Receptor Turnover Research Articles

Computational Modeling and Analysis of the TGF-β-induced ERK and SMAD Pathways.

TGF-β, an important cytokine that plays a key role in many diseases regulates a wide array of cellular and physiologic processes via several TGF-β-driven signaling cascades, including the SMAD and non-SMAD-driven pathways. However, the detailed mechanisms by which TGF-β induces such diverse responses remain poorly understood. In particular, compared to the SMAD-dependent pathway, SMAD-independent pathways such as the ERK/MAPK pathway, which is critical in cancer progression, are less characterized. Here, we develop an integrated mechanistic model of the TGF-β-triggered ERK activation pathway and its crosstalk with the SMAD pathway, an analysis of which demonstrates how SMAD dynamics can be significantly modulated and regulated by the ERK pathway. In particular, SMAD-mediated transcription can be altered and delayed due to expedited phosphorylation of the linker of SMAD by TGF-β-activated ERK; and enhanced ERK activity, but attenuated SMAD activity, can be achieved simultaneously by fast turnover of TGF-β receptors via lipid-rafts. Also, in silico mutations of the TGF-β pathways reveal that the dynamic characteristics of both SMAD and ERK signaling may change significantly during cancer development. Specifically, normal cells may exhibit enhanced and sustained SMAD signaling with transient ERK activation, whereas cancerous cells may produce elevated and prolonged ERK signaling with enervated SMAD activation. These distinctive differences between normal and cancerous signaling behavior provide clues concerning, and potential explanations for, the seemingly contradictory roles played by TGF-β during cancer progression. We demonstrate how crosstalk among various branch pathways of TGF-β can influence overall cellular behavior. Based on model analysis, we hypothesize that aberrant molecular alterations drive changes in the intensity and duration of SMAD and ERK signaling during cancer progression and ultimately lead to an imbalance between the SMAD and ERK pathways in favor of tumor promotion. Thus, to treat cancer patients with a genetic signature of oncogenic Ras effectively may require at least a combination therapy to restore both the expression of TGF-β receptors and the GTPase activity of Ras.

PARP7 Inhibitors and AHR Agonists Act Synergistically Across a Wide-Range of Cancer Models.

Small molecule inhibitors of the mono (ADP) ribosyl transferase PARP7 are being evaluated as a monotherapy for tumors overexpressing PARP7 and in combination with immune checkpoint blockade. We previously showed that sensitivity to the PARP7 inhibitor (PARP7i) RBN-2397 could be enhanced by co-treatment with agonists of the Aryl Hydrocarbon Receptor (AHRa) in cell lines that show strong intrinsic sensitivity to RBN-2397. Here we demonstrate that a range of tumor cell lines that are relatively insensitive to PARP7i or AHRa as individual agents are unexpectedly profoundly sensitive to the combination. Our data show that this synergistic response is dependent on AHR/ARNT and is associated with increased levels of nuclear AHR and increased transcription of AHR target genes. In some hormone receptor-positive cell lines, we find that combination treatment is associated with proteasomal turnover of the steroid hormone receptors, androgen receptor and estrogen receptor. Both wildtype and hormone-resistant mutant forms of these receptors are degraded upon treatment with AHRa and PARP7i in breast and prostate cancer models. These results suggest that combining PARP7i with AHRa may extend the utility of these drugs to a wider range of tumors, including those that are refractory to hormone therapy.

Cholangiocyte ciliary defects induce sustained epidermal growth factor receptor signaling.

The primary cilium, an organelle that protrudes from cell surfaces, is essential for sensing extracellular signals. With disturbed cellular communication and chronic liver pathologies, this organelle's dysfunctions have been linked to disorders, including polycystic liver disease and cholangiocarcinoma. The goal of this study was to elucidate the relationship between primary cilia and the crucial regulator of cellular proliferation, the epidermal growth factor receptor (EGFR) signaling pathway, which has been associated with various clinical conditions. The study identified aberrant EGFR signaling pathways in cholangiocytes lacking functional primary cilia using liver-specific intraflagellar transport 88 knockout mice, a Pkhd1 mutant rat model, and human cell lines that did not have functional cilia. Cilia-deficient cholangiocytes showed persistent EGFR activation because of impaired receptor degradation, in contrast to their normal counterparts, where EGFR localization to the cilia promotes appropriate signaling. Using histone deacetylase 6 inhibitors to restore primary cilia accelerates EGFR degradation, thereby reducing maladaptive signaling. Importantly, experimental intervention with the histone deacetylase 6 inhibitor tubastatin A in an orthotopic rat model moved EGFR to cilia and reduced ERK phosphorylation. Concurrent administration of EGFR and histone deacetylase 6 inhibitors in cholangiocarcinoma and polycystic liver disease cells demonstrated synergistic antiproliferative effects, which were associated with the restoration of functioning primary cilia. This study's findings shed light on ciliary function and robust EGFR signaling with slower receptor turnover. We could use therapies that restore the function of primary cilia to treat EGFR-driven diseases in polycystic liver disease and cholangiocarcinoma.

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.

An Intrinsic Host Defense against HSV-1 Relies on the Activation of Xenophagy with the Active Clearance of Autophagic Receptors.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

A viral effector blocks the turnover of a plant NLR receptor to trigger a robust immune response

Plant intracellular nucleotide-binding and leucine-rich repeat immune receptors (NLRs) play a key role in activating a strong pathogen defense response. Plant NLR proteins are tightly regulated and accumulate at very low levels in the absence of pathogen effectors. However, little is known about how this low level of NLR proteins is able to induce robust immune responses upon recognition of pathogen effectors. Here, we report that, in the absence of effector, the inactive form of the tomato NLR Sw-5b is targeted for ubiquitination by the E3 ligase SBP1. Interaction of SBP1 with Sw-5b via only its N-terminal domain leads to slow turnover. In contrast, in its auto-active state, Sw-5b is rapidly turned over as SBP1 is upregulated and interacts with both its N-terminal and NB-LRR domains. During infection with the tomato spotted wilt virus, the viral effector NSm interacts with Sw-5b and disrupts the interaction of Sw-5b with SBP1, thereby stabilizing the active Sw-5b and allowing it to induce a robust immune response.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7–BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

Neurobeachin regulates hematopoietic progenitor differentiation and survival by modulating Notch activity

AbstractHematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the Beige and Chediak-Higashi domain–containing protein (BDCP), Neurobeachin (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LPS Responsive Beige-Like Anchor Protein (LRBA), Neurobeachin Like 1 (NBEAL1) and Lysosomal Trafficking Regulator (LYST), are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block after transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In summary, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model in which BDCP-regulated vesicle trafficking is required for efficient HSCT.

Umami Altering Salivary Proteome: A Study across a Sensitivity Spectrum on Subjects.

This study delved into the relationship between umami taste sensitivity (UTS) and variations in the salivary proteome among 12 healthy nonsmokers utilizing 4D data-independent acquisition-based proteomics. By assessing UTS through monosodium l-glutamate (MSG) detection thresholds, we discovered notable differences: individuals with high UTS detected umami at significantly lower MSG concentrations (0.20 ± 0.12 mM) compared to their low UTS counterparts (2.51 ± 1.21 mM). Both groups showed an upregulation of the S100A1 protein under MSG stimulation, indicating a potent biochemical response to umami stimuli. The high UTS group exhibited enhanced metabolic pathways including those for amino acid, lipid, and organic acid biosynthesis, essential for maintaining taste receptor functionality and enhancing signal transduction. This group also demonstrated increased activity in cytochrome P450 enzymes and ribonucleoprotein complexes, suggesting a readiness to manage metabolic challenges and optimize umami perception. In contrast, the low UTS group showed adaptive mechanisms, possibly through modulation of receptor availability and function, with an upregulation of structural and ribosomal proteins that may support taste receptor production and turnover. These findings suggest that varying biological mechanisms underpin differences in umami perception, which could significantly influence dietary preferences and nutritional outcomes, highlighting the intricate interplay of genetic, physiological, and metabolic factors in taste sensitivity.

Abstract 6319: Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN

Abstract CAR T cell activity in solid tumors is limited, in part, by off-tumor toxicity. To address this obstacle, AB-1015 contains an “AND” logic gate, consisting of a priming receptor (PrimeR) and an inducible chimeric antigen receptor (CAR) that is upregulated by PrimeR activation. The AB-1015 logic gate targets ALPG/P and MSLN, which are coexpressed in ovarian tumors but not normal tissues. AB-1015 CAR induction within the tumor is expected to be a dynamic process influenced by multiple factors, including antigen density, target cell abundance, as well as tumor microenvironmental factors. To characterize CAR induction and receptor turnover, we developed an in vitro assay with plate-bound ALPG to induce CAR. CAR induction occurred quickly in response to ALPG priming, with most ICT cells expressing CAR within 24 hrs, and plateaued by 72 hrs. Once removed from ALPG, CAR expression returned to baseline within 96 hrs. Similar to conventional CAR T cells, the presence of the cytolytic target antigen (MSLN) was expected to increase the rate of CAR turnover on AB-1015. To assess this, we measured the CAR functional half-life of AB-1015 cells once removed from ALPG priming. The cytotoxic activity of AB-1015 cells rapidly decreased in the absence of ALPG, reaching baseline within 24 hrs. Of note, our in vitro assay did not result in 100% CAR induction from all PrimeR+ AB-1015 cells. To understand whether this reflected biology versus a limitation of our in vitro assay, we conducted a CAR induction, sorted the CAR- cells, then performed a second induction. ICT cells that did not express CAR after the first induction were shown to be able to induce CAR during the second induction assay, thereby demonstrating that all PrimeR+ ICT cells have the potential to induce CAR. To assess the sensitivity of AB-1015 for low density ALPG/P and MSLN, isogenic cell lines were engineered to express each antigen at variable levels. In co-culture, AB-1015 was able to prime and kill targets expressing ALPG and MSLN at or below the limit of detection using immunohistochemistry. In light of these observations, the specificity of AB-1015 was verified using a dual flank in vivo model where one tumor expressed both ALPG and MSLN, and the contralateral tumor expressed MSLN alone. While robust anti-tumor activity was observed against the ALPG+/MSLN+ tumor, no activity was detected against the MSLN+ tumor. In summary, AB-1015 CAR induction and receptor turnover kinetics is a dynamic process. A reductionist in vitro model system was developed, enabling analysis of CAR induction and receptor turnover. Importantly, we demonstrated that all PrimeR+ ICT cells have the potential to induce CAR. Finally, the in vivo specificity of AB-1015 for ALPG+/MSLN+ tumors was established using a dual flank specificity model. Together, these data support the continued development of AB-1015 in a phase I clinical trial (NCT05617755). Citation Format: Xinyan Tang, Marvin Chew, Rakesh Sudhakar, Hongruo Yun, Doreen Sakamoto, Vibhavari Sail, Amanda Fearon, Jasper Williams, Jun Feng, Stephen Santoro. Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6319.

The proteasome modulates endocytosis specifically in glomerular cells to promote kidney filtration

Kidney filtration is ensured by the interaction of podocytes, endothelial and mesangial cells. Immunoglobulin accumulation at the filtration barrier is pathognomonic for glomerular injury. The mechanisms that regulate filter permeability are unknown. Here, we identify a pivotal role for the proteasome in a specific cell type. Combining genetic and inhibitor-based human, pig, mouse, and Drosophila models we demonstrate that the proteasome maintains filtration barrier integrity, with podocytes requiring the constitutive and glomerular endothelial cells the immunoproteasomal activity. Endothelial immunoproteasome deficiency as well as proteasome inhibition disrupt the filtration barrier in mice, resulting in pathologic immunoglobulin deposition. Mechanistically, we observe reduced endocytic activity, which leads to altered membrane recycling and endocytic receptor turnover. This work expands the concept of the (immuno)proteasome as a control protease orchestrating protein degradation and antigen presentation and endocytosis, providing new therapeutic targets to treat disease-associated glomerular protein accumulations.

Disrupted degradative sorting of TLR7 is associated with human lupus.

Hyperactive TLR7 signaling has long been appreciated as driver of autoimmune disease in mouse models. Recently, gain-of-function mutations in TLR7 were identified as a monogenic cause of human lupus. TLR7 is an intracellular transmembrane receptor, sensing RNA breakdown products within late endosomes. Here, we show that endosome dysfunction leads to unrestricted TLR7 signaling and is associated with human lupus. The late endosomal BORC complex together with the small GTPase Arl8b controls intracellular TLR7 levels by regulating receptor turnover. This requires a direct interaction between the TLR7-associated trafficking factor Unc93b1 and Arl8b. We identified an UNC93B1 mutation in a patient with childhood-onset lupus, which results in reduced BORC interaction and endosomal TLR7 accumulation. Therefore, a failure to control TLR7 turnover is sufficient to break immunological tolerance to nucleic acids. Our results highlight the importance of an intact endomembrane system in preventing pathological TLR7 signaling and autoimmune disease.

Suppression of TGFβ-Induced Interleukin-6 Secretion by Sinulariolide from Soft Corals through Attenuation of the p38-NF-kB Pathway in Carcinoma Cells.

Sinulariolide (SC-1) is a natural product extracted from the cultured-type soft coral Sinularia flexibilis and possesses anti-inflammation, anti-proliferative, and anti-migratory in several types of cancer cells. However, the molecular pathway behind its effects on inflammation remains poorly understood. Since inflammatory cytokines such as TGFβ, TNFα, IL-1, IL-6, and IL-8 activate transcription factors such as Smads, NF-κB, STAT3, Snail, Twist, and Zeb that drive the epithelial-to-mesenchymal transition (EMT), in this study, we focus on the investigation in effects of SC-1 on TGFβ-induced interleukin-6 (IL-6) releases in an in vitro cell culture model. We showed that both intracellular IL-6 expression and secretion were stimulated by TGFβ and associated with strong upregulation of IL-6 mRNA and increased transcription in A549 cells. SC-1 blocked TGFβ-induced secretion of IL-6 while showing no effect on the induction of fibronectin and plasminogen activator inhibitor-1 genes, indicating that SC-1 interferes with only a subset of TGFβ activities. In addition, SC-1 inhibits TGFβ-induced IL-6 by suppressing p38 MAPK signaling and subsequently inhibits NF-κB and its nuclear translocation without affecting the canonical Smad pathway and receptor turnover. Overall, these data suggest that p38 may involve in the inhibition of SC-1 in IL-6 release, thus illustrating an inhibitory effect for SC-1 in the suppression of inflammation, EMT phenotype, and tumorigenesis.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.

An insulin-regulated arrestin domain protein controls hepatic glucagon action

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the invivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver.

Levodopa-Induced Dyskinesias in Parkinson's Disease: An Overview on Pathophysiology, Clinical Manifestations, Therapy Management Strategies and Future Directions.

Since its first introduction, levodopa has become the cornerstone for the treatment of Parkinson's disease and remains the leading therapeutic choice for motor control therapy so far. Unfortunately, the subsequent appearance of abnormal involuntary movements, known as dyskinesias, is a frequent drawback. Despite the deep knowledge of this complication, in terms of clinical phenomenology and the temporal relationship during a levodopa regimen, less is clear about the pathophysiological mechanisms underpinning it. As the disease progresses, specific oscillatory activities of both motor cortical and basal ganglia neurons and variation in levodopa metabolism, in terms of the dopamine receptor stimulation pattern and turnover rate, underlie dyskinesia onset. This review aims to provide a global overview on levodopa-induced dyskinesias, focusing on pathophysiology, clinical manifestations, therapy management strategies and future directions.

Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation

Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors.

Abstract 6134: Exploiting the oncogenic state of colorectal cancer for therapeutic development

Abstract Over the past two decades, only incremental progresses have been made in the therapeutic management of colorectal cancer (CRC). This is in part attributed to the fact that the Wnt pathway, the oncogenic pathway that initiates the transformation of CRC and sustains tumor maintenance, has been largely undruggable. While most efforts targeting Wnt signaling have revolved around interfering with Wnt target genes that regulate cell growth, we turned our attention to a subset of genes encoding for negative regulators of the pathway. Of particular interest are the cell-surface E3 ubiquitin ligases, ring finger protein 43 (RNF43) and ZNRF3, two known negative regulators of Wnt signaling that promote the turnover of Wnt receptors Frizzled/low-density lipoprotein receptor-related proteins (FZD/LRPs) via ubiquitination-mediated degradation. Given their exposed extracellular domains (ECDs), we set out to explore whether this intrinsic cellular degradative machinery could be repurposed for an antibody-based targeted protein degradation platform. More specifically, we have devised a technology that exploits the elevated expression of ZNRF3 and RNF43 in colon cancer by generating bispecific antibodies that can tether these E3 ubiquitin ligases to a variety of cell surface receptors to promote tumor receptor degradation. Most notably, these antibody degraders are highly active in vivo and can degrade receptors in a tumor specific manner while sparing normal tissues. This platform, that we have been dubbed Proteolysis Targeting Antibodies (PROTABs) describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins. Citation Format: Felipe de Sousa e Melo. Exploiting the oncogenic state of colorectal cancer for therapeutic development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6134.

Rat model of attention-deficit hyperactivity disorder exhibits delayed recovery from acute incisional pain due to impaired descending noradrenergic inhibition

Chronic pain and attention-deficit hyperactivity disorder (ADHD) frequently coexist. However, the common pathology is still unclear. Attenuated noradrenergic endogenous analgesia can produce acute pain chronification, and dysfunction of noradrenergic systems in the nervous system is relevant to ADHD symptoms. Noxious stimuli-induced analgesia (NSIA) is measured to estimate noradrenergic endogenous analgesia in spontaneously hypertensive rats (SHR) as an ADHD model and control. Recovery of pain-related behaviors after paw incision was assessed. Contributions of noradrenergic systems were examined by in vivo microdialysis and immunohistochemistry. The SHR showed attenuated NSIA and needed a more extended period for recovery from acute pain. These results suggest ADHD patients exhibit acute pain chronification due to pre-existing attenuated noradrenergic endogenous analgesia. Immunohistochemistry suggests abnormal noradrenaline turnover and downregulation of the target receptor (alpha2a adrenoceptor). Standard ADHD treatment with atomoxetine restored NSIA and shortened the duration of hypersensitivity after the surgery in the SHR. NSIA protocol activated the locus coeruleus, the origin of spinal noradrenaline, of both strains, but only the control exhibited an increase in spinal noradrenaline. This result suggests dysfunction in the noradrenaline-releasing process and can be recognized as a novel mechanism of attenuation of noradrenergic endogenous analgesia.

Ubiquitylation of PHYTOSULFOKINE RECEPTOR 1 modulates the defense response in tomato.

Phytosulfokine (PSK) is a danger-associated molecular pattern recognized by PHYTOSULFOKINE RECEPTOR 1 (PSKR1) and initiates intercellular signaling to coordinate different physiological processes, especially in the defense response to the necrotrophic fungus Botrytis cinerea. The activity of peptide receptors is largely influenced by different posttranslational modifications, which determine intercellular peptide signal outputs. To date, the posttranslational modification to PHYTOSULFOKINE RECEPTOR 1 (PSKR1) remains largely unknown. Here, we show that tomato (Solanum lycopersicum) PSKR1 is regulated by the ubiquitin/proteasome degradation pathway. Using multiple protein-protein interactions and ubiquitylation analyses, we identified that plant U-box E3 ligases PUB12 and PUB13 interacted with PSKR1, among which PUB13 caused PSKR1 ubiquitylation at Lys-748 and Lys-905 sites to control PSKR1 abundance. However, this posttranslational modification was attenuated upon addition of PSK. Moreover, the disease symptoms observed in PUB13 knock-down and overexpression lines demonstrated that PUB13 significantly suppressed the PSK-initiated defense response. This highlights an important regulatory function for the turnover of a peptide receptor by E3 ligase-mediated ubiquitylation in the plant defense response.