Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays.

As a dominant oncogenic protein, Ras is well-known to segregate into clusters on the plasma membrane for activating downstream signaling. However, current technologies for direct measurements of Ras clustering are limited to sophisticated high-resolution techniques like electron microscopy and fluorescence lifetime imaging. To further promote fundamental investigations and the related drug development, we hereby introduce a nanobar-based platform which effectively guides Ras clusters into quantifiable patterns in live cells that is resolvable under conventional microscopy. Major Ras isoforms, K-Ras, H-Ras, and N-Ras, were differentiated, as well as their highly prevalent oncogenic mutants G12V and G13D. Moreover, the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized on nanobars. We envision that this nanobar-based platform will serve as an effective tool to read Ras clustering on the plasma membrane, enabling a novel avenue both to decipher Ras regulations and to facilitate anti-Ras drug development.

Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection.

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

Multiomic analysis of papillary thyroid cancers identifies BAIAP2L1-BRAF fusion and requirement of TRIM25, PDE5A and PKCδ for tumorigenesis.

BACKGROUND: Papillary thyroid carcinoma (PTC) is one of the most common forms of thyroid cancer with a cure rate of over 90% after surgery. However, aggressive forms may still occur, and personalized therapeutic strategies are increasingly required. METHODS: We performed integrated genomic and proteomic analysis of PTC tumor samples from patients who did not harbor BRAF or RAS mutations. We validate the analysis and present in-depth molecular analysis of the identified genetic rearrangement by employing biochemical and cell biological assays. Finally, we employ 3D spheroid models, loss of function studies and chemical inhibitors to target the hitherto upregulated factors. The data are analysed with appropriate statistical tests which are mentioned in the legends section. RESULTS: In a 23-year-old patient with thyroiditis, we identified a novel rearrangement leading to a BAIAP2L1-BRAF fusion that transforms immortalized human thyroid cells in a kinase and CC-domain dependent manner. Moreover, quantitative proteomic analysis of the same patient samples revealed the upregulation of several proteins including the Ubiquitin E3 ligase TRIM25, PDE5A, and PKCδ. Further, in a cohort of PTC patients, we observed higher expression of TRIM25 and PKCδ in the tumor and metastatic lesions, when compared to the matched normal tissue. Inhibition of TRIM25, PDE5A and PKCδ with small molecules or RNA interference affected not only viability and proliferation of BAIAP2L1-BRAF transformed cells, but also the viability, growth and invasion of corresponding 3D spheroid cultures. CONCLUSIONS: Apart from unveiling a novel oncogenic BRAF fusion in PTCs, our data may open a novel avenue of therapeutic targeting in human PTCs.

Targeting prohibitins at the cell surface prevents Th17-mediated autoimmunity.

T helper (Th)17 cells represent a unique subset of CD4+ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells. Increased expression of PHBs at the cell surface contributed to enhanced CRAF/MAPK activation in Th17 cells. Targeting surface-expressed PHBs on Th17 cells with ligands such as Vi polysaccharide (Typhim vaccine) inhibited CRAF-MAPK pathway, reduced interleukin (IL)-17 expression and ameliorated disease pathology with an increase in FOXP3+-expressing Tregs in an animal model for multiple sclerosis (MS). Interestingly, we detected a CD4+ T cell population with high PHB1 surface expression in blood samples from MS patients in comparison with age- and sex-matched healthy subjects. Our observations suggest a pivotal role for the PHB-CRAF-MAPK signalling axis in regulating the polarization and pathogenicity of Th17 cells and unveil druggable targets in autoimmune disorders such as MS.

A subset of flavaglines inhibits KRAS nanoclustering and activation.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications.

Regulation of mRNA export through API5 and nuclear FGF2 interaction.

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5-FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids.

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases.

Over the last three decades, the scaffold proteins prohibitins-1 and -2 (PHB1/2) have emerged as key signaling proteins regulating a myriad of signaling pathways in health and diseases. Small molecules targeting PHBs display promising effects against cancers, osteoporosis, inflammatory, cardiac and neurodegenerative diseases. This review provides an updated overview of the various classes of PHB ligands, with an emphasis on their mechanism of action and therapeutic potential. We also describe how these ligands have been used to explore PHB signaling in different physiological and pathological settings.

E4BP4/NFIL3 modulates the epigenetically repressed RAS effector RASSF8 function through histone methyltransferases.

RAS proteins are major human oncogenes, and most of the studies are focused on enzymatic RAS effectors. Recently, nonenzymatic RAS effectors (RASSF, RAS association domain family) have garnered special attention because of their tumor-suppressive properties in contrast to the oncogenic potential of the classical enzymatic RAS effectors. Whereas most members of RASSF family are deregulated by promoter hypermethylation, RASSF8 promoter remains unmethylated in many cancers but the mechanism(s) of its down-regulation remains unknown. Here, we unveil E4BP4 as a critical transcriptional modulator repressing RASSF8 expression through histone methyltransferases, G9a and SUV39H1. In line with these observations, we noticed a negative correlation of RASSF8 and E4BP4 expression in primary breast tumor samples. In addition, our data provide evidence that E4BP4 attenuates RASSF8-mediated anti-proliferation and apoptosis, shedding mechanistic insights into RASSF8 down-regulation in breast cancers. Collectively, our study provides a better understanding on the epigenetic regulation of RASSF8 function and implicates the development of better treatment strategies.

The non-enzymatic RAS effector RASSF7 inhibits oncogenic c-Myc function.

c-Myc is a proto-oncogene controlling expression of multiple genes involved in cell growth and differentiation. Although the functional role of c-Myc as a transcriptional regulator has been intensively studied, targeting this protein in cancer remains a challenge. Here, we report a trimodal regulation of c-Myc function by the Ras effector, Ras-association domain family member 7 (RASSF7), a nonenzymatic protein modulating protein-protein interactions to regulate cell proliferation. Using HEK293T and HeLa cell lines, we provide evidence that RASSF7 destabilizes the c-Myc protein by promoting Cullin4B-mediated polyubiquitination and degradation. Furthermore, RASSF7 competed with MYC-associated factor X (MAX) in the formation of a heterodimeric complex with c-Myc and attenuated its occupancy on target gene promoters to regulate transcription. Consequently, RASSF7 inhibited c-Myc-mediated oncogenic transformation, and an inverse correlation between the expression levels of the RASSF7 and c-Myc genes was evident in human cancers. Furthermore, we found that RASSF7 interacts with c-Myc via its RA and leucine zipper (LZ) domains and LZ domain peptide is sufficient to inhibit c-Myc function, suggesting that this peptide might be used to target oncogenic c-Myc. These results unveil that RASSF7 and c-Myc are functionally linked in the control of tumorigenesis and open up potential therapeutic avenues for targeting the "undruggable" c-Myc protein in a subset of human cancers.

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase-2.

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase-2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase-2 also regulates autophagy, genomic stability and ageing. Caspase-2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase-2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase-2 and impedes dimerization and activation of caspase-2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase-2. Depletion of endogenous API5 leads to an increase in caspase-2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase-2-dependent apoptotic cell death. These results establish API5/AAC-11 as a direct inhibitor of caspase-2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.

Ubiquitin-dependent regulation of MEKK2/3-MEK5-ERK5 signaling module by XIAP and cIAP1.

Mitogen-activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular-signal-regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three-tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5-ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3-ERK5-dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5-MAPK module and human muscle cell differentiation.

IAPs and cell migration.

Inhibitors of apoptosis (IAPs) constitute a family of cell signaling regulators controlling several fundamental biological processes such as innate immunity, inflammation, cell death, cell proliferation, and cell differentiation. Increasing evidence from in vivo and in vitro studies indicate a function for IAPs in the modulation of invasive and migratory properties of cells. Here, we present and discuss the mechanisms whereby IAPs can control cell migration.

Caspase-2 is an initiator caspase responsible for pore-forming toxin-mediated apoptosis.

Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-forming toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus α-toxin- and Aeromonas aerolysin-mediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediated apoptosis. Activation of caspase-2 is PIDDosome-independent, and endogenous caspase-2 is recruited to a high-molecular-weight complex in α-toxin-treated cells. Interestingly, prevention of PFT-induced potassium efflux inhibits the formation of caspase-2 complex, leading to its inactivation, thus resisting apoptosis. These results revealed a thus far unknown, obligatory role for caspase-2 as an initiator caspase during PFT-mediated apoptosis.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

A novel role for inhibitor of apoptosis (IAP) proteins as regulators of endothelial barrier function by mediating RhoA activation.

Inhibitor of apoptosis (IAP) proteins, such as XIAP or cIAP1/2, are important regulators of apoptosis in cancer cells, and IAP antagonists are currently evaluated as antitumor agents. Beyond their function in cancer cells, this study demonstrates a novel role of IAPs as regulators of vascular endothelial permeability. Two structurally different IAP antagonists, ABT and Smac085, as well as silencing of IAPs, reduced the thrombin receptor-activating peptide (TRAP)-induced barrier dysfunction in human endothelial cells as assessed by measuring macromolecular permeability or transendothelial electrical resistance. ABT diminished thrombin-evoked stress fiber formation, activation of myosin light chain 2, and disassembly of adherens junctions independent of calcium signaling, protein kinase C, and mitogen-activated protein kinases. Interestingly, ABT and silencing of IAPs, in particular XIAP, reduced the TRAP-evoked RhoA activation, whereas Rac1 was not affected. XIAP and, to a lesser extent, cIAP1 were found to directly interact with RhoA independently of the RhoA activation status. Under cell-free conditions, XIAP did not induce an ubiquitination of RhoA. In summary, our work discloses IAPs as crucial regulators of endothelial permeability and suggests IAP inhibition as interesting approach for the prevention of endothelial barrier dysfunction.