasma membrane, if the protease TMPRSS2 is present or via the endosome, in a cathepsin dependent fashion. A small number of positive regulators of viral uptake were described in the literature, which are potentially useful targets for host directed antiviral therapy or biomarkers indicating increased or diminished susceptibility to infection. We identified here by cell surface proximity ligation novel proteins, required for efficient virion uptake. Importantly, chemical inhibition of one of these factors, SLC3A2, resulted in robust reduction of viral replication, to that achieved with a TMPRSS2 inhibitor. Our screen identified new host dependency factors for SARS-CoV-2 entry, which could be targeted by novel antiviral therapies.

Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Chromosome-scale Elaeis guineensis and E. oleifera assemblies: comparative genomics of oil palm and other Arecaceae.

Elaeis guineensis and E. oleifera are the two species of oil palm. E. guineensis is the most widely cultivated commercial species, and introgression of desirable traits from E. oleifera is ongoing. We report an improved E. guineensis genome assembly with substantially increased continuity and completeness, as well as the first chromosome-scale E. oleifera genome assembly. Each assembly was obtained by integration of long-read sequencing, proximity ligation sequencing, optical mapping, and genetic mapping. High interspecific genome conservation is observed between the two species. The study provides the most extensive gene annotation to date, including 46,697 E. guineensis and 38,658 E. oleifera gene predictions. Analyses of repetitive element families further resolve the DNA repeat architecture of both genomes. Comparative genomic analyses identified experimentally validated small structural variants between the oil palm species and resolved the mechanism of chromosomal fusions responsible for the evolutionary descending dysploidy from 18 to 16 chromosomes.

Protein Kinase A Regulates Platelet Phosphodiesterase 3A through an A-Kinase Anchoring Protein Dependent Manner.

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.

Proximity-Induced Bipedal DNA Walker for Accurately Visualizing microRNA in Living Cancer Cell.

DNA walker, a type of dynamic DNA device that is capable of moving progressively along prescribed walking tracks, has emerged as an ideal and powerful tool for biosensing and bioimaging. However, most of the reported three-dimensional (3D) DNA walker were merely designed for the detection of a single target, and they were not capable of achieving universal applicability. Herein, we reported for the first time the development of a proximity-induced 3D bipedal DNA walker for imaging of low abundance biomolecules. As a proof of concept, miRNA-34a, a biomarker of breast cancer, is chosen as the model system to demonstrate this approach. In our design, the 3D bipedal DNA walker can be generated only by the specific recognition of two proximity probes for miRNA-34a. Meanwhile, it stochastically and autonomously traveled on 3D tracks (gold nanoparticles) via catalytic hairpin assembly (CHA), resulting in the amplified fluorescence signal. In comparison with some conventional DNA walkers that were utilized for living cell imaging, the 3D DNA walkers induced by proximity ligation assay can greatly improve and ensure the high selectivity of bioanalysis. By taking advantage of these unique features, the proximity-induced 3D bipedal DNA walker successfully realizes accurate and effective monitoring of target miRNA-34a expression levels in living cells, affording a universal, valuable, and promising platform for low-abundance cancer biomarker detection and accurate identification of cancer.

Pharmacological Activation of SIRT3 Modulates the Response of Cancer Cells to Acidic pH.

Cancer cells modulate their metabolism, creating an acidic microenvironment that, in turn, can favor tumor progression and chemotherapy resistance. Tumor cells adopt strategies to survive a drop in extracellular pH (pHe). In the present manuscript, we investigated the contribution of mitochondrial sirtuin 3 (SIRT3) to the adaptation and survival of cancer cells to a low pHe. SIRT3-overexpressing and silenced breast cancer cells MDA-MB-231 and human embryonic kidney HEK293 cells were grown in buffered and unbuffered media at pH 7.4 and 6.8 for different times. mRNA expression of SIRT3 and CAVB, was measured by RT-PCR. Protein expression of SIRT3, CAVB and autophagy proteins was estimated by western blot. SIRT3-CAVB interaction was determined by immunoprecipitation and proximity ligation assays (PLA). Induction of autophagy was studied by western blot and TEM. SIRT3 overexpression increases the survival of both cell lines. Moreover, we demonstrated that SIRT3 controls intracellular pH (pHi) through the regulation of mitochondrial carbonic anhydrase VB (CAVB). Interestingly, we obtained similar results by using MC2791, a new SIRT3 activator. Our results point to the possibility of modulating SIRT3 to decrease the response and resistance of tumor cells to the acidic microenvironment and ameliorate the effectiveness of anticancer therapy.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Monomethyl fumarate attenuates lung Ischemia/Reperfusion injury by disrupting the GAPDH/Siah1 signaling cascade

Monomethyl fumarate (MMF), a potent anti-inflammatory agent used to treat multiple sclerosis, has demonstrated efficacy in various inflammatory and ischemia/reperfusion (IR) models; however, its impact on IR-induced acute lung injury (ALI) has not been explored. We investigated, for the first time, whether MMF attenuates lung IR injury through inhibition of the GAPDH/Siah1 signaling pathway. Rats were subjected to IR injury using an isolated perfused lung model, and proximity ligation assays were employed to evaluate the presence and distribution of the GAPDH/Siah1 complex. In vitro studies involved pretreating human primary alveolar epithelial cells (HPAECs) with MMF and/or inducing GAPDH overexpression or silencing, followed by exposure to hypoxia–reoxygenation. The findings revealed significantly reduced lung damage indicators, including edema, proinflammatory cytokines, oxidative stress and apoptosis, in MMF-treated rats. Notably, MMF treatment inhibited GAPDH/Siah1 complex formation and nuclear translocation, indicating that disruption of the GAPDH/Siah1 cascade was the primary cause of these improvements. Our in vitro studies on pretreated HPAECs corroborate these in vivo findings, further strengthening this interpretation. Our study results suggest that the protective effects of MMF against lung IR injury may be attributed, at least in part, to its ability to disrupt the GAPDH/Siah1 signaling cascade, thereby attenuating inflammatory and apoptotic responses. Given these encouraging results, MMF has emerged as a promising therapeutic candidate for the management of lung IR injury.

Prx1/PHB2 axis mediates mitophagy in oral leukoplakia cellular senescence

BackgroundOral leukoplakia (OLK) is the most common oral potentially malignant disorder (OPMD), which can be malignantly transformed into oral squamous cell carcinoma (OSCC). Peroxiredoxin1(Prx1) has been predicted to bind to Prohibitin2 (PHB2), which confers to affect OLK progression; however, the mechanism of Prx1/PHB2 mediated mitophagy involved in OLK remains unclear. MethodsThis study aimed to explore the mechanism of the Prx1/PHB2 axis on senescence in OLK through mediating mitophagy. The positive rate of Ki67 and the expression of p21, p16, PHB2, and LC3 in human normal, OLK, and OSCC tissues were detected by immunohistochemical staining. The mitophagy and mitochondrial function changes were then analyzed in Prx1 knockdown and Prx1C52S mutations in dysplastic oral keratinocyte (DOK) cells treated with H2O2. In situ Proximity Ligation Assay combined with co-immunoprecipitation was used to detect the interaction between Prx1 and PHB2. ResultsClinically, the positive rate of Ki67 progressively increased from normal to OLK, OLK with dysplasia, and OSCC. Higher p21, p16, PHB2, and LC3 expression levels were observed in OLK with dysplasia than in normal and OSCC tissues. In vitro, PHB2 and LC3II expression gradually increased with the degree of DOK cell senescence. Prx1/PHB2 regulated mitophagy and affected senescence in H2O2-induced DOK cells. Furthermore, Prx1C52S mutation specifically reduced interaction between Prx1 and PHB2. Prx1Cys52 is associated with mitochondrial reactive oxygen species (ROS) accumulated and cell cycle arrest. ConclusionPrx1Cys52 functions as a redox sensor that binds to PHB2 and regulates mitophagy in the senescence of OLK, suggesting its potential as a clinical target.

Citrullinating enzyme PADI4 and transcriptional repressor RING1B bind in cancer cells

Polycomb groups (PcGs) are transcriptional repressors, formed by a complex of several proteins, involved in multicellular development and cancer epigenetics. One of these proteins is the E3 ubiquitin-protein ligase RING1 (or RING1B), associated with the regulation of transcriptional repression and responsible for monoubiquitylation of the histone H2A. On the other hand, PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline, and it is also involved in the development of glioblastoma, among other types of cancers. In this work, we showed the association of PADI4 and RING1B in the nucleus and cytosol in several cancer cell lines by using immunofluorescence and proximity ligation assays. Furthermore, we demonstrated that binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that RING1B could bind to the active site of PADI4, as confirmed by protein-protein docking simulations. In vitro and in silico findings showed that binding to PADI4 occurred for the isolated fragments corresponding to both the N-terminal (residues 1–221) and C-terminal (residues 228–336) regions of RING1B. Binding to PADI4 was also hampered by GSK484, as shown by isothermal titration calorimetry (ITC) experiments for the sole N-terminal region, and by both NMR and ITC for the C-terminal one. The dissociation constants between PADI4 and any of the two isolated RING1B fragments were in the low micromolar range (~2–10 μM), as measured by fluorescence and ITC. The interaction between RING1B and PADI4 might imply citrullination of the former, leading to several biological consequences, as well as being of potential therapeutic relevance for improving cancer treatment with the generation of new antigens.

ATG10S promotes IFNL1 expression and autophagic degradation of multiple viral proteins mediated by IFNL1

ABSTRACT ATG10S is a newly discovered subtype of the autophagy protein ATG10. It promotes complete macroautophagy/autophagy, degrades multiple viral proteins, and increases the expression of type III interferons. Here, we aimed to investigate the mechanism of ATG10S cooperation with IFNL1 to degrade viral proteins from different viruses. Using western blot, immunoprecipitation (IP), tandem sensor RFP-GFP-LC3B and in situ proximity ligation assays, we showed that exogenous recombinant ATG10S protein (rHsATG10S) could enter into cells through clathrin, and ATG10S combined with ATG7 with IFNL1 assistance to facilitate ATG12–ATG5 conjugation, thereby contributing to the autophagosome formation in multiple cell lines containing different virions or viral proteins. The results of DNA IP and luciferase assays also showed that ATG10S was able to directly bind to a core motif (CAAGGG) within a binding site of transcription factor ZNF460 on the IFNL1 promoter, by which IFNL1 transcription was activated. These results clarified that ATG10S promoted autophagosome formation with the assistance of IFNL1 to ensure autophagy flux and autophagic degradation of multiple viral proteins and that ATG10S could also act as a novel transcription factor to promote IFNL1 gene expression. Importantly, this study further explored the antiviral mechanism of ATG10S interaction with type III interferon and provided a theoretical basis for the development of ATG10S into a new broad-spectrum antiviral protein drug. Abbreviation: ATG: autophagy related; ATG10S: the shorter isoform of autophagy-related 10; CC50: half cytotoxicity concentration; CCV: clathrin-coated transport vesicle; CLTC: clathrin heavy chain; CM: core motif; co-IP: co-immunoprecipitation; CPZ: chlorpromazine; ER: endoplasmic reticulum; HCV: hepatitis C virus; HBV: hepatitis B virus; HsCoV-OC43: Human coronavirus OC43; IFN: interferon; PLA: proximity ligation assay; rHsATG10S: recombinant human ATG10S protein; RLU: relative light unit; SQSTM1: sequestosome 1; ZNF: zinc finger protein

Using the Proximity Ligation Assay to Visualize Colocalization of Proteins at the Drosophila Larval Neuromuscular Junction.

In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in the Drosophila NMJ.

Differential Downregulation of β1-Adrenergic Receptor Signaling in the Heart.

Chronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart. We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the β1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening. This study reveals distinct mechanisms for the downregulation of subcellular β1AR signaling in the heart under chronic adrenergic stimulation.

Suppression of smooth muscle cell inflammation by myocardin-related transcription factors involves inactivation of TANK-binding kinase 1

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.