Heterotrimeric Complex Research Articles

Regulation of versican expression in macrophages is mediated by canonical type I interferon signaling via ISGF3.

Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving Toll-like receptor (TLR)4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via interferon-stimulated gene factor 3 (ISGF3), the heterotrimeric transcription factor complex of Irf9, Stat1, and Stat2, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versican's role in regulating inflammation.NEW & NOTEWORTHY We report the novel finding that versican expression is regulated by the interferon-stimulated gene factor 3 (ISGF3) arm of canonical type I Ifn signaling in LPS-stimulated macrophages. This pathway is distinct from mechanisms that control versican expression in other cell types. This suggests that macrophage-derived versican may play a role in limiting a potentially excessive inflammatory response. The detailed understanding of how versican expression is regulated in different cells could lead to unique approaches for enhancing its anti-inflammatory properties.

Chromatin assembly factor subunit CHAF1A as a monogenic cause for oculo-auriculo-vertebral spectrum.

Oculo-auriculo-vertebral spectrum (OAVS) is characterized by abnormal development of the 1st and 2nd branchial arches. Despite arguments against a monogenic condition, a few genes have been involved in a minority of cases. We now report heterozygous, presumably loss-of function variants in the CHAF1A gene in 8 individuals, including 3 members of the same family. Four cases fulfill stringent diagnostic criteria for OAVS, including asymmetric ear dysplasia, preauricular tags, mandibular asymmetry +/- vertebral malformations. Two patients also presented with kidney malformations. CHAF1A encodes a subunit of CAF-1 (chromatin assembly factor-1), a heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3-H4 onto the newly synthetized DNA strand during replication. The identification of loss-of-unction variants in CHAF1A is consistent with the hypothesis of early developmental genes dysregulation driving OAVS and other associations recently lumped under the acronym Recurrent Constellations of Embryonic Malformations (RCEM).

Abstract B092: PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with late-stage, metastatic disease that is often resistant to therapeutics, resulting in the lowest survival rate of all major cancers. Metabolic rewiring in response to oncogenic signals plays a critical role in PDAC cell survival, tumor growth, and metastasis. In contrast to normal epithelial cells, these metabolic alterations make PDAC tumors dependent on glutamine and glucose for survival, highlighting a unique metabolic vulnerability that can be therapeutically exploited. However, during times of nutrient stress, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids, including glutamine, in a process called, macropinocytosis. Macropinocytosis occurs downstream of oncogenic KRAS, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, underscoring the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process and the contribution of macropinocytic signaling to aggressive cancer phenotypes remains poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that reguates a wide variety of cell signaling pathways, inlcuding KRAS, and is commonly deregulated in human PDAC tumors. Recently, PP2A has been implicated as an important regulator of macropinocytosis, but the mechanism by which this occurs is unknown. We demosntrate that suppression of PP2A accelerates the formation of KRAS-driven precursor lesions in an in vivo mouse model of PDAC, implicating PP2A as a critical regulator of mutant KRAS phenotypes. Here, we show that acute PP2A activation prevents the fusion of macropinosomes with the lysosome, robbing PDAC cells of critical metabolic nutrients and driving cell death. Furthermore, we find PP2A associates with the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determined that PP2A activating compounds can function synergistically with metabolic inhibitors, support a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging. Citation Format: Garima Baral, Claire M Pfeffer, Indiraa Doraivel, Sara N Filippelli, Brittany N Heil, Brittany L Allen-Petersen. PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B092.

Molecular Mechanism of PP2A/B55α Phosphatase Inhibition by IER5.

PP2A serine/threonine phosphatases are heterotrimeric complexes that execute many essential physiologic functions. These activities are modulated by additional regulatory proteins, such as ARPP19, FAM122A, and IER5. Here, we report the cryoelectron microscopy structure of a complex of PP2A/B55α with the N-terminal structured region of IER5 (IER5-N50), which occludes a surface on B55α used for substrate recruitment, and show that IER5-N50 inhibits PP2A/B55α catalyzed dephosphorylation of pTau in biochemical assays. Mutations of full-length IER5 that disrupt its PP2A/B55α interface interfere with co-immunoprecipitation of PP2A/B55α. These mutations and deletions that remove the nuclear localization sequence of IER5 suppress cellular events such as KRT1 expression that depend on association of IER5 with PP2A/B55α. Querying the Alphafold2 predicted structure database identified SERTA domain proteins as high-confidence PP2A/B55α-binding structural homologs of IER5-N50. These studies define the molecular basis of PP2A/B55α inhibition by IER5-family proteins and suggest a roadmap for selective pharmacologic modulation of PP2A/B55α complexes.

Cellular N-Myristoyl Transferases Are Required for Mammarenavirus Multiplication.

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress. Meanwhile, heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and the SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect the Z-mediated virus budding and GP2-mediated fusion activity that is required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan-NMT inhibitor DDD85646 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlates with reduced Z budding activity and GP2-mediated fusion activity as well as with proteasome-mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic-fever-causing Junin (JUNV) and Lassa (LASV) mammarenaviruses. Our results support the exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

ATAD5 functions as a regulatory platform for Ub–PCNA deubiquitination

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.

Cellular N-myristoyl transferases Are Required for Mammarenavirus Multiplication.

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress, whereas heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect Z mediated virus budding and GP2 mediated fusion activity required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan NMT inhibitor DDD85464 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlated with reduced Z budding activity and GP2 mediated fusion activity, as well as proteasome mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic fever causing mammarenaviruses Junin (JUNV) and Lassa (LASV) viruses. Our results support exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

EIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding.

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2β subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2β-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2βS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2βS264Y mutation has eIF2β-γ interaction defect. Consistently, the eIF2βT238A intragenic suppressor mutation restored the eIF2β-γ and Met-tRNAiMet binding. The eIF2β-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2βT238A mutation, suggesting the eIF2β-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2βF217A/Q221A double mutation in the HTH domain. The eIF2βT238A suppressor mutation could not rescue the eIF2β binding defect of the eIF2γV281K mutation; however, combining the eIF2βS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2β with the eIF2γ subunit via its α1-helix, the eIF2β-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2β-γ interacts via two distinct binding sites.

Genome and Transcriptome Analysis of NF-Y Transcription Factors in Sweet Potato under Salt Stress

Nuclear factor Y (NF-Y) is a heterotrimeric complex composed of three unique subunits: NF-YA, NF-YB, and NF-YC. This transcription factor complex binds to the CCAAT box of eukaryotic promoters, playing a crucial role in various biological processes in plants. Despite its importance, the NF-Y gene family has not been reported in the sweet potato (Ipomoea batatas) genome, an important food and energy crop. Understanding the role and function of NF-Y in sweet potatoes could provide valuable insights for genetic improvement and yield enhancement. To address this gap, our research aimed to comprehensively catalog and characterize the NF-Y genes in sweet potatoes, which we refer to as ‘IbNF-Y’, where ‘Ib’ denotes Ipomoea batatas. A total of 37 NF-Ys were identified, including 11 NF-YA, 21 NF-YB, and 5 NF-YC members, and their phylogeny, gene structure, chromosomal distribution, and conserved motifs were analyzed. Additionally, we assessed their expression patterns under salt stress in both light and dark conditions using transcriptome sequencing. Notably, we discovered that certain IbNF-Y genes showed significant changes in expression under salt stress, suggesting their potential roles in sweet potato’s adaptation to saline environments. Furthermore, our work enriches the genomics and genetic research on sweet potatoes and contributes valuable knowledge to the broader scientific community of the Convolvulaceae family.

Exploring a New Generation of Pyrimidine and Pyridine Derivatives as Anti-Influenza Agents Targeting the Polymerase PA-PB1 Subunits Interaction.

The limited range of available flu treatments due to virus mutations and drug resistance have prompted the search for new therapies. RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex of three subunits, i.e., polymerase acidic protein (PA) and polymerase basic proteins 1 and 2 (PB1 and PB2). It is widely recognized as one of the most promising anti-flu targets because of its critical role in influenza infection and high amino acid conservation. In particular, the disruption of RdRp complex assembly through protein-protein interaction (PPI) inhibition has emerged as a valuable strategy for discovering a new therapy. Our group previously identified the 3-cyano-4,6-diphenyl-pyridine core as a privileged scaffold for developing PA-PB1 PPI inhibitors. Encouraged by these findings, we synthesized a small library of pyridine and pyrimidine derivatives decorated with a thio-N-(m-tolyl)acetamide side chain (compounds 2a-n) or several amino acid groups (compounds 3a-n) at the C2 position. Interestingly, derivative 2d, characterized by a pyrimidine core and a phenyl and 4-chloro phenyl ring at the C4 and C6 positions, respectively, showed an IC50 value of 90.1 μM in PA-PB1 ELISA, an EC50 value of 2.8 μM in PRA, and a favorable cytotoxic profile, emerging as a significant breakthrough in the pursuit of new PPI inhibitors. A molecular modeling study was also completed as part of this project, allowing us to clarify the biological profile of these compounds.

An antibody to IL-1 receptor 7 protects mice from LPS-induced tissue and systemic inflammation.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.

ZmNF-YA1 Contributes to Maize Thermotolerance by Regulating Heat Shock Response.

Zea mays (maize) is a staple food, feed, and industrial crop. Heat stress is one of the major stresses affecting maize production and is usually accompanied by other stresses, such as drought. Our previous study identified a heterotrimer complex, ZmNF-YA1-YB16-YC17, in maize. ZmNF-YA1 and ZmNF-YB16 were positive regulators of the drought stress response and were involved in maize root development. In this study, we investigated whether ZmNF-YA1 confers heat stress tolerance in maize. The nf-ya1 mutant and overexpression lines were used to test the role of ZmNF-YA1 in maize thermotolerance. The nf-ya1 mutant was more temperature-sensitive than the wild-type (WT), while the ZmNF-YA1 overexpression lines showed a thermotolerant phenotype. Higher malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation were observed in the mutant, followed by WT and overexpression lines after heat stress treatment, while an opposite trend was observed for chlorophyll content. RNA-seq was used to analyze transcriptome changes in nf-ya1 and its wild-type control W22 in response to heat stress. Based on their expression profiles, the heat stress response-related differentially expressed genes (DEGs) in nf-ya1 compared to WT were grouped into seven clusters via k-means clustering. Gene Ontology (GO) enrichment analysis of the DEGs in different clades was performed to elucidate the roles of ZmNF-YA1-mediated transcriptional regulation and their contribution to maize thermotolerance. The loss function of ZmNF-YA1 led to the failure induction of DEGs in GO terms of protein refolding, protein stabilization, and GO terms for various stress responses. Thus, the contribution of ZmNF-YA1 to protein stabilization, refolding, and regulation of abscisic acid (ABA), ROS, and heat/temperature signaling may be the major reason why ZmNF-YA1 overexpression enhanced heat tolerance, and the mutant showed a heat-sensitive phenotype.

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple β-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

In silico structural features of the CgNR5A: CgDAX complex and its role in regulating gene expression of CYP target genes in Crassostrea gigas

Contamination of aquatic environments has been steadily increasing due to human activities. The Pacific oyster Crassostrea gigas has been used as a key species in studies assessing the impacts of contaminants on human health and the aquatic biome. In this context, cytochrome P450 (CYPs) play a crucial role in xenobiotic metabolism. In vertebrates many of these CYPs are regulated by nuclear receptors (NRs) and little is known about the NRs role in C. gigas. Particularly, the CgNR5A represents a homologue of SF1 and LRH-1 found in vertebrates. Members of this group can regulate genes of CYPs involved in lipid/steroid metabolism, with their activity regulated by other NR, called as DAX-1, generating a NR complex on DNA response elements (REs). As C. gigas does not exhibit steroid biosynthesis pathways, CgNR5A may play other physiological roles. To clarify this issue, we conducted an in silico investigation of the interaction between CgNR5A and DNA to identify potential C. gigas CYP target genes. Using molecular docking and dynamics simulations of the CgNR5A on DNA molecules, we identified a monomeric interaction with extended REs. This RE was found in the promoter region of 30 CYP genes and also the NR CgDAX. When the upstream regulatory region was analyzed, CYP2C39, CYP3A11, CYP4C21, CYP7A1, CYP17A1, and CYP27C1 were mapped as the main genes regulated by CgNR5A. These identified CYPs belong to families known for their involvement in xenobiotic and lipid/steroid metabolism. Furthermore, we reconstructed a trimeric complex, previously proposed for vertebrates, with CgNR5A:CgDAX and subjected it to molecular dynamics simulations analysis. Heterotrimeric complex remained stable during the simulations, suggesting that CgDAX may modulate CgNR5A transcriptional activity. This study provides insights into the potential physiological processes involving these NRs in the regulation of CYPs associated with xenobiotic and steroid/lipid metabolism.

Generation of Comprehensive GPCR-Transducer-Deficient Cell Lines to Dissect the Complexity of GPCR Signaling.

G-protein-coupled receptors (GPCRs) compose the largest family of transmembrane receptors and are targets of approximately one-third of Food and Drug Administration-approved drugs owing to their involvement in almost all physiologic processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and β-arrestins, both of which serve as transducers, resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein-coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR genome-editing technology. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, β-arrestins, G-protein-coupled receptor kinases (GRKs), and many other signaling nodes in the GPCR pathways at one's convenience. Here, we review the practicality of using CRISPR-derived knockout (KO) cells in the experimental contexts of unraveling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of β-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies. SIGNIFICANCE STATEMENT: This review emphasizes the practical application of G-protein-coupled receptor (GPCR) transducer knockout (KO) cells in dissecting the intricate regulatory mechanisms of the GPCR signaling network. Currently available cell lines, along with accumulating KO cell lines in diverse cell types, offer valuable resources for systematically elucidating GPCR signaling regulation. Given the association of GPCR signaling with numerous diseases, uncovering the system-based signaling map is crucial for advancing the development of novel drugs targeting specific diseases.

Nuclear factor Y-A3b binds to the SINGLE FLOWER TRUSS promoter and regulates flowering time in tomato.

The control of flowering time is essential for reproductive success and has a major effect on seed and fruit yield and other important agricultural traits in crops. Nuclear factors Y (NF-Ys) are transcription factors that form heterotrimeric protein complexes to regulate gene expression required for diverse biological processes, including flowering time control in plants. However, to our knowledge, there has been no report on mutants of individual NF-YA subunits that promote early flowering phenotype in plants. In this study, we identified SlNF-YA3b, encoding a member of the NF-Y transcription factor family, as a key gene regulating flowering time in tomato. Knockout of NF-YA3b resulted in an early flowering phenotype in tomato, whereas overexpression of NF-YA3b delayed flowering in transgenic tomato plants. NF-YA3b was demonstrated to form heterotrimeric protein complexes with multiple NF-YB/NF-YC heterodimers in yeast three-hybrid assays. Biochemical evidence indicated that NF-YA3b directly binds to the CCAAT cis-elements of the SINGLE FLOWER TRUSS (SFT) promoter to suppress its gene expression. These findings uncovered a critical role of NF-YA3b in regulating flowering time in tomato and could be applied to the management of flowering time in crops.

Abstract 3026: STK11 negatively regulates NFKB signaling in KRAS-driven lung adenocarcinoma

Abstract In the US, more people die each year from lung cancer than breast, colorectal, and prostate cancers combined. Despite advances in lung cancer detection and treatment, the overall prognosis for patients remains poor. Patients whose lung tumors harbor druggable mutations often experience initial responses with targeted therapies, but acquisition of therapy resistance results in 5-year survival rates that have remained near ~20%. Immune check-point inhibitors (ICI) promise to improve outcomes in patients with lung cancer by targeting mechanisms of immune evasion. Drugs that disrupt PD-L1-mediated immune evasion, including pembrolizumab and nivolumab, have revolutionized treatment for some cancers. Unfortunately, only ~30% of lung adenocarcinomas (LUADs) respond to anti-PD-1 therapy and we cannot reliably identify this group prior to treatment. This underscores the need to discriminate which patients will respond to ICI therapies, while simultaneously highlighting the ~70% non-responder rate. To that end, recent clinical studies have linked anti-PD-1 therapy resistance with Serine/Threonine Kinase 11 (STK11) loss of function (LoF). STK11 operates in a heterotrimeric complex with the pseudo-kinase STRADα and the scaffolding protein MO25 where it regulates numerous intracellular signaling networks impacting metabolism, proliferation, transcription, and cell morphology. Why STK11 LoF correlates with anti-PD-1 resistance in the context of KRAS-driven LUAD remains unknown and represents a critical question in lung oncology. As an initial approach to understand this phenomenon, we knocked-out STK11 in multiple KRAS-driven, STK11-competent human LUAD cell lines and performed whole transcriptome analyses to identify STK11-loss-dependent differential gene expression profiles. We assert the transcriptional mediators downstream of STK11 governing the changes we report represent therapeutic targets whose antagonism may restore anti-PD-1 efficacy. Supporting this rationale, our data has identified STK11-loss-dependent activation of the NFκ-B transcriptional network. NFκB is a master transcription factor that regulates numerous cytokines, but whether and/or how STK11 directs NFκB activity remains largely unaddressed. We highlight NFκB activation as a potential mechanistic link between STK11 loss and tumor-intrinsic cytokine upregulation. We speculate that STK11 normally acts as a key player in the negative feedback loop limiting NFκB activity. When STK11 is lost, that activity continues unabated. In summary, we propose STK11 loss drives anti-PD-1 therapy resistance by generating an altered tumor immune microenvironment via constitutive activation of NFκB-mediated cytokine production. In future we intend to assess strategies for reversing anti-PD-1 therapy resistance via disruption of the NFκB transcriptional axis in KRAS-driven LUAD lacking STK11. Citation Format: David Joseph Seward, Sean Lenahan, Allison Racela, Israel Odekunle. STK11 negatively regulates NFKB signaling in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3026.

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis

Tribbles pseudokinase 3 (TRIB3) has been identified recently as a novel oncogene in several cancers. Still, further extensive research is imperative to elucidate its function and the molecular mechanisms underlying its involvement in the progression of head and neck squamous cell carcinoma (HNSCC). In our study, we found that TRIB3 silencing significantly promoted cell death by inducing ferroptosis. The interaction of TRIB3 with Transcription Factor 4 (TCF4) and β-catenin created a heterotrimeric complex, which directly interacts with the ALOXE3 promoter, detrimentally impacting its activation. The consequential partial neutralization of ferroptosis induced by TRIB3 deficiency is observed through the implementation of ALOXE3 knockdown. Furthermore, the study demonstrated that the molecular inhibitor hesperidin, targeting TRIB3, not only reduced cell malignancy but also induced ferroptosis, thereby suppressing tumor growth. Overall, our findings unequivocally validate the proposition that TRIB3 deficiency precipitates the iron death mechanism, thereby indicating that the strategic targeting of TRIB3 could emerge as an innovative therapeutic strategy for HNSCC.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the Ear, Nose and Throat department of a tertiary pediatric hospital linked to the United Kingdom’s National EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wildtype LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof-of-concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.