Ligase Domain Research Articles

A mechanism for hypoxia-induced inflammatory cell death in cancer.

Hypoxic cancer cells resist many antineoplastic therapies and can seed recurrence1,2. We previously found that either deficiency or inhibition of protein-tyrosine phosphatase (PTP1B) promotes human epidermal growth factor receptor 2-positive breast cancer cell death in hypoxia by activation of RNF213 (ref. 3), a large protein with multiple AAA-ATPase domains and two ubiquitin ligase domains (RING and RZ) implicated in Moyamoya disease, lipotoxicity and innate immunity4. Here we report that PTP1B and ABL1/2 reciprocally control RNF213 tyrosine phosphorylation and, consequently, its oligomerization and RZ domain activation. The RZ domain ubiquitylates and induces the degradation of the major NF-κB regulator CYLD/SPATA2. Decreased CYLD/SPATA2 levels lead to NF-κB activation and induction of the NLRP3 inflammasome which, together with hypoxia-induced endoplasmic reticulum stress, triggers pyroptotic cell death. Consistent with this model, CYLD deletion phenocopies, whereas NLRP3 deletion blocks, the effects of PTP1B deficiency on human epidermal growth factor receptor 2-positive breast cancer xenograft growth. Reconstitution studies with RNF213 mutants confirm that the RZ domain mediates tumour cell death. In concert, our results identify a unique, potentially targetable PTP1B-RNF213-CYLD-SPATA2 pathway critical for the control of inflammatory cell death in hypoxic tumours, provide new insights into RNF213 regulation and have potential implications for the pathogenesis of Moyamoya disease, inflammatory disorders and autoimmune disease.

Rabex-5 E3 and Rab5 GEF domains differ in their regulation of Ras, Notch, and PI3K signaling in Drosophila wing development.

Rabex-5 (also called RabGEF1), a protein originally characterized for its Rab5 GEF function, also has an A20-like E3 ubiquitin ligase domain. We and others reported that Rabex-5 E3 activity promotes Ras mono- and di-ubiquitination to inhibit Ras signaling in Drosophila and mammals. Subsequently, we reported that Rabex-5 inhibits Notch signaling in the Drosophila hematopoietic system. Here we report genetic interactions using Rabex-5 transgenes encoding domain-specific mutations that show that Rabex-5 requires an intact E3 domain to inhibit Notch signaling in the epithelial tissue of the developing wing. Surprisingly, we discovered that Rabex-5 with an impaired E3 domain but active Rab5 GEF domain suppresses Notch loss-of-function phenotypes and enhances both Notch duplication phenotypes and activated Ras phenotypes consistent with a model that the Rab5 GEF activity of Rabex-5 might positively regulate Ras and Notch. Positive and negative regulation of developmental signaling by its different catalytic domains could allow Rabex-5 to precisely coordinate developmental signaling to fine-tune patterning. Finally, we report that Rabex-5 also inhibits the overgrowth due to loss of PTEN or activation of PI3K but not activation of AKT. Inhibition of Ras, Notch, and PI3K signaling may explain why Rabex-5 is deleted in some cancers. Paradoxically, Rabex-5 is reported to be an oncogene in other cancers. We propose that Rabex-5 acts as a tumor suppressor via its E3 activity to inhibit Ras, Notch, and PI3K signaling and as an oncogene via its Rab5 GEF activity to enhance Ras and Notch signaling.

Map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5'-OH kinase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO4 and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for anti-fungal drug discovery. Mucorales species, deemed high priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO4 and 5'-PO4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg2+ that illuminates the basis for guanosine nucleotide specificity.

Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis

Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation–immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.

E3 ubiquitin ligase ANKIB1 attenuates antiviral immune responses by promoting K48-linked polyubiquitination of MAVS

Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with mitochondrial antiviral signaling proteins (MAVSs) to activate IRF3 and nuclear factor κB (NF-κB) signaling, initiating innate immune responses. Thus, RLR activation plays a vital role in the removal of invasive RNA viruses while maintaining immune homeostasis. However, inadequate or excessive activation of immunity can cause harm and can even lead to lethal consequences. In this study, we identify an E3 ligase, ankyrin repeat and IBR domain containing 1 (ANKIB1), which suppresses RLR signaling via MAVS. ANKIB1 binds to MAVS to enhance K48-linked polyubiquitination with K311R, causing proteasomal degradation of MAVS. Deficiency of ANKIB1 significantly increases the RLR-mediated production of type I interferon (IFN) along with pro-inflammatory factors. Consequently, ANKIB1 deficiency remarkably increases antiviral immunity and decreases viral replication invivo. Therefore, we reveal that ANKIB1 restricts RLR-induced innate immune activation, indicating its potential role as a therapeutic target for viral infections.

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/β-catenin signaling

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/β-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using invitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/β-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/β-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/β-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/β-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

Programmable Protein Stabilization with Language Model-Derived Peptide Guides.

Dysregulated protein degradation via the ubiquitin-proteasomal pathway can induce numerous disease phenotypes, including cancer, neurodegeneration, and diabetes. Stabilizing improperly ubiquitinated proteins via target-specific deubiquitination is thus a critical therapeutic goal. Building off the major advances in targeted protein degradation (TPD) using bifunctional small-molecule degraders, targeted protein stabilization (TPS) modalities have been described recently. However, these rely on a limited set of chemical linkers and warheads, which are difficult to generate de novo for new targets and do not exist for classically "undruggable" targets. To address the limited reach of small molecule-based degraders, we previously engineered ubiquibodies (uAbs) by fusing computationally-designed "guide" peptides to E3 ubiquitin ligase domains for modular, CRISPR-analogous TPD. Here, we expand the TPS target space by engineering "deubiquibodies" (duAbs) via fusion of computationally-designed guides to the catalytic domain of the potent OTUB1 deubiquitinase. In human cells, duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner. To demonstrate duAb modularity, we swap in new target-binding peptides designed via our generative language models to stabilize diverse target proteins, including key tumor suppressor proteins such as p53 and WEE1, as well as heavily-disordered fusion oncoproteins, such as PAX3::FOXO1. In total, our duAb system represents a simple, programmable, CRISPR-analogous strategy for TPS.

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

Differentially Expressed Proteins in the Serum of Elderly Patients Who Experienced Perioperative Neurocognitive Disorders Following Transurethral Resection of the Prostate.

Perioperative neurocognitive disorders (PND) are a group of prevalent neurological complications that often occur in elderly individuals following major or emergency surgical procedures. The etiologies are not fully understood. This study endeavored to investigate novel targets and prediction methods for the occurrence of PND. A total of 229 elderly patients diagnosed with prostatic hyperplasia who underwent transurethral resection of the prostate (TURP) combined with spinal cord and epidural analgesia were included in this study. The patients were divided into two groups, the PND group and non-PND group, based on the Z-score method. According to the principle of maintaining consistency between preoperative and intraoperative conditions, three patients from each group were randomly chosen for serum sample collection. isobaric tags for relative and absolute quantification (iTRAQ) proteomics technology was employed to analyze and identify the proteins that exhibited differential expression in the serum samples from the two groups. Bioinformatics analysis was performed on the proteins that exhibited differential expression. Among the 1101 serum proteins analyzed in the PND and non-PND groups, eight differentially expressed proteins were identified in PND patients. Of these, six proteins showed up-regulation, while two proteins showed down-regulation. Further bioinformatics analysis of the proteins that exhibited differential expression revealed their predominant involvement in cellular biological processes, cellular component formation, as well as endocytosis and phagocytosis Additionally, these proteins were found to possess the RING domain of E3 ubiquitin ligase. The iTRAQ proteomics technique was employed to analyze the variation in protein expression in serum samples from patients with PND and those without PND. This study successfully identified eight proteins that exhibited differential expression levels between the two groups. Bioinformatics analysis indicates that proteins exhibiting differential expression are primarily implicated in the biological processes associated with microtubules. Investigating the microtubule formation process as it relates to neuroplasticity and synaptic formation may offer valuable insights for enhancing our comprehension and potential prevention of PND. Registered (ChiCTR2000028836). Date (20190306).

Abstract LB052: PTP1B inhibitors activate a novel hypoxia-induced inflammatory cell death pathway in breast cancer

Abstract Dual PTP1B/TC-PTP (PTPN1/2) inhibitors are in clinical trials for multiple cancer types, primarily because they augment anti-tumor immunity, but they can also have cancer cell-autonomous effects. Hypoxic cancer cells resist many anti-neoplastic therapies and can seed recurrence. We found previously that PTP1B deficiency/inhibition promotes HER2+ breast cancer cell death in hypoxia by activating RNF213, an ~600kDa protein containing AAA-ATPase domains and two ubiquitin ligase domains (RING and RZ) that also is implicated in Moyamoya disease (MMD), lipotoxicity, and innate immunity. We now report that PTP1B and ABL1/2 reciprocally control RNF213 phosphorylation on tyrosine-1275. This phosphorylation promotes RNF213 oligomerization and RZ domain activation. The RZ domain ubiquitylates CYLD/SPATA2, and together with the LUBAC complex, induces their degradation. Decreased CYLD/SPATA2 causes NF-κB activation, which together with hypoxia-induced ER-stress triggers GDSMD-dependent pyroptosis. Mutagenesis experiments show that the RING domain negatively regulates the RZ domain. CYLD-deleted HER2+ cell-derived xenografts phencopy the effects of PTP1B deficiency, and reconstituting RNF213 knockout lines with RNF213 mutants shows that the RZ domain mediates PTP1B-dependent tumor cell death. Our results identify a novel, potentially targetable PTP1B/RNF213/CYCLD/SPATA pathway critical for controlling inflammatory cell death in hypoxic tumors that could be exploited to target hypoxic tumor cells, potentially turning “cold” tumors “hot”, thereby increasing the potential clinical utility of PTP1B inhibitors. Citation Format: Benjamin G. Neel, Abhishek Bhardwaj, Maria Antonelli, Beatrix Ueberheide. PTP1B inhibitors activate a novel hypoxia-induced inflammatory cell death pathway in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB052.

Rapid depletion of target proteins in plants by an inducible protein degradation system.

Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella-secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the homology region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the leucine-rich repeat and novel E3 ligase domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system, and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high-temporal resolution and may become an important tool in plant cell biology.

Molecular structure and function of mysterin/RNF213.

Mysterin is a large intracellular protein harboring a RING finger ubiquitin ligase domain and is also referred to as RING finger protein 213 (RNF213). The author performed the first molecular cloning of the mysterin gene as the final step in genetic exploration of cerebrovascular moyamoya disease (MMD) and initiated the next round of exploration to understand its molecular and cellular functions. Although much remains unknown, accumulating findings suggest that mysterin functions in cells by targeting massive intracellular structures, such as lipid droplets (LDs) and various invasive pathogens. In the latter case, mysterin appears to directly surround and ubiquitylate the surface of pathogens and stimulate cell-autonomous antimicrobial reactions, such as xenophagy and inflammatory response. To date, multiple mutations causing MMD have been identified within and near the RING finger domain of mysterin; however, their functional relevance remains largely unknown. Besides the RING finger, mysterin harbors a dynein-like ATPase core and an RZ finger, another ubiquitin ligase domain unique to mysterin, while functional exploration of these domains has also just commenced. In this review, the author attempts to summarize the core findings regarding the molecular structure and function of the mysterin protein, with an emphasis on the perspective of MMD research.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Abstract C118: Design of programmable peptide-guided oncoprotein degraders via generative language models

Abstract Targeted protein degradation of pathogenic proteins represents a powerful new treatment strategy for multiple cancers. Unfortunately, a sizable portion of these proteins are considered “undruggable” by standard small molecule-based approaches, including PROTACs and molecular glues, largely due to their disordered nature, instability, and lack of binding site accessibility. As a more modular strategy, we have developed a genetically-encoded protein architecture by fusing target-specific peptides to E3 ubiquitin ligase domains for selective and potent intracellular degradation of oncoproteins. To enable programmability of our system, we develop a suite of algorithms that enable the design of target-specific peptides via protein language model (pLM) embeddings, without the requirement of 3D structures. First, we train a model that leverages pLM embeddings to efficiently select high-affinity peptides from natural protein interaction interfaces. Next, we develop a high-accuracy discriminator, based on the contrastive language-image pretraining (CLIP) architecture underlying OpenAI's DALL-E model, to prioritize and screen peptides with selectivity to a specified target oncoprotein. As input to the discriminator, we create a Gaussian diffusion generator to sample a pLM latent space, fine-tuned on experimentally-valid peptide sequences. Finally, to enable de novo design of binding peptides, we train an instance of GPT-2 with protein interacting sequences to enable peptide generation conditioned on target oncoprotein sequences. Our models demonstrate low perplexities across both existing and generated peptide sequences, highlighting their robust generative capability. By experimentally fusing model-derived peptides to E3 ubiquitin ligase domains, we reliably identify candidates exhibiting robust and selective endogenous degradation of diverse, "undruggable" oncoproteins in cancer cell models, including tumorigenic regulators such as β-catenin and TRIM8, as well as oncogenic fusion proteins, such as EWS-FLI1, PAX3-FOXO1, and DNAJB1-PRKACA. We further show that our peptide-guided degraders have negligible off-target effects via whole-cell proteomics and demonstrate their modulation of transcriptional and apoptotic pathways, motivating further translation of our therapeutic platform. Together, our work establishes a CRISPR-analogous system for programmable protein degradation applications across the oncoproteome. Citation Format: Suhaas Bhat, Garyk Brixi, Kalyan Palepu, Lauren Hong, Vivian Yudistyra, Tianlai Chen, Sophia Vincoff, Lin Zhao, Pranam Chatterjee. Design of programmable peptide-guided oncoprotein degraders via generative language models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C118.

SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding “guide” peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous β-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation.

E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

BackgroundAlterations in several tripartite motif-containing (TRIM) family proteins have been implicated in the pathogenesis of lung cancer. TRIM28, a member of the TRIM E3 ligase family, has been associated with tumorigenesis, cell proliferation, and inflammation. However, little is known about TRIM28 expression and its role in the immune microenvironment of non-small cell lung cancer (NSCLC).MethodsWe assessed the clinical significance of TRIM28 in tissue microarrays and TCGA cohorts. We investigated the function of TRIM28 in syngeneic mouse tumor models, the KrasLSL−G12D/+; Tp53fl/fl (KP) mouse model, and humanized mice. Immune cell composition was analyzed using flow cytometry and immunohistochemistry.ResultsOur findings revealed a positive correlation between TRIM28 expression and the infiltration of suppressive myeloid-derived suppressor cells (MDSCs) in NSCLC. Moreover, silencing TRIM28 enhanced the efficacy of anti-PD-1 immunotherapy by reshaping the inflamed tumor microenvironment. Mechanistically, we demonstrated that TRIM28 could physically interact with receptor-interacting protein kinase 1 (RIPK1) and promote K63-linked ubiquitination of RIPK1, which is crucial for sustaining activation of the NF-κB pathway. Mutagenesis of the E3 ligase domain corroborated the essential role of E3 ligase activity in TRIM28-mediated NF-κB activation. Further experiments revealed that TRIM28 could upregulate the expression of CXCL1 by activating NF-κB signaling. CXCL1 could bind to CXCR2 on MDSCs and promote their migration to the tumor microenvironment. TRIM28 knockdown increased responsiveness to anti-PD-1 therapy in immunocompetent mice, characterized by increased CD8+T tumor-infiltrating lymphocytes and decreased MDSCs.ConclusionThe present study identified TRIM28 as a promoter of chemokine-driven recruitment of MDSCs through RIPK1-mediated NF-κB activation, leading to the suppression of infiltrating activated CD8+T cells and the development of anti-PD-1 resistance. Understanding the regulation of MDSC recruitment and function by TRIM28 provides crucial insights into the association between TRIM28 signaling and the development of an immunosuppressive tumor microenvironment. These insights may inform the development of combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in NSCLC.

An enhanced activity and thermostability of chimeric Bst DNA polymerase for isothermal amplification applications.

Loop-mediated isothermal amplification (LAMP) is a widely used method for clinical diagnosis, customs quarantine, and disease prevention. However, the low catalytic activity of Bst DNA polymerase has made it challenging to develop rapid and reliable point-of-care testing. Herein, we developed a series of Bst DNA polymerase mutants with enhanced activity by predicting and analyzing the activity sites. Among these mutants, single mutants K431D and K431E showed a 1.93- and 2.03-fold increase in catalytic efficiency, respectively. We also created a chimeric protein by fusing the DNA-binding domain of DNA ligase from Pyrococcus abyssi (DBD), namely DBD-K431E, which enabled real-time LAMP at high temperatures up to 73 ℃ and remained active after heating at 70 ℃ for 8h. The chimeric DBD-K431E remained active in the presence of 50 U/mL heparin, 10% ethanol, and up to 100mM NaCl, and showed higher activity in 110mM (NH4)2SO4, 110mM KCl, and 12mM MgSO4. Notably, it generated a fluorescence signal during the detection of Salmonella typhimurium at 2 × 102 ag/μL of genomic DNA and 1.24CFU/mL of bacterial colony, outperforming the wild type and the commercial counterpart Bst 2.0. Our results suggest that the DBD-K431E variant could be a promising tool for general molecular biology research and clinical diagnostics. KEY POINTS: • Residue K431 is probably a key site of Bst DNA polymerase activity • The chimeric DBD-K431E is more inhibitor tolerant and thermostable than Bst-LF • The DBD-K431E variant can detect Salmonella typhimurium at 102 ag/μL or 100CFU/mL.

The emerging role of E3 ubiquitin ligase RNF213 as an antimicrobial host determinant.

Ring finger protein 213 (RNF213) is a large E3 ubiquitin ligase with a molecular weight of 591 kDa that is associated with moyamoya disease, a rare cerebrovascular disease. It is located in the cytosol and perinuclear space. Missense mutations in this gene have been found to be more prevalent in patients with moyamoya disease compared with that in healthy individuals. Understanding the molecular function of RNF213 could provide insights into moyamoya disease. RNF213 contains a C3HC4-type RING finger domain with an E3 ubiquitin ligase domain and six AAA+ adenosine triphosphatase (ATPase) domains. It is the only known protein with both AAA+ ATPase and ubiquitin ligase activities. Recent studies have highlighted the role of RNF213 in fighting against microbial infections, including viruses, parasites, bacteria, and chlamydiae. This review aims to summarize the recent research progress on the mechanisms of RNF213 in pathogenic infections, which will aid researchers in understanding the antimicrobial role of RNF213.