Dominant Negative Inhibitor Research Articles

Identifying the amino acid domains of ORF59 responsible for interactions with ORF57 and PAN RNA during KSHV lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor, ORF59, is a lytic protein essential for viral DNA synthesis as part of the core replication complex. The multifunctional nature of ORF59 has prompted the investigation into its various functional domains. Prior studies of ORF59 have identified dimerization, DNA interaction, and polymerase interaction domains. The regions of ORF59 responsible for the interaction with the viral mRNA transport accumulation protein (MTA/ORF57) and the viral long non-coding polyadenylated nuclear (PAN) RNA have not been explored. Using a series of previously characterized ORF59 deletion KSHV BACmid mutants, we identified the domains of ORF59 that interact with ORF57 and PAN RNA. Interestingly, amino acids 51-100 were essential for interacting with both ORF57 and PAN RNA. Using this information, we generated a plasmid that expresses a DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59. When the 30-100 aa DsRed-tagged polypeptide expression plasmid was transfected into KSHV wild-type iSLK cells prior to lytic reactivation, a dominant-negative inhibition of virus replication was observed, resulting in a decrease of infectious virus production. Our data suggest that interactions between ORF59 with ORF57 and PAN RNA are important to successful lytic replication.IMPORTANCETo better understand the Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor ORF59, we investigated the interaction of ORF59 with ORF57 and polyadenylated nuclear (PAN) RNA. We used a previously characterized KSHV BACmid containing internal deletions of ORF59 to identify the domains of ORF59 that interact with ORF57 and PAN RNA. Our study revealed multiple domains of ORF59 that are essential for its association with PAN RNA. These domains span amino acids 51-100, 251-300, and 351-396. Additional experiments confirmed amino acids 51-100 are critical for the interaction between ORF59 and ORF57. Using this information, we generated an expression plasmid encompassing the ORF57 and PAN RNA interaction domains of ORF59. The ORF59 polypeptide expression plasmid of amino acids 30-100 functioned as a dominant negative inhibitor during viral reactivation and caused a decrease in virus production. These findings provide valuable insights into the key domains of ORF59, essential for its functionality, and ultimately the production of infectious viruses.

An improved TEAD dominant-negative protein inhibitor to study Hippo YAP1/TAZ-dependent transcription.

Hippo signaling is one of the top pathways altered in human cancer, and intensive focus has been devoted to developing therapies targeting Hippo-dependent transcription mediated by YAP1 and TAZ interaction with TEAD proteins. However, a significant challenge in evaluating the efficacy of these approaches is the lack of models that can precisely characterize the consequences of TEAD inhibition. To address this gap, our laboratory developed a strategy that utilizes a fluorescently traceable, dominant-negative protein named TEADi. TEADi specifically blocks the nuclear interactions of TEAD with YAP1 and TAZ, enabling precise dissection of Hippo TEAD-dependent and independent effects on cell fate. In this study, we aimed to enhance TEADi effectiveness by altering post-transcriptional modification sites within its TEAD-binding domains (TBDs). We demonstrate that a D93E mutation in the YAP1 TBD significantly increases TEADi inhibitory capacity. Additionally, we find that TBDs derived from VGLL4 and YAP1 are insufficient to block TAZ-induced TEAD activity, revealing crucial differences in YAP1 and TAZ displacement mechanisms by dominant-negative TBDs. Structural differences in YAP1 and TAZ TBDs were also identified, which may contribute to the distinct binding of these proteins to TEAD. Our findings expand our understanding of TEAD regulation and highlight the potential of an optimized TEADi as a more potent, specific, and versatile tool for studying TEAD-transcriptional activity.

Abstract Tu121: Investigating Mechanisms Of MCUB Inhibition Of Mitochondrial Calcium Uptake

The mitochondrial calcium uniporter mediates Ca 2+ entry into the matrix, and matches energetic supply to demand during cardiac activity. However, many cardiac diseases are characterized by mitochondrial Ca 2+ overload, such as ischemia/reperfusion (I/R) injury. The uniporter channel is typically formed by a tetramer of MCU subunits. These subunits have two transmembrane domains and create a pore lined with negative charges to allow rapid and selective Ca 2+ permeation. Intriguingly, many studies have shown that a close homolog, MCUB can potentially oligomerize with MCU subunits to alter the pore. When the tetramer is made of MCU Ca 2+ transmission is facilitated, but the presence of MCUB inhibits Ca 2+ intake through the channel, suggesting that it is a dominant negative subunit. Moreover, recent studies in animals subject to ischemic cardiovascular injury has revealed endogenous upregulation of the MCUB subunit, proposed as an endogenous mechanism to prevent Ca 2+ overload. Deletion of MCUB enhances activation and uptake when exposed to high Ca 2+ , making MCUB −/− cells prone to mitochondrial Ca 2+ overload and pore opening. Here, we investigate the mechanisms by which MCUB regulates the behavior of the channel, focusing on how MCUB may influence complex composition and regulate its targeting. For this study, we performed structure-function studies of reconstituted MCUB in MCU -/- MCUB -/- double knockout HEK 293T cells. Using modified MCUB constructs and a range of MCU to MCUB transfection ratios, we queried how many MCUB subunits can successfully incorporate into uniporter complex. We subsequently created a range of tetrameric concatemers featuring different ratios of MCU to MCUB, allowing us to observe that increasing amounts of MCUB incorporation lead to greater inhibition of mitochondrial Ca 2+ uptake. Finally, we examined whether replacing portions of MCUB with the corresponding MCU sequence was able to rescue Ca 2+ uptake, finding that these substitutions failed to significantly increase mitochondrial Ca 2+ uptake. Our findings clarify how MCUB incorporation into the uniporter complex leads to dominant-negative inhibition of mitochondrial Ca 2+ uptake.

Abstract 3101: Developing a drug repurposing screen for head and neck squamous cell carcinoma using a novel c-Rel bioassay

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Approximately 890,000 new cases are diagnosed each year, while incidence continues to rise and is anticipated to increase by 30% by 2030. HNSCC occurs most often in men in their 50-60s, although the incidence among younger individuals is increasing. Current standard of care practices include surgical resection, followed by adjuvant radiation or chemoradiation. HNSCC tumors exhibit frequent mutation of CDKN2A (22% of tumors) and TP53 (72% of tumors). Additionally, two members of the TP53 gene family, TP63 and TP73, are frequently altered in HNSCC. TP63 encodes two major isoforms, ΔNp63 which lacks the main transcription activation domain and acts as a dominant-negative inhibitor of transactivation (TA), and TAp63 (contains a p53-like TA domain). ΔNp63 is overexpressed in a majority of HNSCC tumors. Our lab has reported ΔNp63α interacts with activated c-Rel (a nuclear factor-KB family member) in HNSCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers. Consistent with a role in growth regulation, ΔNp63α:c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human HNSCCs and normal keratinocytes overexpressing ΔNp63α. The direct relationship between ΔNp63α and activated c-Rel is observed by strong nuclear co-localization in the proliferating compartment of primary head and neck SCC. Stimulation of HNSCC cells with TNFα results in the induction of the c-Rel oncoprotein that binds to ΔNp63, displacing and inactivating the tumor suppressor TAp73 from ΔNp63-TAp73 complexes. Using western blot analysis in human HNSCC cell lines, we confirmed that stimulation with TNFα enhances nuclear c-Rel localization. We then set out to design a high throughput screening bioassay for the purpose of evaluating compounds that can inhibit c-Rel accumulation in the nucleus under TNFα stimulation. By preventing activated c-Rel from interacting with ΔNp63α in the nucleus we aim to decrease uncontrolled proliferation and restore the tumor-suppressive functionality of the ΔNp63-TAp73 complex. A 384-well immunocytochemistry assay was developed using a confocal microscopy readout and an automated image analysis algorithm to quantify nuclear translocation. The optimized assay will be used to screen the NCATS collection of approved drugs and investigational oncology agents. Top candidates will undergo further investigation in vitro, ex vivo, and in vivo to explore the possibility of repurposing the small molecules for HNSCC therapeutics. Citation Format: Kristin Ann Altwegg, Kathryn E. King, Dvir Blivis, Ty C. Voss, Natalia J. Martinez, Wendy C. Weinberg, Mark J. Henderson. Developing a drug repurposing screen for head and neck squamous cell carcinoma using a novel c-Rel bioassay [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 3101.

Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1

The role of glycan-binding proteins as an activator of immune regulatory receptors has gained attention recently. We report that galectin 7 reduced CD4+ T cell percentage in both in vitro culture and mouse tumor models. Immunohistochemical staining of esophageal cancer patient samples showed a lower percentage of CD4+ cells in the galectin 7 high area. The lack of CD4+ T cell depletion by galectin 7 in PD-1 knockout mice supports the role of PD-1 in mediating the effects of galectin 7. The binding assays demonstrate that galectin 7 binds to the N-glycosylation of PD-1 on N74 and N116 sites and leads to the recruitment of SHP-2. NFAT suppressive activity of galectin 7 was abrogated upon overexpression of the dominant negative SHP-2 mutant or inhibition of PD-1 by siRNA. Glycosylation of PD-1 has been reported to play a critical role in surface expression, stability, and interaction with its ligand PD-L1. This report further expands the significance of PD-1 glycosylation and suggests that galectin 7, a glycan-binding protein, interacts with the immune regulatory receptor PD-1 through glycosylation recognition.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

RNA Binding Proteins that Mediate LPS-induced Alternative Splicing of the MyD88 Innate Immune Regulator

Inflammation driven by Toll-like receptor (TLR) signaling pathways is required to combat infection. However, inflammation can damage host tissues; thus it is essential that TLR signaling ultimately is terminated to prevent chronic inflammatory disorders. One mechanism that terminates persistent TLR signaling is alternative splicing of the MyD88 signaling adaptor, which functions in multiple TLR signaling pathways. While the canonical long isoform of MyD88 (MyD88-L) mediates TLR signaling and promotes inflammation, an alternatively-spliced shorter isoform of MyD88 (MyD88-S) produces a dominant negative inhibitor of TLR signaling. MyD88-S production is induced by inflammatory agonists including lipopolysaccharide (LPS), and thus MyD88-S induction is thought to act as a negative feedback loop that prevents chronic inflammation. Despite the potential role that MyD88-S production plays in inflammatory disorders, the mechanisms controlling MyD88 alternative splicing remain unclear. Here, we identify two RNA binding proteins, SRSF1 and HNRNPU, that regulate LPS-induced alternative splicing of MyD88.

Aquaporin splice variation differentially modulates channel function during marine teleost egg hydration.

Aquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.

A transcriptional activator effector of Ustilago maydis regulates hyperplasia in maize during pathogen-induced tumor formation

Ustilago maydis causes common smut in maize, which is characterized by tumor formation in aerial parts of maize. Tumors result from the de novo cell division of highly developed bundle sheath and subsequent cell enlargement. However, the molecular mechanisms underlying tumorigenesis are still largely unknown. Here, we characterize the U. maydis effector Sts2 (Small tumor on seedlings 2), which promotes the division of hyperplasia tumor cells. Upon infection, Sts2 is translocated into the maize cell nucleus, where it acts as a transcriptional activator, and the transactivation activity is crucial for its virulence function. Sts2 interacts with ZmNECAP1, a yet undescribed plant transcriptional activator, and it activates the expression of several leaf developmental regulators to potentiate tumor formation. On the contrary, fusion of a suppressive SRDX-motif to Sts2 causes dominant negative inhibition of tumor formation, underpinning the central role of Sts2 for tumorigenesis. Our results not only disclose the virulence mechanism of a tumorigenic effector, but also reveal the essential role of leaf developmental regulators in pathogen-induced tumor formation.

TAp73 Inhibits EMT and Cell Migration in Pancreatic Cancer Cells through Promoting SMAD4 Expression and SMAD4-Dependent Inhibition of ERK Activation.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-β. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-β/Smad signaling, while preventing non-canonical TGF-β signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-β1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73-but not the β isoform-interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-β-stimulated pro-invasive ERK activation as an underlying mechanism.

Heterozygosity for cervid S138N polymorphism results in subclinical CWD in gene-targeted mice and progressive inhibition of prion conversion

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

Blocking soluble TNFα sensitizes HER2-positive breast cancer to trastuzumab through MUC4 downregulation and subverts immunosuppression

BackgroundThe success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields...

Abstract P1-11-12: Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model

Abstract Background. Clinical trials have demonstrated that trastuzumab deruxtecan (T-DXd) provides a durable responses for patients with HER2-positive and HER2 low metastatic breast cancer (BC). With T-DXd treatment, approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We previously shown that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2-positive BC patients. We also showed that MUC4 is upregulated by soluble TNFα (sTNFα) secreted by the tumor, which confers primary trastuzumab resistance since it hides trastuzumab epitope on the HER2 molecule, reducing its binding and diminishing its therapeutic effects. In preclinical models of de novo trastuzumab-resistant tumors, we proved that administration of the sTNFα blocking agent INB03 (DN) together with trastuzumab inhibited tumor growth and induced an innate immune response in the tumor microenvironment (TME). DN is a dominant-negative inhibitor of sTNFα that is not immunosuppressive because it does not affect transmembrane TNFα. Our goal is to study whether neutralizing sTNFα can improve T-DXd effects in a multiple HER2-targeted therapy-resistant model. Methods JIMT-1 is a HER2-positive BC cell line resistant to trastuzumab, pertuzumab and lapatinib, which expresses MUC4. JIMT-1 tumor-bearing nude mice were treated with (1) IgG 5 mg/kg, (2) T-DXd 5 mg/kg, (3) T-DXd 2.5 mg/kg, (4) T-DXd 1.25 mg/kg, (5) DN 10 mg/kg, (6) T-DXd 5 mg/kg +DN, (7) T-DXd 2.5 mg/kg +DN and (8) T-DXd 1.25 mg/kg +DN. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored regularly. Tumor-infiltrating macrophages, NK cells and myeloid-derived suppressor cells (MDSCs) were evaluated by immunofluorescence and flow cytometry. Results The dose-response curve of T-DXd exhibited tumor growth inhibitions of 83% (5 mg/kg), 61% (2.5 mg/kg) and 37% (1.25 mg/kg) vs IgG-treated tumors. DN alone had no antitumor effect. Combination of T-DXd with DN resulted in a reinforced antitumor effect, as the tumor growth inhibition escalated to 98% (T-DXd 5 mg/kg+ DN), 81% (T-DXd 2.5 mg/kg+DN) and 73% (T-DXd 1.25 mg/kg+DN). Moreover, we observed that addition of DN to T-DXd 1.25 and 5 mg/kg enhances the infiltration of resident macrophages (p < 0.05 and p<0.01, respectively) and promotes polarization to the M1-like phenotype (p < 0.05 and p<0.001, respectively). While T-DXd 2.5 and 5 mg/kg alone achieved a decrease in M2-like macrophages (p < 0.05), combination of T-DXd 1.25mg/kg+DN impaired M2-like polarization at similar levels of that observed with 2.5 and 5 mg/kg T-DXd (p < 0.05). Notably, the M1/M2 ratio escalated from 10.9% to 51.5% when DN was added to the lowest T-DXd dose (p < 0.01). In addition, T-DXd 2.5 and 5 mg/kg induce an increase in the proportion of NK cells in TME (p < 0.001 and p<0.05, respectively), which T-DXd 1.25 mg/kg+ DN treatment mimicked (p < 0.05). Although an increase in NK cell activation was observed with 1.25 and 2.5 mg/kg treatments (p < 0.05 and p<0.001, respectively), adding DN did not further improved this effect. Only the highest dose of T-DXd in combination with DN was able to increase NK cell degranulation (p < 0.05), compared to T-DXd alone. Finally, the percentage of MDSCs population decreases with the addition of DN to the T-DXd 2.5 and 5 mg/kg. Conclusions Our results suggest that sTNFα blockade is able to enhance T-DXd effect in a multiple HER2-targeted therapy resistant model. Notably, the administration of T-DXd 1.25 mg/kg+DN achieved a similar antitumor effect to T-DXd 5 mg/kg alone and also transforms the TME to an antitumor one with a reinforced immune response. This finding highlights that sTNFα and MUC4 expression are important variables in the response to T-DXd. Neutralization of sTNFα may open new therapeutic strategies for treatment of patients who present with MUC4 expression or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Cecilia Proietti, Rosalia Cordo-Russo, Mercogliano María Florencia, Roxana Schillaci. Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-12.

Mapping functional regions of essential bacterial proteins with dominant-negative protein fragments.

Fragments of native proteins can act as dominant negative inhibitors, representing a substantial stress for cells. Massively-parallel measurements of growth inhibition by protein fragments have been used to discover peptide inhibitors and map protein interaction sites. However, the underlying principles governing fragment-based inhibition have thus far remained unclear. Here, we adapt a high-throughput inhibitory fragment assay for use in Escherichia coli, applying it to a set of ten essential proteins. This approach yielded single amino acid resolution maps of inhibitory activity, with peaks localized to functionally important interaction sites, including oligomerization interfaces and folding contacts. Leveraging these data, we perform a systematic analysis to uncover principles of fragment-based inhibition. We determine a robust negative correlation between susceptibility to inhibition and cellular protein concentration, demonstrating that inhibitory fragments likely act primarily by titrating native protein interactions. We also characterize a series of trade-offs related to fragment length, showing that shorter peptides allow higher-resolution mapping but suffer from lower activity. We employ an unsupervised statistical analysis to show that the inhibitory activities of protein fragments are largely driven not by generic properties such as charge, hydrophobicity, and secondary structure, but by the more specific characteristics of their bespoke macromolecular interactions. AlphaFold computational modeling of peptide complexes with one protein shows that the inhibitory activity of peptides is associated with their predicted ability to form native-like interactions. Overall, this work demonstrates fundamental characteristics of inhibitory protein fragment function and provides a foundation for understanding and controlling protein interactions in vivo.

Inhibition of Foxp4 Disrupts Cadherin-based Adhesion of Radial Glial Cells, Leading to Abnormal Differentiation and Migration of Cortical Neurons in Mice.

Heterozygous loss-of-function variants of FOXP4 are associated with neurodevelopmental disorders (NDDs) that exhibit delayed speech development, intellectual disability, and congenital abnormalities. The etiology of NDDs is unclear. Here we found that FOXP4 and N-cadherin are expressed in the nuclei and apical end-feet of radial glial cells (RGCs), respectively, in the mouse neocortex during early gestation. Knockdown or dominant-negative inhibition of Foxp4 abolishes the apical condensation of N-cadherin in RGCs and the integrity of neuroepithelium in the ventricular zone (VZ). Inhibition of Foxp4 leads to impeded radial migration of cortical neurons and ectopic neurogenesis from the proliferating VZ. The ectopic differentiation and deficient migration disappear when N-cadherin is over-expressed in RGCs. The data indicate that Foxp4 is essential for N-cadherin-based adherens junctions, the loss of which leads to periventricular heterotopias. We hypothesize that FOXP4 variant-associated NDDs may be caused by disruption of the adherens junctions and malformation of the cerebral cortex.

Alternative pre-mRNA splicing as a mechanism for terminating Toll-like Receptor signaling.

While inflammation induced by Toll-like receptor (TLR) signaling is required to combat infection, persistent inflammation can damage host tissues and contribute to a myriad of acute and chronic inflammatory disorders. Thus, it is essential not only that TLR signaling be activated in the presence of pathogens but that TLR signaling is ultimately terminated. One mechanism that limits persistent TLR signaling is alternative pre-mRNA splicing. In addition to encoding the canonical mRNAs that produce proteins that promote inflammation, many genes in the TLR signaling pathway also encode alternative mRNAs that produce proteins that are dominant negative inhibitors of signaling. Many of these negative regulators are induced by immune challenge, so production of these alternative isoforms represents a negative feedback loop that limits persistent inflammation. While these alternative splicing events have been investigated on a gene by gene basis, there has been limited systemic analysis of this mechanism that terminates TLR signaling. Here we review what is known about the production of negatively acting alternative isoforms in the TLR signaling pathway including how these inhibitors function, how they are produced, and what role they may play in inflammatory disease.

Identification of genomic binding sites and direct target genes for the transcription factor DDIT3/CHOP

DDIT3 is a tightly regulated basic leucine zipper (bZIP) transcription factor and key regulator in cellular stress responses. It is involved in a variety of pathological conditions and may cause cell cycle block and apoptosis. It is also implicated in differentiation of some specialized cell types and as an oncogene in several types of cancer. DDIT3 was originally believed to act as a dominant-negative inhibitor by forming heterodimers with other bZIP transcription factors, preventing their DNA binding and transactivating functions. DDIT3 has, however, been reported to bind DNA and regulate target genes. Here, we employed ChIP sequencing combined with microarray-based expression analysis to identify direct binding motifs and target genes of DDIT3. The results reveal DDIT3 binding to motifs similar to other bZIP transcription factors, known to form heterodimers with DDIT3. Binding to a class III satellite DNA repeat sequence was also detected. DDIT3 acted as a DNA-binding transcription factor and bound mainly to the promotor region of regulated genes. ChIP sequencing analysis of histone H3K27 methylation and acetylation showed a strong overlap between H3K27-acetylated marks and DDIT3 binding. These results support a role for DDIT3 as a transcriptional regulator of H3K27ac-marked genes in transcriptionally active chromatin.

ODP571 Blockade of ErbB-2 Nuclear Function Induces the Interferon Signaling Pathway in Breast Cancer Models Resistant to Trastuzumab

Abstract ErbB-2, a member of ErbB family of receptor tyrosine kinases, is a key oncogenic driver in breast cancer. Despite clinical efficiency of ErbB-2-targeted therapies (trastuzumab, TZ), resistance to drugs is a major issue in the clinic. While ErbB-2 is mainly a plasma membrane-bound receptor, it also migrates to the nucleus (NErbB-2) where it can act as a transcription factor or coactivator. We previously reported that NErbB-2 is a major proliferation driver in TZ-resistant breast cancer. To investigate the NErbB-2 dependent transcriptome, RNAseq was performed using a TZ-resistant breast cancer model (JIMT-1 cells) with high constitutive levels of NErbB-2. JIMT-1 cells were transfected with an ErbB-2 nuclear localization domain mutant (hErbB-2ΔNLS), which also acts as a dominant-negative inhibitor of endogenous NErbB-2 migration. Exclusion of ErbB-2 from the nucleus resulted in up-regulation of 280 genes and down-regulation of 33 genes. Functional analysis revealed that NErbB-2 blockade enriched the expression of genes involved in type-I interferon (IFN) signaling pathway. IFNB1 and its downstream effectors OAS2 and TRIM22 were among the top up-regulated genes. In an independent breast cancer model (i. e., HCC-1569 cells), exclusion of NErbB-2 from the nucleus also induced expression of these genes. Blockade of NErbB-2 localization by injection of the hErbB-2ΔNLS mutant into JIMT-1 tumor xenografts significantly inhibited in vivo tumor growth and induced mRNA expression of IFNB1, OAS2 and TRIM22. Interestingly, blockade of NErbB-2 localization by treatment with Retro-2, an inhibitor of the retrograde transport, showed similar effects consistent with modulation of the IFN signaling pathway by NErbB-2. Bioinformatic analyses showed that both the promoter and the coding region of the IFNB1 gene contain ErbB-2 associated sequences (HAS sites). ChIP-PCR analyses revealed ErbB-2 recruitment to the HAS sites of the IFNB1 promoter and coding regions in normal growth conditions. Transfection of JIMT-1 cells with the hErbB-2ΔNLS mutant abolished the recruitment of ErbB-2 at the IFNB1 gene and also caused an increase in histone H4 acetylation, a marker of active gene transcription. NErbB-2 immunostaining in a cohort of 32 primary invasive ErbB-2-positive breast carcinomas treated with TZ revealed that NErbB-2 expression correlated with a poor disease-free survival. While this cohort is small, the findings suggest that NErbB-2 could be used as a biomarker of poor response to TZ in the clinic. In summary, our findings indicate that NErbB-2 drives the growth of TZ-resistant breast cancer cells via transcriptional repression of the IFNB1 signaling pathway, and highlight NErbB-2 as a therapeutic target and biomarker in TZ-resistant breast cancer. Presentation: No date and time listed

The Pathological Mechanisms of Hearing Loss Caused by KCNQ1 and KCNQ4 Variants.

Deafness-associated genes KCNQ1 (also associated with heart diseases) and KCNQ4 (only associated with hearing loss) encode the homotetrameric voltage-gated potassium ion channels Kv7.1 and Kv7.4, respectively. To date, over 700 KCNQ1 and over 70 KCNQ4 variants have been identified in patients. The vast majority of these variants are inherited dominantly, and their pathogenicity is often explained by dominant-negative inhibition or haploinsufficiency. Our recent study unexpectedly identified cell-death-inducing cytotoxicity in several Kv7.1 and Kv7.4 variants. Elucidation of this cytotoxicity mechanism and identification of its modifiers (drugs) have great potential for aiding the development of a novel pharmacological strategy against many pathogenic KCNQ variants. The purpose of this review is to disseminate this emerging pathological role of Kv7 variants and to underscore the importance of experimentally characterizing disease-associated variants.

Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma

Histone H3 mutations at amino acids 27 (H3K27M) and 34 (H3G34R) are recurrent drivers of pediatric-type high-grade glioma (pHGG). H3K27M mutations lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, their broader oncogenic mechanisms remain unclear. We characterized the H3.1K27M, H3.3K27M and H3.3G34R interactomes, finding that H3K27M is associated with epigenetic and transcription factor changes; in contrast H3G34R removes a break on cryptic transcription, limits DNA methyltransferase access, and alters mitochondrial metabolism. All 3 mutants had altered interactions with DNA repair proteins and H3K9 methyltransferases. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. H3K9 methyltransferase inhibition was lethal to H3.1K27M, H3.3K27M and H3.3G34R pHGG cells, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it as an attractive therapeutic target.