Non-canonical Interaction Research Articles

An acidic loop in the forkhead-associated domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a 5-amino acid sequence, RPSKR, located between the DNA-binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full-length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a noncanonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt 2-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint and, in certain circumstances, exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

An acidic loop in the FHA domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

14-3-3θ, a novel player in TDP-43 pathophysiology: Implications for ALS/FTD

In this issue of Neuron, Ke etal.1 report a novel non-canonical interaction between 14-3-3θ and TDP-43 that impacts loss-of-function and gain-of-toxic pathology in TDP-43 proteinopathies. The authors further provide proof of principle for a 14-3-3θ-targeted gene therapy to reduce TDP-43-induced deficits in transgenic TDP-43 mutant mice.

Differential contribution of canonical and noncanonical NLGN3 pathways to early social development and memory performance

Neuroligin (NLGN) 3 is a postsynaptic cell adhesion protein organizing synapse formation through two different types of transsynaptic interactions, canonical interaction with neurexins (NRXNs) and a recently identified noncanonical interaction with protein tyrosine phosphatase (PTP) δ. Although, NLGN3 gene is known as a risk gene for neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID), the pathogenic contribution of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ pathways to these disorders remains elusive. In this study, we utilized Nlgn3 mutant mice selectively lacking the interaction with either NRXNs or PTPδ and investigated their social and memory performance. Neither Nlgn3 mutants showed any social cognitive deficiency in the social novelty recognition test. However, the Nlgn3 mutant mice lacking the PTPδ pathway exhibited significant decline in the social conditioned place preference (sCPP) at the juvenile stage, suggesting the involvement of the NLGN3-PTPδ pathway in the regulation of social motivation and reward. In terms of learning and memory, disrupting the canonical NRXN pathway attenuated contextual fear conditioning while disrupting the noncanonical NLGN3-PTPδ pathway enhanced it. Furthermore, disruption of the NLGN3-PTPδ pathway negatively affected the remote spatial reference memory in the Barnes maze test. These findings highlight the differential contributions of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ synaptogenic pathways to the regulation of higher order brain functions associated with ASD and ID.

Targeting 14-3-3θ-mediated TDP-43 pathology in amyotrophic lateral sclerosis and frontotemporal dementia mice

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by cytoplasmic deposition of the nuclear TAR-binding protein 43 (TDP-43). Although cytoplasmic re-localization of TDP-43 is a key event in the pathogenesis of ALS/FTD, the underlying mechanisms remain unknown. Here, we identified a non-canonical interaction between 14-3-3θ and TDP-43, which regulates nuclear-cytoplasmic shuttling. Neuronal 14-3-3θ levels were increased in sporadic ALS and FTD with TDP-43 pathology. Pathogenic TDP-43 showed increased interaction with 14-3-3θ, resulting in cytoplasmic accumulation, insolubility, phosphorylation, and fragmentation of TDP-43, resembling pathological changes in disease. Harnessing this increased affinity of 14-3-3θ for pathogenic TDP-43, we devised a gene therapy vector targeting TDP-43 pathology, which mitigated functional deficits and neurodegeneration in different ALS/FTD mouse models expressing mutant or non-mutant TDP-43, including when already symptomatic at the time of treatment. Our study identified 14-3-3θ as a mediator of cytoplasmic TDP-43 localization with implications for ALS/FTD pathogenesis and therapy.

Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin.

Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of β-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.

N-terminal ectodomain of BTNL2 inhibits T cell activation via a non-canonical interaction with its putative receptor that results in a delayed progression of DSS-induced ulcerative colitis

Butyrophilin-like 2 (BTNL2) is a T cell inhibitory molecule that interacts with unknown binding partners to modulate the immune response in a number of inflammatory and autoimmune diseases. In this study, we found that the inhibitory effects of BTNL2 on T cell activation and effector functions can be executed by its N-terminal IgV domain (BTNL2 IgV1) alone. Structure-guided mutation of key residues on BTNL2 IgV1 based on known receptor-ligand interfaces involving immunoglobulin superfamily members revealed that BTNL2 uses a non-canonical binding interface with its putative receptor. A high avidity BTNL2 IgV1 probe revealed that in an inducible model of ulcerative colitis, severe colitis was accompanied by a selective enrichment of BTNL2-receptor expressing effector-memory CD4+ and CD8+ T cells in the Peyer’s patches. Intraperitoneal administration of BTNL2 IgV1 resulted in a significant delay in the progression of DSS-induced colitis and also showed reduced activation of the BTNL2-receptor-expressing T cells in the Peyer’s patches. Thus, this study demonstrates that the BTNL2-receptor-expressing T cells in the Peyer’s patches participate in the disease pathogenesis and can serve as a novel therapeutic target in ulcerative colitis, which can be modulated by BTNL2 IgV1.

Let’s Talk about Sex Hormone Receptors and Their Physical Interaction with Sonic Hedgehog Protein: A Computational Study with Emphasis on Progesterone Receptor

The mature form of the sonic hedgehog protein (SHH-N) is the main canonical activator of the Hedgehog-GLI signaling pathway whose aberrant activity can lead to the development of hormone-dependent cancers like breast or prostate cancer. In this study, we employed computational methods to explore the potential binding of SHH-N with the progesterone receptor (PR), the sole member of the nuclear sex hormone receptor (SHRs) subfamily not previously linked to SHH-N. Through a combination of molecular docking, robust molecular dynamics (MD) simulations, and free energy calculations, we predicted a stable binding between SHH-N-cholesterol and PR. To validate our findings, we extended our in silico investigation to encompass the complexes between SHH-N-cholesterol and estrogen receptor alpha (ERα) and androgen receptor (AR)—complexes that have been experimentally confirmed in our prior studies. The calculations not only confirmed the stable binding of SHH-N-cholesterol with both ERα and AR but also revealed the strongest binding occurred with ERα, followed by AR and PR, suggesting a non-canonical interaction with potential biological significance. Microsecond-long MD simulations unveiled tight cholesterol binding in the SHRs’ binding sites, and we gained insights into sub-molecular interactions contributing to protein-protein stabilization in complexes involving PR and ERα for the first time. The MM/PBSA calculations indicated comparable binding affinities of PR for progesterone and SHH-N-cholesterol, with ERα exhibiting a more favorable enthalpy of binding with SHH-N-cholesterol than with estradiol.

Canonical binding of Chaetomium thermophilum DNA polymerase δ/ζ subunit PolD3 and flap endonuclease Fen1 to PCNA.

The sliding clamp PCNA is a key player in eukaryotic genome replication and stability, acting as a platform onto which components of the DNA replication and repair machinery are assembled. Interactions with PCNA are frequently mediated via a short protein sequence motif known as the PCNA-interacting protein (PIP) motif. Here we describe the binding mode of a PIP motif peptide derived from C-terminus of the PolD3 protein from the thermophilic ascomycete fungus C. thermophilum, a subunit of both DNA polymerase δ (Pol δ) and the translesion DNA synthesis polymerase Pol ζ, characterised by isothermal titration calorimetry (ITC) and protein X-ray crystallography. In sharp contrast to the previously determined structure of a Chaetomium thermophilum PolD4 peptide bound to PCNA, binding of the PolD3 peptide is strictly canonical, with the peptide adopting the anticipated 310 helix structure, conserved Gln441 inserting into the so-called Q-pocket on PCNA, and Ile444 and Phe448 forming a two-fork plug that inserts into the hydrophobic surface pocket on PCNA. The binding affinity for the canonical PolD3 PIP-PCNA interaction determined by ITC is broadly similar to that previously determined for the non-canonical PolD4 PIP-PCNA interaction. In addition, we report the structure of a PIP peptide derived from the C. thermophilum Fen1 nuclease bound to PCNA. Like PolD3, Fen1 PIP peptide binding to PCNA is achieved by strictly canonical means. Taken together, these results add to an increasing body of information on how different proteins bind to PCNA, both within and across species.

OR33-04 Mechanistic Dissection Of Circadian-Specific Glucocorticoid Effects On Exercise Tolerance And Muscle Glucose Utilization

Abstract Disclosure: A. Prabakaran: None. M. Wintzinger: None. K. Piczer: None. K. Miz: None. A. Walton: None. H. Durumutla: None. M. Quattrocelli: None. Glucocorticoids (GCs) are circadian hormones regulating metabolism by activating the glucocorticoid receptor (GR) as pleiotropic transcription factor. Timing of exogenous GC drug regimens is emerging as a key determinant of their metabolic effects. This was shown by the surprising finding that once-weekly GC intermittence reverses many of the pro-obesogenic side effects of once-daily GC dosing in mice and humans. However, despite the intrinsic circadian nature of this signaling, the impact of time-of-day of exogenous GC dosing, i.e. chrono-pharmacology, remains remarkably unknown for metabolic physiology and exercise tolerance. Relevance of this question for humans is supported by the initial findings of increased lean mass and motor function with circadian-restricted prednisone intermittence in a recent pilot clinical trial in patients with genetic myopathies. However, mechanisms to support significance of these effects for metabolic diseases and aging remain unknown. We studied 12-week-long intermittent regimens of the exogenous GC prednisone in mice in conditions of diet-induced obesity or advanced aging, comparing dosing at light-phase start (ZT0) versus dark-phase start (ZT12). We found that, compared to dark-phase, light-phase dosing enhanced the regimen-driven increase in body-wide lean mass, muscle mass and muscle function in both obese and aged mice. Intriguingly, experiments with adiponectin-knockout mice showed that these effects were dependent on adiponectin, which promotes muscle insulin sensitivity. We then used tissue-specific inducible knockout models to dissect non-muscle versus muscle-autonomous effects of GC chrono-pharmacology. We found that the GC effects on total and high-molecular-weight adiponectin upregulation were dependent on regimen-specific engagement of the adipocyte-specific GR on the adiponectin gene promoter. Complementary to adipose-driven adiponectin production, we found that adiponectin receptor AdipoR1 upregulation in muscle depended on circadian-specific engagement of the myocyte-specific GR on the AdipoR1 promoter. Consistent with the adiponectin action on muscle insulin sensitivity, ablation of myocyte-specific GR blocked the regimen-driven effects on insulin-sensitive muscle glucose uptake and muscle mass. Furthermore, we found that light-phase GC dosing engaged the muscle GR for a non-canonical interaction with the clock factor BMAL1 to upregulate the mitochondrial regulator PGC1alpha. Indeed, we found that the regimen-driven increase in glucose oxidation and amino acid biogenesis from TCA cycle intermediates in muscle was dependent on the myocyte-specific PGC1alpha. In summary, we present here novel circadian and molecular mechanisms reconverting glucocorticoid drugs from deleterious to re-energizing agents for potential chrono-treatment of metabolic conditions and aging. Presentation: Sunday, June 18, 2023

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

The challenge of nascent chain folding at the ribosome is met by the conserved ribosome-associated complex (RAC), which forms a chaperone triad with the Hsp70 protein Ssb in fungi, and consists of the non-canonical Hsp70 Ssz1 and the J domain protein Zuotin (Zuo1). Here we determine cryo-EM structures of Chaetomium thermophilum RAC bound to 80S ribosomes. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. It is held together by a tight interaction between the Ssz1 substrate-binding domain and the Zuo1 N terminus, and additional contacts between the Ssz1 nucleotide-binding domain and Zuo1 J- and Zuo1 homology domains, which form a rigid unit. The Zuo1 HPD motif conserved in J-proteins is masked in a non-canonical interaction by the Ssz1 nucleotide-binding domain, and allows the positioning of Ssb for activation by Zuo1. Overall, we provide the basis for understanding how RAC cooperates with Ssb in a dynamic nascent chain interaction and protein folding.

The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies invitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

Biochemical and Neurobehavioral Changes in the Rat Model of Parkinson�s Disease with Suprathreshold Exercise and Usnic Acid

Finding a solution for Parkinson's disease has been a challenge to researchers despite advances in medicinal research. None of the options available so far has been able to reverse dopaminergic damage or increase endogenous dopamine production from existing dopaminergic cells in Parkinson's disease. Research in Parkinson�s disease has not so far resulted in finding better solutions for reversing dopaminergic damage or increasing dopamine production. Above-the-threshold (suprathreshold) exercises producing improvement have been documented with supporting clinical and imaging findings but without any biochemical evidence. Above-the-threshold (suprathreshold) exercises producing improvement in Parkinson's disease have been documented with supporting clinical and imaging findings. To the best of our knowledge, the biochemical basis of this improvement has not been studied in detail. Recently, biomolecules like usnic acid have been shown to improve dopamine levels through protective and regenerative effects. This study aimed at understanding the biochemical basis of the improvement following suprathreshold exercises and usnic acid. The objectives were to assess whether the suprathreshold exercise produces improvement in PD rat models and, if so, to understand the biochemical basis of improvement and compare the mechanism with that of usnic acid.The effect of suprathreshold exercise and usnic acid on Parkinson�s disease Hence, this study was conceived to understand whether the exercise produces results due to a similar mechanism that had never been reported before. Parkinson's disease was compared with rotenone alone, rotenone and high-fat diet models, and control. Induced with rotenone and a high-fat diet, the effect of suprathreshold exercise was compared to that of usnic acid, the rotenone alone model, and control. Locomotor behaviour was assessed with an actophotometer and rotarod. Western blotting assessed the blood biochemical parameters, antioxidant and dopamine levels in addition to a-synuclein, tyrosine hydroxylase, Nurr1, and HSP70 levels. Usnic acid decreased oxidative stress markers like MDA, peroxynitrite and nitric oxide, indicating lesser oxidative stress, increased dopamine and serotonin levels and decreased a-synuclein expression. But the suprathreshold exercise did not significantly affect oxidative stress markers nor increased dopamine levels. However, a-synuclein expression was decreased. Compared with usnic acid, lower Nurr1 levels in the suprathreshold group demonstrate lesser protective and regenerative effects on dopaminergic neurons. HSP70 levels indicate that improvement in suprathreshold exercise resulted from preventing misfolding of a-synuclein or through a noncanonical interaction, and comparable locomotor behaviour was observed in both groups. Biomolecules like brain-derived neurotropic factors produced by suprathreshold exercises acting centrally and in muscles might have also contributed to improvement in this group. When usnic acid increased dopamine levels in the brain through protective effects on dopaminergic neurons, antioxidant effects, and reduced expression of a-synuclein, suprathreshold exercise did not increase dopamine, decreased a-synuclein and had less antioxidant effects. This study biochemically proves that suprathreshold exercise alleviates Parkinson�s disease symptoms by preventing a-synuclein misfolding and not by mechanisms other than increasing dopamine, neuroprotection or regeneration of dopaminergic neurons, which require further studies in humans.

Noncanonical β-catenin interactions promote leukemia-initiating activity in early T-cell acute lymphoblastic leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography–mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin– and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

A cryptic transactivation domain of EZH2 binds AR and AR's splice variant, promoting oncogene activation and tumorous transformation.

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2's multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of bothEZH2's canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2TAD for mediating EZH2's non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancerthat rely on the circuits wired by EZH2 and AR.

Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA.

The binding interaction between the DNA repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the VEGF promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the VEGF promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of ∼100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the VEGF G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.

Ezh2Y641F mutations co-operate with Stat3 to regulate MHC class I antigen processing and alter the tumor immune response in melanoma.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic component of the Polycomb Repressive Complex 2, a chromatin modifying complex, which mediates methylation of lysine 27 on histone 3 (H3K27me3), a repressive chromatin mark. Genetic alterations in EZH2 in melanoma include amplifications and activating point mutations at tyrosine 641 (Y641) whose underlying oncogenic mechanisms remain largely unknown. Here, we found that expression of Ezh2Y641F causes upregulation of a subset of interferon-regulated genes in melanoma cells. Upregulation of these genes was not a direct effect of changes in H3K27me3, but via a non-canonical interaction between Ezh2 and Signal Transducer and Activator of Transcription 3 (Stat3). Ezh2 and Stat3 together function as transcriptional activators to mediate gene activation of numerous genes, including MHC Class 1b antigen processing genes. Furthermore, expression of Stat3 is required to maintain an anti-tumor immune response in Ezh2Y641F melanomas and to prevent melanoma progression and recurrence.

Afadin couples RAS GTPases to the polarity rheostat Scribble

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS complex with AFDN are unknown. We used proximity-based proteomics to generate an interaction map for two isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with AFDN and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in epithelial cells disrupts MAPK and PI3K activation kinetics and inhibits motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects and implicate AFDN as a genuine RAS effector.

Dual Leucine Zipper Kinase Regulates Dscam Expression through a Noncanonical Function of the Cytoplasmic Poly(A)-Binding Protein.

Dual leucine zipper kinase (DLK) plays a pivotal role in the development, degeneration, and regeneration of neurons. DLK can regulate gene expression post-transcriptionally, but the underlying mechanism remains poorly understood. The Drosophila DLK, Wallenda (Wnd), regulates the expression of Down syndrome cell adhesion molecule (Dscam) to control presynaptic arbor growth. This regulation is mediated by the 3' untranslated region (3'UTR) of Dscam mRNA, which suggests that RNA binding proteins (RBPs) mediate DLK function. We performed a genome-wide cell-based RNAi screen of RBPs and identified the cytoplasmic poly(A)-binding protein, pAbp, as an RBP that mediates Wnd-induced increase in Dscam expression. Genetic analysis shows that Wnd requires pAbp for promoting presynaptic arbor growth and for enhancing Dscam expression. Our analysis revealed that Dscam mRNAs harbor short poly(A) tails. We identified a region in Dscam 3'UTR that specifically interacts with pAbp. Removing this region significantly reduced Wnd-induced increase in Dscam expression. These suggest that a noncanonical interaction of PABP with the 3'UTR of target transcripts is essential for DLK functions.SIGNIFICANCE STATEMENT The kinase DLK plays key roles in a multitude of neuronal responses, including axon development, neurodegeneration, and nerve injury. Previous studies show that DLK acts via mRNAs to regulate protein synthesis, but how DLK does so is poorly understood. This study demonstrates that DLK regulates the synthesis of Dscam through the poly(A)-binding protein PABP-C. Whereas PABP-C is known as a general translational activator, our study shows that DLK-mediated Dscam expression involves a noncanonical interaction between PABP-C and the Dscam mRNA, which leads to a selective regulation of Dscam translation by PABP-C. Thus, our study provides novel insights into the mechanisms that underlie the function of DLK and regulation of gene expression of PABP-C.

Membrane extraction by calmodulin underpins the disparate signalling of RalA and RalB.

Both RalA and RalB interact with the ubiquitous calcium sensor, calmodulin (CaM). New structural and biophysical characterisation of these interactions strongly suggests that, in the native membrane-associated state, only RalA can be extracted from the membrane by CaM and this non-canonical interaction could underpin the divergent signalling roles of these closely related GTPases. The isoform specificity for RalA exhibited by CaM is hypothesised to contribute to the disparate signalling roles of RalA and RalB in mitochondrial dynamics. This would lead to CaM shuttling RalA to the mitochondrial membrane but leaving RalB localisation unperturbed, and in doing so triggering mitochondrial fission pathways rather than mitophagy.