Interaction Motif Research Articles

EML2-S constitutes a new class of proteins that recognizes and regulates the dynamics of tyrosinated microtubules

Tubulin post-translational modifications (PTMs) alter microtubule properties by affecting the binding of microtubule-associated proteins (MAPs). Microtubule detyrosination, which occurs by proteolytic removal of the C-terminal tyrosine from ɑ-tubulin, generates the oldest known tubulin PTM, but we lack comprehensive knowledge of MAPs that are regulated by this PTM. We developed a screening pipeline to identify proteins that discriminate between Y- and ΔY-microtubules and found that echinoderm microtubule-associated protein-like 2 (EML2) preferentially interacts with Y-microtubules. This activity depends on a Y-microtubule interaction motif built from WD40 repeats. We show that EML2 tracks the tips of shortening microtubules, a behavior not previously seen among human MAPs invivo, and influences dynamics to increase microtubule stability. Our screening pipeline is readily adapted to identify proteins that specifically recognize a wide range of microtubule PTMs.

Identification of a non-canonical G3BP-binding sequence in a Mayaro virus nsP3 hypervariable domain.

Ras-GTPase-activating SH3 domain-binding-proteins 1 (G3BP1) and 2 (G3BP2) are multifunctional RNA-binding proteins involved in stress granule nucleation, previously identified as essential cofactors of Old World alphaviruses. They are recruited to viral replication complexes formed by the Chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) via an interaction with a duplicated FGxF motif conserved in the hypervariable domain (HVD) of virus-encoded nsP3. According to mutagenesis studies, this FGxF duplication is strictly required for G3BP binding and optimal viral growth. Contrasting with this scenario, nsP3 encoded by Mayaro virus (MAYV), an arthritogenic virus grouped with Old World alphaviruses, contains a single canonical FGxF sequence. In light of this unusual feature, we questioned MAYV nsP3/G3BPs relationships. We report that G3BP1 and G3BP2 are both required for MAYV growth in human cells and bind nsP3 protein. In infected cells, they are recruited to nsP3-containing cytosolic foci and active replication complexes. Unexpectedly, deletion of the single FGxF sequence in MAYV nsP3 did not abolish these phenotypes. Using mutagenesis and in silico modeling, we identify an upstream FGAP amino acid sequence as an additional MAYV nsP3/G3BP interaction motif required for optimal viral infectivity. These results, therefore, highlight a non-conventional G3BP binding sequence in MAYV nsP3.

Many ways to TOPLESS - manipulation of plant auxin signalling by a cluster of fungal effectors.

Plant biotrophic pathogens employ secreted molecules, called effectors, to suppress the host immune system and redirect the host's metabolism and development in their favour. Putative effectors of the gall-inducing maize pathogenic fungus Ustilago maydis were analysed for their ability to induce auxin signalling in plants. Using genetic, biochemical, cell-biological, and bioinformatic approaches we functionally elucidate a set of five, genetically linked effectors, called Topless (TPL) interacting protein (Tips) effectors that induce auxin signalling. We show that Tips induce auxin signalling by interfering with central corepressors of the TPL family. CRISPR-Cas9 mutants and deletion strain analysis indicate that the auxin signalling inducing subcluster effectors plays a redundant role in virulence. Although none of the Tips seem to have a conserved interaction motif, four of them bind solely to the N-terminal TPL domain and, for Tip1 and Tip4, we demonstrate direct competition with auxin/indole-3-acetic acid transcriptional repressors for their binding to TPL class of corepressors. Our findings reveal that TPL proteins, key regulators of growth-defence antagonism, are a major target of the U. maydis effectome.

Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition.

The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

What a Difference an Amino Acid Makes: An All-Atom Simulation Study of Nonameric Peptides in Inhibitory HLA-E/NKG2A/CD94 Immune Complexes.

MHC class I antigen E (HLA-E), a ligand for the inhibitory NKG2A/CD94 receptor of the immune system, is responsible for evading the immune surveillance in several settings, including senescent cell accumulation and tumor persistence. The formation of this ligand-receptor interaction promotes the inhibition of the cytolytic action of immune system natural killer (NK) cells and CD8+ T-cells expressing this receptor. The final outcome of the HLA-E/NKG2A/CD94 interaction on target cells is also highly dependent on the identity of the nonameric peptide incorporated into the HLA-E ligand. To better understand the role played by a nonameric peptide in these immune complexes, we performed a series of multi-microsecond all-atom molecular dynamics simulations. We generated natural and alternative variants of the nonameric peptide bound to the HLA-E ligand alone or in the HLA-E/NKG2A/CD94 complexes. A systematic study of molecular recognition between HLA-E and peptides led to the development of new variants that differ at the strategic 6th position (P6) of the peptide and have favorable in silico properties comparable to those of natural binding peptides. Further examination of a selected subset of peptides in full complexes revealed a new variant that, according to our previously derived atomistic model, can interfere with the signal transduction via HLA-E/NKG2A/CD94 and thus prevent the target cell from evading immune clearance by NK and CD8+ T-cells. These simulations provide an atomistic picture of how a small change in amino acid sequence can lead to a profound effect on binding and molecular recognition. Furthermore, our study also provides new data on the peptide interaction motifs as well as the energetic and conformational properties of the binding interface, laying the structure-based foundation for future development of potential therapeutic peptides, peptidomimetics, or even small molecules that would bind to the HLA-E ligand and abrogate NKG2A/CD94 recognition. Such external intervention would be useful in the emerging field of targeting senescent cells in a variety of age-related diseases, as well as in novel cancer immunotherapies.

Molecular Compatibility and Hydrogen Bonding Mechanism of PES/PEI Blends.

The development of high-performance polymer membranes has sparked a lot of attention in recent years. Polymer blending is a potential method of modification. A limitation, however, is the compatibility of blends at the molecular level. In this investigation, polyethersulfone/polyetherimide hollow fiber membranes were prepared by the solution blending method. Compatibility, hydrogen bonding, crystallinity, microstructure, hydrophilicity, mechanical properties, and transmissibility of blended membranes were also characterized. The compatibility and hydrogen bonding action of the two components were confirmed by DSC, FTIR, XPS, and XRD. The structure exhibits a C−H···O interaction motif with the sulfone group acting as a hydrogen bond acceptor from a methyl C−H donor. The π–π stacking between the two polymers arranged molecules more orderly, resulting in enhanced intermolecular interactions. Compared to polyethersulfone hollow fiber membranes, the hydrophilic, mechanical properties, and rejection rate of the blended membranes are more effectively enhanced. Self-assembly of the host polymer with a polymer capable of forming hydrogen bonds to construct controllable blends is a crucial and proven method.

Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f. sp. hordei.

In barley (Hordeum vulgare), signalling rat sarcoma homolog (RHO) of plants guanosine triphosphate hydrolases (ROP GTPases) support the penetration success of Blumeria graminis f. sp. hordei but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of ROP‐bound GDP for GTP and thereby turn ROPs into a signalling‐activated ROP‐GTP state. Plants possess a unique class of GEFs harbouring a plant‐specific ROP nucleotide exchanger domain (PRONE). Here, we performed phylogenetic analyses and annotated barley PRONE‐GEFs. The leaf epidermal‐expressed PRONE‐GEF HvGEF14 undergoes a transcriptional down‐regulation on inoculation with B. graminis f. sp. hordei and directly interacts with the ROP GTPase and susceptibility factor HvRACB in yeast and in planta. Overexpression of activated HvRACB or of HvGEF14 led to the recruitment of ROP downstream interactor HvRIC171 to the cell periphery. HvGEF14 further supported direct interaction of HvRACB with a HvRACB‐GTP‐binding CRIB (Cdc42/Rac Interactive Binding motif) domain‐containing HvRIC171 truncation. Finally, the overexpression of HvGEF14 caused enhanced susceptibility to fungal entry, while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells during fungal penetration.

A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4–NOT deadenylase complex subunits to repress target mRNAs

Pumilio is a sequence-specific RNA-binding protein that controls development, stem cell fate, and neurological functions in Drosophila. Pumilio represses protein expression by destabilizing target mRNAs in a manner dependent on the CCR4–NOT deadenylase complex. Three unique repression domains in the N-terminal region of Pumilio were previously shown to recruit CCR4–NOT, but how they do so was not well understood. In this study, we identified the motifs that are necessary and sufficient for the activity of the third repression domain of Pumilio, designated RD3, which is present in all isoforms and has conserved regulatory function. We identified multiple conserved regions of RD3 that are important for repression activity in cell-based reporter gene assays. Using yeast two-hybrid assays, we show that RD3 contacts specific regions of the Not1, Not2, and Not3 subunits of the CCR4–NOT complex. Our results indicate that RD3 makes multivalent interactions with CCR4–NOT mediated by conserved short linear interaction motifs. Specifically, two phenylalanine residues in RD3 make crucial contacts with Not1 that are essential for its repression activity. Using reporter gene assays, we also identify three new target mRNAs that are repressed by Pumilio and show that RD3 contributes to their regulation. Together, these results provide important insights into the mechanism by which Pumilio recruits CCR4–NOT to regulate the expression of target mRNAs.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

Environment Constrains Fitness Advantages of Division of Labor in Microbial Consortia Engineered for Metabolite Push or Pull Interactions.

ABSTRACTFitness benefits from division of labor are well documented in microbial consortia, but the dependency of the benefits on environmental context is poorly understood. Two synthetic Escherichia coli consortia were built to test the relationships between exchanged organic acid, local environment, and opportunity costs of different metabolic strategies. Opportunity costs quantify benefits not realized due to selecting one phenotype over another. The consortia catabolized glucose and exchanged either acetic or lactic acid to create producer-consumer food webs. The organic acids had different inhibitory properties and different opportunity costs associated with their positions in central metabolism. The exchanged metabolites modulated different consortial dynamics. The acetic acid-exchanging (AAE) consortium had a “push” interaction motif where acetic acid was secreted faster by the producer than the consumer imported it, while the lactic acid-exchanging (LAE) consortium had a “pull” interaction motif where the consumer imported lactic acid at a comparable rate to its production. The LAE consortium outperformed wild-type (WT) batch cultures under the environmental context of weakly buffered conditions, achieving a 55% increase in biomass titer, a 51% increase in biomass per proton yield, an 86% increase in substrate conversion, and the complete elimination of by-product accumulation all relative to the WT. However, the LAE consortium had the trade-off of a 42% lower specific growth rate. The AAE consortium did not outperform the WT in any considered performance metric. Performance advantages of the LAE consortium were sensitive to environment; increasing the medium buffering capacity negated the performance advantages compared to WT.IMPORTANCE Most naturally occurring microorganisms persist in consortia where metabolic interactions are common and often essential to ecosystem function. This study uses synthetic ecology to test how different cellular interaction motifs influence performance properties of consortia. Environmental context ultimately controlled the division of labor performance as shifts from weakly buffered to highly buffered conditions negated the benefits of the strategy. Understanding the limits of division of labor advances our understanding of natural community functioning, which is central to nutrient cycling and provides design rules for assembling consortia used in applied bioprocessing.

Dynamics of Protein Phosphorylation during Arabidopsis Seed Germination.

Seed germination is critical for early plantlet development and is tightly controlled by environmental factors. Nevertheless, the signaling networks underlying germination control remain elusive. In this study, the remodeling of Arabidopsis seed phosphoproteome during imbibition was investigated using stable isotope dimethyl labeling and nanoLC-MS/MS analysis. Freshly harvested seeds were imbibed under dark or constant light to restrict or promote germination, respectively. For each light regime, phosphoproteins were extracted and identified from dry and imbibed (6 h, 16 h, and 24 h) seeds. A large repertoire of 10,244 phosphopeptides from 2546 phosphoproteins, including 110 protein kinases and key regulators of seed germination such as Delay Of Germination 1 (DOG1), was established. Most phosphoproteins were only identified in dry seeds. Early imbibition led to a similar massive downregulation in dormant and non-dormant seeds. After 24 h, 411 phosphoproteins were specifically identified in non-dormant seeds. Gene ontology analyses revealed their involvement in RNA and protein metabolism, transport, and signaling. In addition, 489 phosphopeptides were quantified, and 234 exhibited up or downregulation during imbibition. Interaction networks and motif analyses revealed their association with potential signaling modules involved in germination control. Our study provides evidence of a major role of phosphosignaling in the regulation of Arabidopsis seed germination.

Signalling mechanisms and cellular functions of SUMO.

Sumoylation is an essential post-translational modification that is catalysed by a small number of modifying enzymes but regulates thousands of target proteins in a dynamic manner. Small ubiquitin-like modifiers (SUMOs) can be attached to target proteins as one or more monomers or in the form of polymers of different types. Non-covalent readers recognize SUMO-modified proteins via SUMO interaction motifs. SUMO simultaneously modifies groups of functionally related proteins to regulate predominantly nuclear processes, including gene expression, the DNA damage response, RNA processing, cell cycle progression and proteostasis. Recent progress has increased our understanding of the cellular and pathophysiological roles of SUMO modifications, extending their functions to the regulation of immunity, pluripotency and nuclear body assembly in response to oxidative stress, which partly occurs through the recently characterized mechanism of liquid-liquid phase separation. Such progress in understanding the roles and regulation of sumoylation opens new avenues for the targeting of SUMO to treat disease, and indeed the first drug blocking sumoylation is currently under investigation in clinical trials as a possible anticancer agent.

Molecular basis of Tick Born encephalitis virus NS5 mediated subversion of apico-basal cell polarity signalling.

The Scribble (Scrib) protein is a conserved cell polarity regulator with anti-tumorigenic properties. Viruses like the Tick-born encephalitis virus (TBEV) target Scribble to establish a cellular environment supporting viral replication, which is ultimately associated with poor prognosis upon infection. The TBEV NS5 protein has been reported to harbour both an internal as well as a C-terminal PDZ binding motif (PBM), however only the internal PBM was shown to be an interactor with Scribble, with the interaction being mediated via the Scribble PDZ4 domain to antagonize host interferon responses. We examined the NS5 PBM motif interactions with all Scribble PDZ domains using isothermal titration calorimetry, which revealed that the proposed internal PBM did not interact with any Scribble PDZ domains. Instead, the C-terminal PBM of NS5 interacted with Scrib PDZ3. We then established the structural basis of these interactions by determining crystal structures of Scrib PDZ3 bound to the NS5 C-terminal PBM. Our findings provide a structural basis for Scribble PDZ domain and TBEV NS5 interactions and provide a platform to dissect the pathogenesis of TBEV and the role of cell polarity signalling using structure guided approaches.

Z-RNA and the Flipside of the SARS Nsp13 Helicase: Is There a Role for Flipons in Coronavirus-Induced Pathology?

We present evidence suggesting that the severe acute respiratory syndrome (SARS) coronavirus non-structural protein 13 (Nsp13) modulates the Z-RNA dependent regulated cell death pathways . We show that Z-prone sequences [called flipons] exist in coronavirus and provide a signature (Z-sig) that enables identification of the animal viruses from which the human pathogens arose. We also identify a potential RIP Homology Interaction Motif (RHIM) in the helicase Nsp13 that resembles those present in proteins that initiate Z-RNA-dependent cell death through interactions with the Z-RNA sensor protein ZBP1. These two observations allow us to suggest a model in which Nsp13 down regulates Z-RNA activated innate immunity by two distinct mechanisms. The first involves a novel ATP-independent Z-flipon helicase (flipase) activity in Nsp13 that differs from that of canonical A-RNA helicases. This flipase prevents formation of Z-RNAs that would otherwise activate cell death pathways. The second mechanism likely inhibits the interactions between ZBP1 and the Receptor Interacting Proteins Kinases RIPK1 and RIPK3 by targeting their RHIM domains. Together the described Nsp13 RHIM and flipase activities have the potential to alter the host response to coronaviruses and impact the design of drugs targeting the Nsp13 protein. The Z-sig and RHIM domains may provide a way of identifying previously uncharacterized viruses that are potentially pathogenic for humans.

Structural basis for tunable affinity and specificity of LxCxE-dependent protein interactions with the retinoblastoma protein family

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their "pocket domain" to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity.

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

Abstract 2919: The structural basis of BCR-ABL recruitment of GRB2 in chronic myelogenous leukemia

Abstract Philadelphia chromosomes 9 and 22 encode BCR and ABL1 genes, respectively. Translocation mutation of two chromosomes generates the fused BCR-ABL1 gene. The resulting fused gene encodes the chimeric BCR-ABL oncoprotein with constitutively enhanced and dysregulated tyrosine activity to disturb different cell signaling. Ras/MAPK is a significant pathway in mediating cell proliferation and its hyperactivation is responsible for malignant transformation in cancer. Autophosphorylation Y177 (pY177) of BCR protein stimulates the direct binding of an adaptor protein GRB2, which is involved in Ras/MAPK pathway. In BCR-ABL-associated Ras/MAPK signaling pathway, BCR-ABL can target GRB2 through the interaction of its pY motif with GRB2 SH2 domain, followed by SOS recruitment to the plasma membrane by GRB2. The resulting BCR-ABL/GRB2/SOS assembly stimulates the transformation of inactive GDP-bound form of Ras to the active GTP-bound state, which causes the constitutive activation of the downstream components of Raf/MEK/ERK. Despite a number of biophysical experiments have showed that BCR-ABL is able to recruit GRB2, the molecular-level understanding of how the two proteins interact with each other remains to be elucidated. In this work, we used molecular modeling and molecular dynamics (MD) simulations to study the structural basis for the binding between BCR-ABL and GRB2 SH2 domain to explain CML oncogenesis at the atomic level. Different pY/Y-peptide-SH2 complex models have been constructed and their binding specificity and affinity were compared. Simulation results showed that phosphorylated BCR (pBCR) specifically binds to GRB2 SH2 domain through its five-residue 176FpYVNV180 motif. GRB2 SH2 domain contains two binding pockets: the pY-binding pocket for the phosphate recognition and the specificity pocket for the selection of Asn. pY177 triggers the binding through the interaction in pY-binding pocket. The interaction in specificity pocket determines the binding specificity, which is regulated by N179 in pBCR and W121 in EF loop of GRB2 SH2 domain. In addition, the interaction in the specificity pocket experiences conformation selection for both the bound motif in pBCR and GRB2 SH2 domain; the binding motif adopts type I β-turn conformation and the specificity pocket of GRB2 SH2 domain displays the closed conformation. This specific binding mode between pBCR and GRB2 SH2 domain is similar with that between phosphorylated EGFR (pEGFR) and GRB2 SH2 domain. Residues adjacent to the pY and Arg residue at the +2 position C-terminal to pY in the binding motifs influence the binding affinity of a pY-peptide to GRB2 SH2 domain, explaining why pBCR shows stronger affinity to GRB2 SH2 domain than pEGFR. Our study supports the BCR-ABL oncoprotein recruitment of GRB2 for the induction of CML through Ras/MAPK pathway and provides deeply understanding of the specific interaction of the pY motif with GRB2 SH2 domain at atomic level. Citation Format: Yonglan Liu, Hyunbum Jang, Mingzhen Zhang, Chung-Jung Tsai, Ryan Maloney, Ruth Nussinov. The structural basis of BCR-ABL recruitment of GRB2 in chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2919.

LncNSPL facilitates influenza A viral immune escape by restricting TRIM25-mediated K63-linked RIG-I ubiquitination

SummaryLong noncoding RNAs (lncRNAs) participate in host antiviral responses; however, how viruses exploit host lncRNAs for immune evasion remains largely unexplored. Functional screening of differentially expressed lncRNA profile in patients infected with influenza A virus (IAV) revealed that lncNSPL (Gene Symbol: LOC105370355) was highly expressed in monocytes. Deregulated lncNSPL expression in infected monocytes significantly increased type I interferon (IFN-I) production and inhibited IAV replication. Moreover, lncNSPL overexpression in mice increased the susceptibility to IAV infection and impaired IFN-I production. LncNSPL directly bound to retinoic acid-inducible gene I (RIG-I) and blocked the interaction between RIG-I and E3 ligase tripartite interaction motif 25 (TRIM25), reducing TRIM25-mediated lysine 63 (K63)-linked RIG-I ubiquitination and limiting the downstream production of antiviral mediators during the late stage of IAV infection. Our findings provide mechanistic insights into the means by which lncNSPL promotes IAV replication and immune escape via restricting the TRIM25-mediated RIG-I K63-linked ubiquitination. Thus, lncNSPL may represent a promising pharmaceutical target for anti-IAV therapy.

Abstract 2369: The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression

Abstract Basal-like breast cancer (BLBC) is an aggressive malignancy with a poor prognosis. Persistent activation of NRF2 and STAT3 is considered to stimulate the aggressive behavior of BLBC. An online database, NRF2-ome predicted the possibility of interplay between NRF2 and STAT3 based on domain motif interactions. However, cooperation between NRF2 and STAT3 and its implications for breast cancer progression remain elusive. Immunoprecipitation and in situ proximity analyses revealed that NRF2 and STAT3 directly interact with each other, which predominantly occurred in the nucleus of human breast cancer cells. After confirming the physical interaction between STAT3 and NRF2, we examined whether this could influence the expression or stability of each transcription factor. While silencing of NRF2 failed to alter the STAT3 expression, STAT3 knockdown facilitated degradation of NRF2. Of note, silencing of STAT3 did not affect the mRNA expression of NRF2, suggesting that STAT3-induced accumulation of NRF2 is attributable to stabilization of the preexisting protein. As the NRF2-STAT3 complex was found to accumulate predominantly in the nucleus, we speculate that NRF2 could bind to a functionally active phosphorylated form of STAT3 (P-STAT3Y705). The substitution of Y705 of STAT3 with the non-phosphorylatable phenylalanine (STAT3Y705F) abolished interaction with NRF2. Notably, the dimeric form, but not a monomeric form, of STAT3 interacts directly with the Neh1 and Neh3 domains of NRF2. Next, we investigated the functional significance of STAT3- NRF2 interaction in BLBC growth in vivo. STAT3 knockdown significantly reduced expression of NRF2 in the xenograft tumor tissues derived from MDA-MB-231 human breast cancer cells. Tumors derived from cells transfected with the non-specific control siRNA showed a pronounced interaction between STAT3 and NRF2, whereas such interaction was abolished in the STAT3-silenced xenograft tumor. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and NRF2 mRNA transcripts were correlated with elevated expression of P-STAT3Y705 and NRF2 target proteins in breast cancer patients. Next generation RNA sequencing analysis identified the gene encoding interleukin-23A (IL-23A) upregulated by concurrent binding of STAT3 and NRF2 to its promoter. IL-23A is markedly overexpressed in BLBC, compared with other subtypes of breast cancer. Combined silencing of NRF2 and STAT3 inhibited BLBC growth to a greater extent than that achieved with single siRNA transfection. IL-23A depletion by siRNA also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, these findings suggest that the STAT3-NRF2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer. Citation Format: Su-Jung Kim, Soma Saeidi, Nam-Chul Cho, Seung Hyeon Kim, Han-Byoel Lee, Wonshik Han, Hye-Kyung Na, Young-Joon Surh. The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2369.

RING finger 138 deregulation distorts NF-кB signaling and facilities colitis switch to aggressive malignancy

Prolonged activation of nuclear factor (NF)-кB signaling significantly contributes to the development of colorectal cancer (CRC). New therapeutic opportunities are emerging from targeting this distorted cell signaling transduction. Here, we discovered the critical role of RING finger 138 (RNF138) in CRC tumorigenesis through regulating the NF-кB signaling, which is independent of its Ubiquitin-E3 ligase activity involved in DNA damage response. RNF138−/− mice were hyper-susceptible to the switch from colitis to aggressive malignancy, which coincided with sustained aberrant NF-кB signaling in the colonic cells. Furthermore, RNF138 suppresses the activation of NF-кB signaling pathway through preventing the translocation of NIK and IKK-Beta Binding Protein (NIBP) to the cytoplasm, which requires the ubiquitin interaction motif (UIM) domain. More importantly, we uncovered a significant correlation between poor prognosis and the downregulation of RNF138 associated with reinforced NF-кB signaling in clinical settings, raising the possibility of RNF138 dysregulation as an indicator for the therapeutic intervention targeting NF-кB signaling. Using the xenograft models built upon either RNF138-dificient CRC cells or the cells derived from the RNF138-dysregulated CRC patients, we demonstrated that the inhibition of NF-кB signaling effectively hampered tumor growth. Overall, our work defined the pathogenic role of aberrant NF-кB signaling due to RNF138 downregulation in the cascade events from the colitis switch to colonic neoplastic transformation and progression, and also highlights the possibility of targeting the NF-кB signaling in treating specific subtypes of CRC indicated by RNF138-ablation.