Binding Partners Research Articles

Dissecting OGT’s TPR domain to identify determinants of cellular function

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.

The genome of Lespedeza potaninii reveals biased subgenome evolution and drought adaptation.

Lespedeza potaninii, a xerophytic subshrub belonging to the legume family, is native to the Tengger Desert and is highly adapted to drought. It has important ecological value due to its drought adaptability, but the underlying molecular mechanisms remain largely unknown. Here, we report a 1.24 Gb chromosome-scale assembly of the L. potaninii genome (contig N50 = 15.75 Mb). Our results indicate that L. potaninii underwent an allopolyploid event with 2 subgenomes, A and B, presenting asymmetric evolution and B subgenome dominance. We estimate that the 2 diploid progenitors of L. potaninii diverged around 3.6 million years ago (MYA) and merged around 1.0 MYA. We revealed that the expansion of hub genes associated with drought responses, such as the binding partner 1 of accelerated cell death 11 (ACD11) (BPA1), facilitated environmental adaptations of L. potaninii to desert habitats. We found a novel function of the BPA1 family in abiotic stress tolerance in addition to the known role in regulating the plant immune response, which could improve drought tolerance by positively regulating reactive oxygen species homeostasis in plants. We revealed that bZIP transcription factors could bind to the BPA1 promoter and activate its transcription. Our work fills the genomic data gap in the Lespedeza genus and the tribe Desmodieae, which should provide theoretical support both in the study of drought tolerance and in the molecular breeding of legume crops.

Structure-guided mutagenesis targeting interactions between pp150 tegument protein and small capsid protein identify five lethal and two live-attenuated HCMV mutants

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150 kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.

LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses.

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell-specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1+ CD62Lhi PD-1low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1+ PD-1+). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8+ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.

Isolation of short RNAs with homogenous 3’-ends using quaternary-amine anion exchange chromatography

Abstract Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein binding partner.

Abstract A024: Genomic peroxisome proliferator-activated receptor gamma (PPARG) study: A sponsored testing program to identify patients harboring genetic alterations in advanced UC in support of the FX-909 Phase 1 study

Abstract The transcription factor PPARG is associated with the luminal lineage subtype representing ~65% of advanced urothelial cancer (UC) patients. Recurrent genetic alterations in PPARG, fibroblast growth factor receptor 3 (FGFR3), and the heterodimeric binding partner for PPARG, retinoid X receptor alpha (RXRA), are characteristic of this luminal subtype. FX-909, a covalent PPARG inverse agonist, represents a promising first-in-class therapy for patients with advanced UC. FX-909-CLINPRO-1 [NCT05929235] is an open-label Phase 1 study to evaluate the safety, tolerability, pharmacokinetics, biologic activity, and preliminary clinical activity of FX-909. This study includes a Phase 1b expansion cohort, which will enroll advanced UC patients with genetic alterations in PPARG, RXRA, and/or FGFR3. FlareTx is sponsoring a testing program called Genomic PPARG Study (GPS) to enable investigators to identify patients with these relevant genetic alterations. Tumors collected from patients with advanced UC were sequenced using the Tempus xT assay (DNA-seq of 648 genes at 500x coverage; RNA-seq). A clinical report is generated and returned to the physician to guide selection of approved therapies, and if appropriate to support eligibility for inclusion in the study. FlareTx is provided with de-identified genomic data, where assessment of the prevalence of genetic alternations such as copy number variation (CNV), fusions and mutations (single nucleotide variants) in PPARG, RXRA and FGFR3 are undertaken. Additionally, luminal molecular classification is performed using the Robertson method [1]. PPARG mean expression value are normalized by transcripts-per-million (TPM) and reported as Log2[TPM+1]. In the initial data set of 27 patients, 3 patients had PPARG amplification (CN >/= 3), 3 patients had RXRA hotspot mutation (S427F) [2], 6 patients had FGFR3 mutation(s), and 1 patient had both PPARG amplification and a FGFR3 mutation. Twenty of 27 patients tested had sufficient starting material of good quality to also perform whole transcriptome analysis. The mean mRNA expression level of PPARG was 6.77 Log2[TPM+1] (range 2.91 Log2[TPM+1] – 9.22 Log2[TPM+1]). Consistent with the Robertson subtype distribution, 65% of patients were luminal and 35% were non-luminal (basal-squamous and neuronal); 10 of 13 luminal patients had one or more genetic alteration in PPARG, RXRA and/or FGFR3. High PPARG expression was associated with luminal subtype compared to non-luminal subtype (7.90 Log2[TPM+1] vs 4.67 Log2[TPM+1]; p=0.00042). GPS is open at two active Phase 1 sites, with several more planned as the study advances towards the Phase 1B dose expansion stage. GPS runs concurrently with the FX-909 study and is a novel precision strategy to maximize identifying advanced UC patients with actionable genetic alterations to potentially enroll in the clinical study.

Chaperoning the chaperones: Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.

Molecular chaperones and co-chaperones are highly conserved cellular components that perform variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an RNP assembly chaperone and serves as a paradigm for studying how specific small nuclear (sn)RNAs are identified and paired with their client substrate proteins. SMN protein is the eponymous component of a large complex required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs) and localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN forms the oligomeric core of this complex, and missense mutations in its YG box self-interaction domain are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. Here, we carried out affinity purification mass spectrometry (AP-MS) of SMN using stable fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially interacted with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the β-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

HER3 V104 mutations regulate cell signaling, growth, and drug sensitivity in cancer.

HER3 is mutated in ~2%-10% of cancers depending on the cancer type. We found the HER3-V104L mutation to be activating from patient-derived mutations introduced via lentiviral transduction in HER3KO HER2 + HCC1569 breast cancer cells in which endogenous HER3 was eliminated by CRISPR/Cas9. Cells expressing HER3-V104L showed higher p-HER3 and p-ERK1/2 expression versus cells expressing wild-type HER3 or HER3-V104M. Patients whose tumor expressed the HER3 V104L variant had a reduced probability of overall survival compared to patients lacking a HER3 mutation whereas we did not find a statistically significant difference in overall survival of various cancer patients with the HER3 V104M mutation. Our data showed that HER2 inhibitors suppressed cell growth of HCC1569HER3KO cells stably expressing the HER3-V104L mutation. Cancer cell lines (SNU407, UC15 and DV90) with endogenous HER3-V104M mutation showed reduced cell proliferation and p-HER2/p-ERK1/2 expression with HER2 inhibitor treatment. Knock down of HER3 abrogated cell proliferation in the above cell lines which were overall more sensitive to the ERK inhibitor SCH779284 versus PI3K inhibitors. HER3-V104L mutation stabilized HER3 protein expression in COS7 and SNUC5 cells. COS7 cells transiently transfected with the HER3-V104L mutation in the presence of HER binding partners showed higher expression of p-HER3, p-ERK1/2 versus HER3-WT in a NRG-independent manner without any change in AKT signaling. Overall, this study shows the clinical relevance of the HER3 V104L and the V104M mutations and its response to HER2, PI3K and ERK inhibitors.

A conserved switch controls virulence, sporulation, and motility in C. difficile.

Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, 3D structural analyses of Spo0E revealed specific and exclusive interactions between Spo0E and binding partners in C. difficile and B. subtilis that provide insight into the conservation of this regulatory mechanism among different species.

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis.

Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

A Computational Pipeline for Accurate Prioritization of Protein-Protein Binding Candidates in High-Throughput Protein Libraries.

Identifying the interactome for a protein of interest is challenging due to the large number of possible binders. High-throughput experimental approaches narrow down possible binding partners but often include false positives. Furthermore, they provide no information about what the binding region is (e.g., the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competitive Binding Assay (AF-CBA) to identify proteins that bind a target of interest from a pull-down experiment and the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide-protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies of AF and AF-CBA to help users identify scenarios where the approach will be most useful. Given the method's speed and accuracy, we anticipate its broad applicability to identify binding epitope regions among potential partners, setting the stage for experimental verification.

A Computational Pipeline for Accurate Prioritization of Protein‐Protein Binding Candidates in High‐Throughput Protein Libraries

AbstractIdentifying the interactome for a protein of interest is challenging due to the large number of possible binders. High‐throughput experimental approaches narrow down possible binding partners but often include false positives. Furthermore, they provide no information about what the binding region is (e.g., the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competitive Binding Assay (AF‐CBA) to identify proteins that bind a target of interest from a pull‐down experiment and the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide‐protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies of AF and AF‐CBA to help users identify scenarios where the approach will be most useful. Given the method‘s speed and accuracy, we anticipate its broad applicability to identify binding epitope regions among potential partners, setting the stage for experimental verification.

Intramolecular autoinhibition regulates the selectivity of PRPF40A tandem WW domains for proline-rich motifs

PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.

Unveiling Actin Cytoskeleton Role in Mediating Chikungunya-Associated Arthritis: An Integrative Proteome-Metabolome Study.

Background: Chikungunya is a zoonotic disease caused by the Chikungunya virus (CHIKV), primarily transmitted to humans through infected Aedes mosquitoes. The infection is characterized by symptoms such as high fever, musculoskeletal pain, polyarthritis, and a rash, which can lead to severe complications such as encephalitis, meningitis, and even fatalities. While many disease manifestations resemble those of other viral infections, chronic arthritis caused by CHIKV is unique, and its molecular mechanisms remain ill-defined. Materials and Methods: Proteomics data from both cellular and patient levels of CHIKV infection were curated from PubMed and screened using inclusion and exclusion criteria. Patient serum proteomics data obtained from P RIDE underwent reanalysis using Proteome Discoverer 2.2. Enrichment and protein-protein interaction network analysis were conducted on differentially expressed proteins from both serum and cellular datasets. Metabolite data from CHIKV-infected patients were further retrieved, and their protein binding partners were identified using BindingDB. The protein-metabolite interaction pathway was further developed using MetaboAnalyst. Results: The proteomics data analysis revealed differential expression of proteins involved in critical host mechanisms, such as cholesterol metabolism and mRNA splicing, during CHIKV infection. Consistent upregulation of two actin cytoskeleton proteins, TAGLN2 and PFN1, was noted in both serum and cellular datasets, and their upregulations are associated with arthritis. Furthermore, alterations in purine metabolism were observed in the integrative proteome-metabolome analysis, correlating with cytoskeletal remodelling. Conclusion: Collectively, this integrative view sheds light on the involvement of actin cytoskeleton remodeling proteins and purine metabolic pathways in the development of arthritis during CHIKV infection.

Unveiling the molecular basis of paracetamol-induced hepatotoxicity: Interaction of N-acetyl-p-benzoquinone imine with mitochondrial succinate dehydrogenase

Background and aimN-acetyl-p-benzoquinoneimine (NAPQI), a toxic byproduct of paracetamol (Acetaminophen, APAP), can accumulate and cause liver damage by depleting glutathione and forming protein adducts in the mitochondria. These adducts disrupt the respiratory chain, increasing superoxide production and reducing ATP. The goal of this study was to provide computational proof that succinate dehydrogenase (SDH), a subunit of complex II in the mitochondrial respiratory chain, is a favorable binding partner for NAPQI in this regard. MethodMolecular docking, molecular dynamics simulation, protein-protein interaction networks (PPI), and KEGG metabolic pathway analysis were employed to identify binding characteristics, interaction partners, and their associations with metabolic pathways. A lipid membrane was added to the experimental apparatus to mimic the natural cellular environment of SDH. This modification made it possible to develop a context for investigating the role and interactions of SDH within a cellular ecosystem that was more realistic and biologically relevant. ResultThe molecular binding affinity score for APAP and NAPQI with SDH was predicted −6.5 and −6.7 kcal/mol, respectively. Furthermore, RMSD, RMSF, and Rog from the molecular dynamics simulations study revealed that NAPQI has slightly higher stability and compactness compared to APAP at 100 ns timeframe with mitochondrial SDH. ConclusionThis study serves to predict the mechanistic process of paracetamol toxicity by using different computational approaches. In addition, this study will provide information about the drug target against APAP hepatotoxicity.

Abstract PO2-24-03: Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer known for its rapid progression and high metastatic potential without known distinctive drivers. Currently, there are no FDA-approved targeted therapies specifically for IBC patients, highlighting the urgent need for novel and effective treatments. Through our investigation of metastatic xenograft IBC sublines, we have identified soluble E-cadherin (sEcad), an extracellular proteolytic fragment of full-length E-cadherin, as a protein associated with IBC tumor progression. We hypothesize that sEcad promotes IBC tumorigenesis by suppressing ferroptosis. Methods: MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) IBC cell lines were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Cell death for ferroptosis was measured by propidium iodide staining using flow cytometry. Mass spectrometry and Bio-ID-based proteomics assays were used to identify sEcad-interacting proteins and potential mechanisms. For in vivo studies, control and sEcad -overexpressing SUM149 and MDA-IBC3 cells were injected into the cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Serum sEcad levels from IBC patients (n=301) were analyzed by ELISA. Results: High serum sEcad levels in IBC patients correlated with poorer OS (p=0.02) and earlier development of metastasis (p=0.006). Overexpression of sEcad in IBC cell lines enhanced cell proliferation and colony formation in vitro. Mice injected with sEcad-overexpressing SUM149 and MDA-IBC3 cells had significantly higher tumor growth rates compared to controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, mass spectrometry and Bio-ID assays identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Additionally, sEcad increased the expression of PDIA4, an enzyme that plays a role in cellular redox regulation and protein folding. PDIA4 has been shown to regulate key players of ferroptosis such as ATF4, SLC7A11. Our results showed that, compared to controls, sEcad-overexpressing IBC cells displayed significant resistance to ferroptosis inducers, including RSL3, FIN56, and sulfasalazine. Knockdown of PDIA4 in sEcad high-expressing cells significantly increased their sensitivity to ferroptosis-promoting drugs. Moreover, we found that PDIA4 regulates ATF4 to impact ferroptosis-mediated cell death in IBC cells. Conclusions: Our study shows that sEcad affects ferroptosis through PDIA4, impacting tumorigenesis in IBC. These findings uncover a novel and pivotal role for sEcad in IBC tumor growth and progression, providing new insights and potential therapeutic targets for IBC patients. Citation Format: Xiaoding Hu, Yun Xiong, Emilly Villodre, Juhee Song, Debu Tripathy, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-03.

Evaluation of cyclin D3 as a transcriptional regulator in a neutrophil differentiation model.

Abstract Cyclin D3 is known to regulate cell cycle entry during the G1 to S transition by associating with its binding partners CDK4/6 upon mitogenic stimulation. Cyclin D3 has also been implicated as a transcriptional regulator, but the indirect mechanism employed is not known. The HL60 cell line was used as a model to investigate cyclin D3’s role in altered gene transcription as DMSO-driven differentiation into neutrophils is induced. We have validated this system by evaluating changes in gene expression indicative of progression to the terminal neutrophil state. Interestingly, ectopic expression of cyclin D3 in undifferentiated HL60 (uHL60) cells causes changes in gene expression to mirror that seen in differentiated (dHL60) cells without the need to treat with DMSO. We have also assessed a cohort of target genes hypothesized to be sensitive to cyclin D3-impacted gene transcription as cells transition from uHL60 to dHL60. For those genes that are differentially expressed between uHL60 and dHL60, many are also sensitive to ectopic expression of cyclin D3. Forced expression of cyclin D3 mutants in uHL60 cells suggests that a C-terminal domain within cyclin D3 is responsible for this observed regulation of gene expression, as three mutations (L215M, Q244R, P278R) abrogate the wild-type cyclin D3-induced differentiation gene signature. Ongoing studies aim to elucidate transcription factor binding partners that interact in complex with cyclin D3 to cause differential gene expression.

Stabilization of β-Catenin directs HEB to limit Thymic Selection

Abstract Activation of β-catenin in CD4+CD8+ (DP) thymocytes stalls progression through thymic selection and predisposes to transformation. Leukemogenesis but not the developmental block depends on Tcf-1, β-catenin’s DNA binding partner. Here we show that β-catenin deregulation directs the DNA-binding protein HEB to stall thymocyte development at the thymic selection stage. Conditional loss of HEB along with β-catenin activation restores the frequency of TCRβ+/CCR7+ DP thymocytes, with reduced CXCR4 expression indicating their potential to migrate to the thymic medulla. It also restores the emergence of TCRβ+/CD69+ DPs and enhances the overall CD69+DP expression profile that were diminished by activating β-catenin, indicating a functional response to TCR stimulation. β-Catenin stabilization redirects the binding of HEB to ~11,000 novel DNA sites throughout the genome. A major fraction of these sites occupy promoter regions of genes associated with cell cycle regulation and mitotic processes. HEB binding to those regions corelates with downregulation of the associated genes, and HEB inactivation restores their expression to physiologic levels. These findings highlight the complex molecular interplay between HEB and β-catenin in impairing thymic development.

Abstract 1039: Endothelial Deletion Of Nck1 Reduces Atherosclerosis In Apoe-/- Mice Through The Nck1-atf3 Axis

The Nck family of signaling adaptors, including both Nck1 and Nck2, classically regulate cell motility during vascular development and postnatal angiogenesis. While Nck1 and Nck2 play redundant roles in this regard with deletion of both isoforms required to affect tissue remodeling, we demonstrated that global Nck1 deletion was sufficient to reduce atherosclerotic plaque formation and selective Nck1 inhibition reduces proinflammatory endothelial activation in response to oscillatory shear stress. Therefore, we sought to determine the role of endothelial Nck1 in atherosclerosis and ischemic angiogenesis using novel endothelial-specific Nck1 knockout mice. Endothelial-specific Nck1 deletion significantly reduced Western diet-induced atherosclerotic plaque formation at multiple sites compared to mice expressing endothelial-specific Cre alone or to endothelial-specific Nck2 knockout mice. This reduced plaque formation was characterized by diminished macrophage and smooth muscle incorporation despite enhanced weight gain in endothelial Nck1 knockout mice. RNA Sequencing analysis of endothelial cells in culture showed that Nck1 depletion reduces the expression of a variety of proinflammatory pathways but enhances the expression of genes involved in central carbon metabolism and proliferation. While Nck1 deletion is sufficient to blunt atherogenic inflammation, Nck1 inhibition did not affect angiogenesis or limb perfusion in the femoral artery ligation model of hind limb ischemia. To further characterized Nck1’s proinflammatory role, we utilized affinity precipitation-mass spectrometry and identified Activating Transcription Factor 3 ATF3 as a Nck1-selective binding partner. While no studies to date have examined endothelial ATF3 signaling in atherosclerosis, our preliminary data show that Nck1 is required for ATF3 expression in response to disturbed flow in vitro and in vivo , but the mechanism by which disturbed flow stimulates ATF3 expression remains unknown. ATF3 knockdown reduces disturbed flow-induced proinflammatory gene expression, suggesting that the Nck1-ATF3 signaling axis is a novel regulator of flow-induced inflammation.