Assembly Of Signaling Complexes Research Articles

Targeting caspase-8/c-FLIPL heterodimer in complex II promotes DL-mediated cell death

Death receptor (DR) networks are controlled by the assembly of the Death-Inducing Signaling Complex (DISC) and complex II. The family of small molecules FLIPins (FLIP interactors) were developed to target the caspase-8/c-FLIPL heterodimer. FLIPin compounds were shown to promote apoptosis and caspase-8 activation at the DISC upon stimulation with death ligands (DLs) such as CD95L and TRAIL. To further investigate the role of FLIPin compounds in the DL-mediated cell death response, we analyzed their effects in combination with DLs and SMAC mimetics treatment. FLIPins were found to enhance cell viability loss and cell death induced by DL and SMAC mimetics in acute myeloid leukemia (AML), colon and pancreatic cancer cells. FLIPins enhanced both DL/BV6-induced apoptosis and DL/BV6/zVAD-fmk-induced necroptosis via an increase in complex II formation. Our results indicate that targeting the caspase-8/c-FLIPL heterodimer plays a prominent role in enhancing cell death induced by co-stimulation of DL/SMAC mimetics and opens new therapeutic strategies for targeting DR networks.

Structure and assembly of the human IL-12 signaling complex

Interleukin (IL)-12 is a heterodimeric pro-inflammatory cytokine. Our cryoelectron microscopy structure determination of human IL-12 in complex with IL-12Rβ1 and IL-12Rβ2 at a resolution of 3.75Å reveals that IL-12Rβ2 primarily interacts with the IL-12p35 subunit via its N-terminal Ig-like domain, while IL-12Rβ1 binds to the p40 subunit with its N-terminal fibronectin III domain. This binding mode of IL-12 with its receptors is similar to that of IL-23 but shows notable differences with other cytokines. Through structural information and biochemical assays, we identified Y62, Y189, and K192 as key residues in IL-12p35, which bind to IL-12Rβ2 with high affinity and mediate IL-12 signal transduction. Furthermore, structural comparisons reveal two distinctive conformational states and structural plasticity of the heterodimeric interface in IL-12. As a result, our study advances our understanding of IL-12 signal initiation and opens up new opportunities for the engineering and therapeutic targeting of IL-12.

FAK Family Kinases: A Potential Therapeutic Target for Atherosclerosis.

Atherosclerosis (AS) is a chronic progressive inflammatory disease of the vascular wall and the primary pathological basis of cardiovascular and cerebrovascular disease. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), two highly homologous members of the FAK family kinases, play critical roles in integrin signaling. They also serve as scaffolding proteins that contribute to the assembly of cellular signaling complexes that regulate cell survival, cell cycle progression, and cell motility. Research indicates that the FAK family kinases is involved in the gene regulation of vascular cells and that aberrant expression of this family is associated with pathological changes in vascular disease. These findings establish the FAK family kinases as a critical signaling mediator in atherosclerotic lesions and inhibition of its activity has the potential to attenuate the pathological progression of AS. This review highlights the indispensable role of the FAK family kinases in abnormal vascular smooth muscle cell proliferation, endothelial cell dysfunction, inflammation, and lipid metabolism associated with AS. We also summarize therapeutic targets against the FAK family kinases, providing valuable insights into therapeutic strategies for AS.

The phage shock protein (PSP) envelope stress response: discovery of novel partners and evolutionary history.

Bacterial phage shock protein (PSP) systems stabilize the bacterial cell membrane and protect against envelope stress. These systems have been associated with virulence, but despite their critical roles, PSP components are not well characterized outside proteobacteria. Using comparative genomics and protein sequence-structure-function analyses, we systematically identified and analyzed PSP homologs, phyletic patterns, domain architectures, and gene neighborhoods. This approach underscored the evolutionary significance of the system, revealing that its core protein PspA (Snf7 in ESCRT outside bacteria) was present in the last universal common ancestor and that this ancestral functionality has since diversified into multiple novel, distinct PSP systems across life. Several novel partners of the PSP system were identified: (i) the Toastrack domain, likely facilitating assembly of sub-membrane stress-sensing and signaling complexes, (ii) the newly defined HTH-associated α-helical signaling domain-PadR-like transcriptional regulator pair system, and (iii) multiple independent associations with ATPase, CesT/Tir-like chaperone, and Band-7 domains in proteins thought to mediate sub-membrane dynamics. Our work also uncovered links between the PSP components and other domains, such as novel variants of SHOCT-like domains, suggesting roles in assembling membrane-associated complexes of proteins with disparate biochemical functions. Results are available at our interactive web app, https://jravilab.org/psp.IMPORTANCEPhage shock proteins (PSP) are virulence-associated, cell membrane stress-protective systems. They have mostly been characterized in Proteobacteria and Firmicutes. We now show that a minimal PSP system was present in the last universal common ancestor that evolved and diversified into newly identified functional contexts. Recognizing the conservation and evolution of PSP systems across bacterial phyla contributes to our understanding of stress response mechanisms in prokaryotes. Moreover, the newly discovered PSP modularity will likely prompt new studies of lineage-specific cell envelope structures, lifestyles, and adaptation mechanisms. Finally, our results validate the use of domain architecture and genetic context for discovery in comparative genomics.

Synaptotagmin-11 facilitates assembly of a presynaptic signaling complex in post-Golgi cargo vesicles

GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

A52 THE LGR5 ACTIVITY MODULATES CELL METABOLISM TO FAVOR WOUND HEALING

Abstract Background The stem cell marker leucine-rich G-protein-coupled receptor-5 (LGR5) acts as an R-spondin (RSPO) signal transducer, facilitating WNT signaling to support tissue development and renewal. In GI diseases, LGR5+intestinal stem cells play a critical role in wound healing, while its expression by cancer stem cells correlates with cancer progression and chemoresistance. Surprisingly, only a handful of studies have investigated the functional role of LGR5 as a receptor. Demonstrating LGR5 receptor function and identifying signaling determinants will be a significant breakthrough in understanding stem cell biology under normal and pathological conditions. The project hypothesis is that LGR5 scaffolds the assembly of signaling complexes that modulate cell metabolism to accommodate cell fate, growth, and migration. Aims The project aims to characterize the molecular determinants linking LGR5 to cell metabolism and wound healing. Methods As a working model, recombinant LGR5 and mutant constructs were expressed in HEK293 cells. Wound healing was determined using scratch and single-cell tracking assays to measure cell migration. Cellular metabolism was measured using Seahorse assays complemented by metabolomic studies. Results Proteomic analysis revealed a rich LGR5 protein interactome associated with amino acid, glucose, purine metabolism, scaffold proteins (e.g., 14-3-3), and GPCR signaling. Using Seahorse assays, we showed that oxygen consumption and glycolysis were increased in LGR5-expressing cells, while metabolic profiling revealed enrichment in metabolites associated with glutamate, purine, pyrimidine metabolism, pentose phosphate pathway, glycolysis, and TCA cycle, among others. Finally, LGR5 expression promoted wound healing and a single cell's migration distance. Conclusions The results describe for the first time a functional role of LGR5 as a receptor, but foremost as an effector modulating cell metabolism. The findings may pave the way for new therapeutics targeting the LGR5 interactome to restore tissue homeostasis in diseases such as inflammatory bowel disease or to block cell adaptation and dissemination in the context of cancer. Funding Agencies CCCNSERC

Nanojunctions: Specificity of Ca2+ signaling requires nano-scale architecture of intracellular membrane contact sites

Spatio-temporal definition of Ca2+ signals involves the assembly of signaling complexes within the nano-architecture of contact sites between the sarco/endoplasmic reticulum (SR/ER) and the plasma membrane (PM). While the requirement of precise spatial assembly and positioning of the junctional signaling elements is well documented, the role of the nano-scale membrane architecture itself, as an ion-reflecting confinement of the signalling unit, remains as yet elusive. Utilizing the Na+/Ca2+ Exchanger-1 / SR/ER Ca2+ ATPase-2-mediated ER Ca2+ refilling process as a junctional signalling paradigm, we provide here the first evidence for an indispensable cellular function of the junctional membrane architecture. Our stochastic modeling approach demonstrates that junctional ER Ca2+ refilling operates exclusively at nano-scale membrane spacing, with a strong inverse relationship between junctional width and signaling efficiency. Our model predicts a breakdown of junctional Ca2+ signaling with loss of reflecting membrane confinement. In addition we consider interactions between Ca2+ and the phospholipid membrane surface, which may support interfacial Ca2+ transport and promote receptor targeting. Alterations in the molecular and nano-scale membrane organization at organelle-PM contacts are suggested as a new concept in pathophysiology.

S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

Abstract 18340: Several De-Regulated Chemokine Pathways Characterize Long COVID Syndrome

Introduction: The diagnosis of the Long COVID multi-organ syndrome is impeded by lack of circulating biomarkers. Hypothesis: We hypothesized, that post-COVID syndrome is associated with circulating protein de-regulation, enabling diagnosis of long COVID syndrome. Methods: Consecutive patients (70% female, 55±8y) with long COVID syndrome (n=70, 64.3% female, 49±6y) and non-diseased, non-vaccinated healthy controls (n=23, 70% female, 55±8y) of the Vienna POSTCOV Registry (EC 1008/2021) were included, and blood samples were collected. Proteomics was performed by using the Olink proteomics technology (Olink Proteomics, Uppsala, Sweden), by using cardiovascular, Immunologic, inflammation and neurologic protein (3x96 protein) panels. Protein-protein interaction network were built by selecting the significantly dysregulated proteins from the 4 panels, and were classified into functional groups. Results: Multiplex protein panel revealed 34 significantly de-regulated proteins as compared to controls. Gene ontology categorized the 29 upregulated proteins into several pathways with significant (false discovery rate <0.05) functional enrichment in biological processes (eg. death-inducing signaling complex assembly or positive regulation of tumor necrosis factor-mediated signaling pathway), and in molecular function (catalytic activity). Downregulated proteins were in association with chemokine-mediated signaling pathway and chemokine activity (Figure). KEGG pathway analyses revealed upregulated apoptosis, TNF- and NF-κB signaling pathways, but unchanged ACE2 receptors in patients with long COVID syndrome. Conclusions: Several de-regulated chemokine pathways characterize long COVID syndrome and may serve as a combined biomarker panel for long COVOD diagnosis and target drug prediction.

TBK1-stabilized ZNF268a recruits SETD4 to methylate TBK1 for efficient interferon signaling

Sufficient activation of interferon signaling is critical for the host to fight against invading viruses, in which post-translational modifications have been demonstrated to play a pivotal role. Here, we demonstrate that the human KRAB-zinc finger protein ZNF268a is essential for virus-induced interferon signaling. We find that cytoplasmic ZNF268a is constantly degraded by lysosome and thus remains low expressed in resting cell cytoplasm. Upon viral infection, TBK1 interacts with cytosolic ZNF268a to catalyze the phosphorylation of Serine 178 of ZNF268a, which prevents the degradation of ZNF268a, resulting in the stabilization and accumulation of ZNF268a in the cytoplasm. Furthermore, we provide evidence that stabilized ZNF268a recruits the lysine methyltransferase SETD4 to TBK1 to induce the mono-methylation of TBK1 on lysine 607, which is critical for the assembly of the TBK1 signaling complex. Notably, ZNF268 S178 is conserved among higher primates but absent in rodents. Meanwhile, rodent TBK1 607th aa happens to be replaced by arginine, possibly indicating a species-specific role of ZNF268a in regulating TBK1 during evolution. These findings reveal novel functions of ZNF268a and SETD4 in regulating antiviral interferon signaling.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by β-arrestin

β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. β-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. β-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, β-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by β-arrestins. Here, we demonstrate that β-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that β-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which β-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state β-arrestin 2 is more robust than by active-state β-arrestin 1, highlighting differential capacities of β-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which β-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

Interleukin-1β (IL-1β)-induced signaling is one of the most important pathways in regulating inflammation and immunity. The assembly of the receptor complex, consisting of the ligand IL-1β, the IL-1 receptor (IL-1R) type 1 (IL1R1), and the IL-1R accessory protein (IL1RAP), initiates this signaling. However, how the IL1R1-associated complex is regulated remains elusive. Angiopoietin like 3 (ANGPTL3), a key inhibitor of plasma triglyceride clearance, is mainly expressed in the liver and exists in both intracellular and extracellular secreted forms. Currently, ANGPTL3 has emerged as a highly promising drug target for hypertriglyceridemia and associated cardiovascular diseases. However, most studies have focused on the secreted form of ANGPTL3, while its intracellular role is still largely unknown. Here, we report that intracellular ANGPTL3 acts as a negative regulator of IL-1β-triggered signaling. Overexpression of ANGPTL3 inhibited IL-1β-induced NF-κB activation and the transcription of inflammatory genes in HepG2, THP1, and HEK293T cells, while knockdown or knockout of ANGPTL3 resulted in opposite effects. Mechanistically, ANGPTL3 interacted with IL1R1 and IL1RAP through its intracellular C-terminal fibrinogen-like domain and disrupted the assembly of the IL1R1-associated complex. Taken together, our study reveals a novel role for ANGPTL3 in inflammation, whereby it inhibits the physiological interaction between IL1R1 and IL1RAP to maintain immune tolerance and homeostasis in the liver.

Vemurafenib inhibits necroptosis in normal and pathological conditions as a RIPK1 antagonist

Necroptosis, a programmed cell death with necrotic-like morphology, has been recognized as an important driver in various inflammatory diseases. Inhibition of necroptosis has shown potential promise in the therapy of multiple human diseases. However, very few necroptosis inhibitors are available for clinical use as yet. Here, we identified an FDA-approved anti-cancer drug, Vemurafenib, as a potent inhibitor of necroptosis. Through direct binding, Vemurafenib blocked the kinase activity of receptor-interacting protein kinases 1 (RIPK1), impeded the downstream signaling and necrosome complex assembly, and inhibited necroptosis. Compared with Necrostain-1, Vemurafenib stabilized RIPK1 in an inactive DLG-out conformation by occupying a distinct allosteric hydrophobic pocket. Furthermore, pretreatment with Vemurafenib provided strong protection against necroptosis-associated diseases in vivo. Altogether, our results demonstrate that Vemurafenib is an effective RIPK1 antagonist and provide rationale and preclinical evidence for the potential application of approved drug in necroptosis-related diseases.

Identification of Host PDZ-Based Interactions with the SARS-CoV-2 E Protein in Human Monocytes.

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus's pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response.

Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK1R), Gαq/i, and βarrestin-2. Whereas the FDA-approved NK1R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK1R+ve neurons in knockin mice expressing human NK1R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK1R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK1R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK1R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

Phosphorylation-dependent recognition of diverse protein targets by the cryptic GK domain of MAGI MAGUKs

Dynamic signal transduction requires the rapid assembly and disassembly of signaling complexes, often mediated by phosphoprotein binding modules. The guanylate kinase-like (GK) domain of the membrane-associated guanylate kinases (MAGUKs) is such a module orchestrating signaling at cellular junctions. The MAGI subfamily of MAGUKs contains a truncated GK domain with unknown structure and function, although they participate in diverse physiological and pathological processes. Here, we demonstrate that the truncated GK domain of MAGI2 interacts with its adjacent PDZ0 domain to form a structural supramodule capable of recognizing phosphoproteins. A conserved phosphorylation-dependent binding motif for PDZ0-GK is delineated, which leads to identification of a set of previously unknown binding partners. We explore the structure and function of the MAGI2-target complex with an inhibitory peptide derived from the consensus motif. Our work reveals an action mechanism of the cryptic MAGI GKs and broadens our understanding of the target recognition rules of phosphoprotein binding modules.

Stress-induced pseudokinase TRB3 augments IL1β signaling by interacting with Flightless homolog 1

Interleukin-1β is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1β-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1β-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1β signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1β receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1β signaling to augment the proinflammatory response in beta cells.

Abstract A015: Dynamics and lipid interactions of the RAS-RBD-CRD protein complex

Abstract The high-resolution structure of the RAS G-domain complexed to the RBD and CRD domains of RAF provides critical insights into the assembly and conformation of the RAS-RAF signaling complex[1]. The influence and impact of the membrane on both the RAS and RAF proteins is a factor that we are just beginning to understand and appreciate. Molecular simulation is an ideal methodology to further study the complicated relationship between the membrane and associated proteins. Our recent work, using MuMMI[2] (Multiscale Machine-learned Modeling Infrastructure), investigated the different lipid compositions around KRAS4b and the interplay between the protein behavior and these membrane environments[3]. MuMMI uses machine learning to couple adjacent simulation scales and can be effectively scaled across some of the world’s largest high performance computing systems. The MuMMI multi-resolution framework has been expanded to include the RAF RBD-CRD domains. Here we present the overall simulation results from this latest MuMMI campaign. Tens of thousands of coarse-grained molecular dynamics simulations were completed, sampled from a variety of protein/lipid composition configurations. The orientations of the RAS-RBD-CRD complex on the membrane occupies distinct configurational states. Furthermore, the spatial patterns of lipid arrangements around these different protein states are also unique to each state. Interactions of the CRD with the membrane indicate the enrichment of very specific lipids in precise locations act to stabilize the complex. The extent, and size of the lipid ‘fingerprint’ imposed on the membrane by the RAS-RBD-CRD protein complex is significantly larger than observed for just the RAS protein on its own. We do not observe any statistically significant defined protein-protein orientations within the simulation ensemble. These observations indicate that spatial co-localize the RAS-RBD-CRD proteins in the same vicinity may be assisted by specific membrane environments. [1] Tran, Timothy H., et al. "KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation." Nature communications 12.1 (2021): 1-16. [2] Di Natale, Francesco, et al. "A massively parallel infrastructure for adaptive multiscale simulations: modeling RAS initiation pathway for cancer." Proceedings of the International Conference for High Performance Computing, Networking, Storage and Analysis. 2019. [3] Ingólfsson, Helgi I., et al. "Machine learning–driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins." Proceedings of the National Academy of Sciences 119.1 (2022): e2113297119. Citation Format: Felice C. Lightstone, Timothy S. Carpenter. Dynamics and lipid interactions of the RAS-RBD-CRD protein complex [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A015.