Direct Substrate Research Articles

TENT5 cytoplasmic noncanonical poly(A) polymerases regulate the innate immune response in animals.

Innate immunity is the first line of host defense against pathogens. Here, through global transcriptome and proteome analyses, we uncover that newly described cytoplasmic poly(A) polymerase TENT-5 (terminal nucleotidyltransferase 5) enhances the expression of secreted innate immunity effector proteins in Caenorhabditis elegans. Direct RNA sequencing revealed that multiple mRNAs with signal peptide-encoding sequences have shorter poly(A) tails in tent-5-deficient worms. Those mRNAs are translated at the endoplasmic reticulum where a fraction of TENT-5 is present, implying that they represent its direct substrates. Loss of tent-5 makes worms more susceptible to bacterial infection. Notably, the role of TENT-5 in innate immunity is evolutionarily conserved. Its orthologs, TENT5A and TENT5C, are expressed in macrophages and induced during their activation. Analysis of macrophages devoid of TENT5A/C revealed their role in the regulation of secreted proteins involved in defense response. In summary, our study reveals cytoplasmic polyadenylation to be a previously unknown component of the posttranscriptional regulation of innate immunity in animals.

Src-mediated phosphorylation of the ribosome biogenesis factor hYVH1 affects its localization, promoting partitioning to the 60S ribosomal subunit

Yeast VH1-related phosphatase (YVH1) (also known as DUSP12) is a member of the atypical dual-specificity phosphatase subfamily. Although no direct substrate has been firmly established, human YVH1 (hYVH1) has been shown to protect cells from cellular stressors, regulate the cell cycle, disassemble stress granules, and act as a 60S ribosome biogenesis factor. Despite knowledge of hYVH1 function, further research is needed to uncover mechanisms of its regulation. In this study, we investigate cellular effects of a Src-mediated phosphorylation site at Tyr179 on hYVH1. We observed that this phosphorylation event attenuates localization of hYVH1 to stress granules, enhances shuttling of hYVH1 to the nucleus, and promotes hYVH1 partitioning to the 60S ribosomal subunit. Quantitative proteomics reveal that Src coexpression with hYVH1 reduces formation of ribosomal species that represent stalled intermediates through the alteration of associating factors that mediate translational repression. Collectively, these results implicate hYVH1 as a novel Src substrate and provide the first demonstrated role of tyrosine phosphorylation regulating the activity of a YVH1 ortholog. Moreover, the ribosome proteome alterations point to a collaborative function of hYVH1 and Src in maintaining translational fitness.

PPM1G promotes the progression of lung adenocarcinoma by inhibiting p38 activation via dephosphorylation of MEK6.

The p38 MAP kinase (MAPK) signaling pathway is a key signal transduction cascade that cancer cells employ to sense and adapt to a plethora of environmental stimuli and has attracted much attention as a promising target for cancer therapy. Although the kinases that phosphorylate p38 have been extensively studied, the negative regulation of p38 phosphorylation remains to be elucidated. Here, we found that PPM1G was highly expressed in lung adenocarcinoma (LUAD) compared to normal tissues, and higher levels of PPM1G were observed in adverse staged LUAD. Furthermore, the higher levels of PPM1G were highly correlated with poor prognosis, according to the Cancer Genome Atlas cohort. Most importantly, we identified phospho-MEK6 as a direct substrate of PPM1G. PPM1G, a metal-dependent protein phosphatase family phosphatase, could reduce p38 phosphorylation via MEK6 dephosphorylation and contribute to the proliferation, invasion and metastasis of LUAD. Our study highlighted the essential role of PPM1G in LUAD and shed new light on unveiling the regulation of p38 activity via direct dephosphorylation of MEK6 in malignant transformation. Together, this study provides new insight into the complexity of regulating the versatile p38 signaling and suggests new directions in intervening in p38 MAPK signaling.

Abstract 15001: Correlating Site of Origin in Ventricular Arrhythmias With Site of Inflammation in Patients With Underlying Myocardial Inflammation via FDG-PET Scan

Introduction: Patients with underlying myocardial inflammation often present with otherwise unexplained ventricular arrhythmias. We seek to further elucidate if these arrhythmias originate from the areas of inflammatory FDG uptake, suggesting a direct arrhythmogenic substrate. Hypothesis: To determine if sites of inflammation on Fluorodeoxyglucose (FDG)-PET scan correlate with the site of origin on electroanatomic mapping for PVC/VT ablation in underlying myocardial inflammation. Methods: We retrospectively reviewed patients presenting with frequent ventricular arrhythmias (PVC/VT) with positive FDG-PET scans indicative of underlying myocardial inflammation, and included patients who had persisting, symptomatic, burdensome arrhythmias despite multiple cycles of immunosuppressive therapy or resolution of inflammation that eventually required catheter ablation for PVC/VT. Patients were then reviewed to see if their site of origin (SOO) of PVCs or VT correlated with the site of inflammation (SOI) seen on FDG-PET scan. Results: We studied 42 patients with documented PVCs or VT, a positive FDG-PET scan and eventual PVC/VT ablation. Mean age was 58.9 years + 14.5. Mean EF was 42.6 + 8.6%. Average PET SUV Max was 3.8 + 1.2, with an average involvement of 2.7 + 2.1 segments. Nearly all patients had multiple sites of FDG uptake (95.2%), with 69.% localized vs 31.0% diffuse distributions. The 42 patients had SOO in the following distribution: RVOT (30.1%), Inferoseptal (26.2%), RCC area, (19.0%), LVOT: (14.3%), Anteroseptal: (14.3%), Basal (11.9%), LV Summit (9.5%), and anterolateral (4%). 35.7% of patients had multiple SOO. Exact correlation between SOO-SOI was seen in 23/42 (54.8%), whereas partial correlation (matching sites with additional PVC locations vs uptake distributions, or additional uptake distributions vs PVC locations) was seen in 10/42 (23.8%). No correlation was seen in 9/42 (21.4%). Of the 19 that had partial or no correlation, the most frequent distributive area of uptake without PVC correlation was anterolateral, present in 7/19 (36.8%). Conclusions: In our retrospective study, patients undergoing cardiac FDG-PET scans to confirm myocarditis, had a positive correlation of site of PVC/VT origin and site of inflammation.

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Calcium Channel α2δ1 is Essential for Pancreatic Tumor-Initiating Cells through Sequential Phosphorylation of PKM2

Tumor-initiating cells (TICs) drive pancreatic cancer tumorigenesis, therapeutic resistance, and metastasis. However, TICs are highly plastic and heterogenous, which impede the robust identification and targeted therapy of such a population. The aim of this study is to identify the surface marker and therapeutic target for pancreatic TICs. We isolated voltage-gated calcium channel α2δ1 subunit (isoform 5)-positive subpopulation from pancreatic cancer cell lines and freshly resected primary tissues by fluorescence-activated cell sorting and evaluated their TIC properties by spheroid formation and tumorigenic assays. Coimmunoprecipitation was used to identify the direct substrate of CaMKⅡδ. We demonstrate that the voltage-gated calcium channel α2δ1 subunit (isoform 5) marks a subpopulation of pancreatic TICs with the highest TIC frequency among the known pancreatic TIC markers tested. Furthermore, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca2+ influx, which activates CaMKⅡδ to directly phosphorylate PKM2 at T454 that results in subsequent phosphorylation at Y105 to translocate into nucleus, enhancing the stem-like properties. Interestingly, blocking α2δ1 with its specific antibody has remarkably therapeutic effects on pancreatic cancer xenografts by reducing TICs. α2δ1 promotes pancreatic TIC properties through sequential phosphorylation of PKM2 mediated by CaMKⅡδ, and targeting α2δ1 provides a therapeutic strategy against TICs for pancreatic cancer.

Transgenic mice harboring direct repeat substrates reveal key underlying causes of homologous recombination in vivo

Homology directed repair is a critical process for maintaining the genome of mammalian cells. While considered to be relatively error free, homologous recombination (HR) can lead to insertions, deletions, translocations, and loss of heterozygosity. Furthermore, it is known that conditions that cause HR events are often carcinogenic, and that HR modulates susceptibility to cancer chemotherapeutics, making HR relevant to both cancer etiology and treatment. To learn about HR in vivo, several laboratories have developed mouse models that harbor direct repeat substrates for which HR yields a phenotype that can be visualized by either a change in pigmentation or by expression of a fluorescent protein. An exciting aspect of this work is that it is possible to detect recombinant cells within intact tissues and thus learn about how physiological changes, genes, and exposures modulate HR susceptibility in vivo, as well as which cell types are most susceptible to HR, and the extent to which recombinant cells have undergone clonal expansion. Here, we review progress in studying HR in vivo for four different mouse strains that harbor direct repeat substrates for HR detection, namely the pun mice, the fluorescent yellow direct repeat (FYDR) mice, the EGFP-DR mice, and the ROSA26 direct repeat (RaDR) mice. Key advances in our understanding of fundamental processes that modulate HR susceptibility in vivo are the focus of this review.

Reactive Additive Capillary Stamping with Double Network Hydrogel-Derived Aerogel Stamps under Solvothermal Conditions

Integration of solvothermal reaction products into complex thin-layer architectures is frequently achieved by combinations of layer transfer and subtractive lithography, whereas direct additive substrate patterning with solvothermal reaction products has remained challenging. We report reactive additive capillary stamping under solvothermal conditions as a parallel contact-lithographic access to patterns of solvothermal reaction products in thin-layer configurations. To this end, corresponding precursor inks are infiltrated into mechanically robust mesoporous aerogel stamps derived from double-network hydrogels. The stamp is then brought into contact with a substrate to be patterned under solvothermal reaction conditions inside an autoclave. The precursor ink forms liquid bridges between the topographic surface pattern of the stamp and the substrate. Evaporation-driven enrichment of the precursors in these liquid bridges, along with their liquid-bridge-guided conversion into the solvothermal reaction products, yields large-area submicron patterns of the solvothermal reaction products replicating the stamp topography. For example, we prepared thin hybrid films, which contained ordered monolayers of superparamagnetic submicron nickel ferrite dots prepared by solvothermal capillary stamping surrounded by nickel electrodeposited in a second orthogonal substrate functionalization step. The submicron nickel ferrite dots acted as a magnetic hardener, halving the remanence of the ferromagnetic nickel layer. In this way, thin-layer electromechanical systems, transformers, and positioning systems may be customized.

An Integrated Proteomic Strategy to Identify SHP2 Substrates.

Protein phosphatases play an essential role in normal cell physiology and the development of diseases such as cancer. The innate challenges associated with studying protein phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly coordinated networks. Here, we introduce a novel strategy using substrate-trapping mutants coupled with quantitative proteomics methods to identify physiological substrates of Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. The technique integrates three parallel mass spectrometry-based proteomics experiments, including affinity isolation of substrate-trapping mutant complex using wild-type and SHP2 KO cells, in vivo global quantitative phosphoproteomics, and in vitro phosphatase reaction. We confidently identified 18 direct substrates of SHP2 in the epidermal growth factor receptor signaling pathways, including both known and novel SHP2 substrates. Docking protein 1 was further validated using biochemical assays as a novel SHP2 substrate, providing a mechanism for SHP2-mediated Ras activation. This advanced workflow improves the systemic identification of direct substrates of protein phosphatases, facilitating our understanding of the equally important roles of protein phosphatases in cellular signaling.

Activation and turnover of the plant immune signaling kinase BIK1: a fine balance.

Mechanisms to sense and respond to pathogens have evolved in all species. The plant immune pathway is initiated by the activation of transmembrane receptor kinases that trigger phosphorylation relays resulting in cellular reprogramming. BOTRYTIS-INDUCED KINASE 1 (BIK1) is a direct substrate of multiple immune receptors in Arabidopsis thaliana and is a central regulator of plant immunity. Here, we review how BIK1 activity and protein stability are regulated by a dynamic interplay between phosphorylation and ubiquitination.

CDC-like kinase 4 deficiency contributes to pathological cardiac hypertrophy by modulating NEXN phosphorylation

Kinase-catalyzed phosphorylation plays a crucial role in pathological cardiac hypertrophy. Here, we show that CDC-like kinase 4 (CLK4) is a critical regulator of cardiomyocyte hypertrophy and heart failure. Knockdown of Clk4 leads to pathological cardiomyocyte hypertrophy, while overexpression of Clk4 confers resistance to phenylephrine-induced cardiomyocyte hypertrophy. Cardiac-specific Clk4-knockout mice manifest pathological myocardial hypertrophy with progressive left ventricular systolic dysfunction and heart dilation. Further investigation identifies nexilin (NEXN) as the direct substrate of CLK4, and overexpression of a phosphorylation-mimic mutant of NEXN is sufficient to reverse the hypertrophic growth of cardiomyocytes induced by Clk4 knockdown. Importantly, restoring phosphorylation of NEXN ameliorates myocardial hypertrophy in mice with cardiac-specific Clk4 deletion. We conclude that CLK4 regulates cardiac function through phosphorylation of NEXN, and its deficiency may lead to pathological cardiac hypertrophy. CLK4 is a potential intervention target for the prevention and treatment of heart failure.

Membrane Targeting and GTPase Activity of Rab7 Are Required for Its Ubiquitination by RNF167

Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7′s membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other’s lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot–Marie–Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7.

Oncoprotein DJ-1 interacts with mTOR complexes to effect transcription factor Hif1α-dependent expression of collagen I (α2) during renal fibrosis

Proximal tubular epithelial cells respond to transforming growth factor β (TGFβ) to synthesize collagen I (α2) during renal fibrosis. The oncoprotein DJ-1 has previously been shown to promote tumorigenesis and prevent apoptosis of dopaminergic neurons; however, its role in fibrosis signaling is unclear. Here, we show TGFβ-stimulation increased expression of DJ-1, which promoted noncanonical mTORC1 and mTORC2 activities. We show DJ-1 augmented the phosphorylation/activation of PKCβII, a direct substrate of mTORC2. In addition, coimmunoprecipitation experiments revealed association of DJ-1 with Raptor and Rictor, exclusive subunits of mTORC1 and mTORC2, respectively, as well as with mTOR kinase. Interestingly, siRNAs against DJ-1 blocked TGFβ-stimulated expression of collagen I (α2), while expression of DJ-1 increased expression of this protein. In addition, expression of dominant negative PKCβII and siRNAs against PKCβII significantly inhibited TGFβ-induced collagen I (α2) expression. In fact, constitutively active PKCβII abrogated the effect of siRNAs against DJ-1, suggesting a role of PKCβII downstream of this oncoprotein. Moreover, we demonstrate expression of collagen I (α2) stimulated by DJ-1 and its target PKCβII is dependent on the transcription factor hypoxia-inducible factor 1α (Hif1α). Finally, we show in the renal cortex of diabetic rats that increased TGFβ was associated with enhanced expression of DJ-1 and activation of mTOR and PKCβII, concomitant with increased Hif1α and collagen I (α2). Overall, we identified that DJ-1 affects TGFβ-induced expression of collagen I (α2) via an mTOR-, PKCβII-, and Hif1α-dependent mechanism to regulate renal fibrosis.

Polygonum multiflorum Thunb. Induces hepatotoxicity in SD rats and hepatocyte spheroids by Disrupting the metabolism of bilirubin and bile acid

Ethnopharmacological relevanceThe liver damage associated with Polygonum multiflorum Thunb. (P. multiflorum) and its preparations have aroused widespread concern. Opinions on the toxicity mechanisms and targets of P. multiflorum vary, and the toxic components are even more controversial. However, based on the current research results, we believed that any single component in P. multiflorum could not directly lead to liver injury, but may be the synergistic effect of multiple components. In addition, the toxicity mechanism also involved multiple targets. Aim of the studyThis study aimed to elucidate the mechanism and target of the hepatotoxicity of P. multiflorum. Materials and methodsIn this study, the manifestations of liver injury triggered by P. multiflorum and the associated metabolic enzymes/transporters in the metabolic pathways of bilirubin and bile acid were investigated to elucidate the mechanism and target of the hepatotoxicity of P. multiflorum and related components. First, the hepatotoxicity and potential effect of P. multiflorum on both metabolic pathways were studied in rats administered P. multiflorum extracts (in 70% ethanol) for 42 days. Then, in vitro cultured hepatocyte spheroids were used to determine the hepatotoxicity of monomer components. ResultsThis revealed that P. multiflorum could simultaneously block bilirubin(BIL) and bile acid(BA) metabolism pathways, subsequently leading to liver damage. The targets and modes of action include reducing the activity of UGT1A1, the only metabolic enzyme of BIL, downregulating BIL and BA uptake transporters NTCP, OATP1B1, OATP1B3, efflux transporters MRP2, and BSEP, and upregulating efflux transporter MRP3. Furthermore, our data indicated that 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucoside (TSG) and emodin-8-O-β-D-glucoside (EG) are the main toxic components in P. multiflorum. TSG accounts for 3.71% of the total content of P. multiflorum. In addition to markedly downregulating UGT1A1, TSG can upregulate OATP1B1/3 and promote the uptakes of bilirubin and bile acid, producing synergistic toxicity. EG accounts for 0.29% of the total content and demonstrates direct hepatotoxicity and extensive substrate overlap with bilirubin and bile acids. It can affect these two metabolic pathways simultaneously, promoting the accumulation of both bilirubin and bile acid for further toxic effects. Emodin is other major component, accounting for 0.01% of the total content, and its hepatotoxicity mechanisms include direct toxicity and inhibitory effects on bilirubin metabolizing enzymes. However, emodin is mainly distributed in the kidneys, so its hepatotoxicity risk is relatively low. ConclusionThe simultaneous blockade of bilirubin and bile acid metabolic pathways as the critical toxic mechanism of P. multiflorum-induced liver injury, and potential toxic components were TSG and EG.

Abstract 154: Novel AKT isoform-preferred substrates as potentially targetable drivers of metastatic prostate cancer

Abstract PI3K/AKT pathway activating mutations are the most prevalent in all of human cancers, and yet drugs that target the PI3K lipid kinase or AKT serine/threonine kinase families have performed poorly in clinical trials on solid cancers. PTEN loss, one of the most frequent mutations in prostate cancer (PC), results in AKT activation, an assumed driver of aggressive metastatic disease (mPC). The poor efficacy of PI3K/AKT inhibitors in PC likely relate to multiple PI3K and AKT isoforms which have compensatory or even oppositional roles. Whereas PI3K-p110 isoform-specific inhibitors are being used in clinical trials, no clinical AKT isoform-specific inhibitors exist. Data from our lab and others indicate that progression to aggressive mPC is dependent on AKT2-, and possibly AKT3-specific pathways. However, previous screens could not distinguish between direct vs. indirect AKT isoform-preferred substrates, and they only focused on substrates with canonical phosphorylation motifs (RxRxxS*/T*). We addressed this gap using a novel phosphoproteomics analysis that identifies direct AKT isoform substrates from PC lysates in which substrates are in endogenous protein-protein complexes. Scores of novel and previously known substrates were identified that are preferred by individual or combinations of AKT isoforms, conforming to both the canonical and four novel non-canonical motifs; >95% of these sites exist in phosphoproteome databases. By comparing data from PC cell lines isogenic for PTEN loss or expression, we identified PTEN-influenced substrate phosphorylation or AKT isoform preference. However, most of these cases did not correlate with changes in substrate abundance, suggesting novel roles for PTEN in regulating substrate modifications or cellular compartmentalization. We hypothesize that AKT2 and AKT3 substrate phosphorylation drives parameters of in vitro and in vivo mPC progression, suggesting that these AKT isoforms may be therapeutic targets if specific inhibitors can be produced. Data will be shown i) confirming AKT isoform preference of specific substrates, ii) identifying novel mechanisms by which PTEN influences substrate usage or isoform preference via altering cellular compartmentalization or post-translational modification, and iii) validating our identified non-canonical AKT isoform-specific phosphorylation sites. We also show that the phosphorylation of AKT2/3-preferred substrates drives metastatic progression in vitro and in vivo. Our data identify a much broader array of AKT isoform-specific, -preferred and -shared substrates, including those encoding previously unidentified non-canonical phosphorylation motifs. We also show novel function for PTEN, namely, to influence AKT isoform substrate usage and preference. These data will allow us to develop AKT isoform-specific inhibitors that could be used to suppress mPC progression. Citation Format: Seamus Degan, Renae Holtz, Henry G. Withers, Jun Qu, Sailee Suryakant Rasam, Irwin H. Gelman. Novel AKT isoform-preferred substrates as potentially targetable drivers of metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 154.

Williams-Beuren Syndrome Related Methyltransferase WBSCR27: From Structure to Possible Function

Williams-Beuren syndrome (WBS) is a genetic disorder associated with the hemizygous deletion of several genes in chromosome 7, encoding 26 proteins. Malfunction of these proteins induce multisystemic failure in an organism. While biological functions of most proteins are more or less established, the one of methyltransferase WBSCR27 remains elusive. To find the substrate of methylation catalyzed by WBSCR27 we constructed mouse cell lines with a Wbscr27 gene knockout and studied the obtained cells using several molecular biology and mass spectrometry techniques. We attempted to pinpoint the methylation target among the RNAs and proteins, but in all cases neither a direct substrate has been identified nor the protein partners have been detected. To reveal the nature of the putative methylation substrate we determined the solution structure and studied the conformational dynamic properties of WBSCR27 in apo state and in complex with S-adenosyl-L-homocysteine (SAH). The protein core was found to form a canonical Rossman fold common for Class I methyltransferases. N-terminus of the protein and the β6–β7 loop were disordered in apo-form, but binding of SAH induced the transition of these fragments to a well-formed substrate binding site. Analyzing the structure of this binding site allows us to suggest potential substrates of WBSCR27 methylation to be probed in further research.

Abstract 589: Discovery of NTX-001, a potent Cbl-b inhibitor with antitumor activity in syngeneic models

Abstract Introduction: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase and a member of a highly conserved family of Cbl proteins, catalyzes the ubiquitination of substrate proteins to regulate multiple signaling events in a variety of cell types, including immune cells. In T cells, Cbl-b negatively regulates adaptive immune system signaling by establishing the threshold for the activation of antigen receptors. Additionally, Cbl-b regulates the function of other immune cell types, including NK cells, dendritic cells (DC) and monocytes. Cbl-b deficient T cells no longer require a costimulatory signal to be fully activated. Cbl-b KO mice spontaneously reject tumors via an enhanced immune response. Taken together, these findings point to Cbl-b inhibitors as having the potential to be highly efficacious immuno-oncology agents. Experimental Procedures: A structure-based drug design approach was used to identify potent inhibitors of Cbl-b. Biochemical and cellular binding assays, a cellular NFAT-luciferase reporter assay, as well as primary in vitro human and mouse T cell activation assays measuring IL-2 production were used to profile inhibitor compounds. In vivo activity of Cb-b inhibitors was evaluated using an anti-CD3 mouse model and a CT-26 syngeneic mouse model Results: Cbl-b inhibitor NTX-001 potently binds to Cbl-b, preventing Cbl-b phosphorylation and binding to E2. In cells, NTX-001 treatment results in enhanced phosphorylation of ZAP-70, a protein proximal to the TCR and reported to be a direct substrate of Cbl-b. Additionally, inhibition of Cbl-b potently enhances cytokine secretion from primary human and mouse T cells. In vivo, an increase in cytokines and T cell activation markers was observed after a single dose of NTX-001. Repeated dosing of NTX-001 showed dose-dependent anti-tumor activity in the colorectal CT-26 syngeneic model. Conclusions: NTX-001 is a potent Cbl-b inhibitor that demonstrated strong T cell activation and anti-tumor activity in a syngeneic tumor model. These data support Cbl-b inhibitors as a promising therapeutic opportunity for cancer treatment. Citation Format: Fred Csibi-Levin, Silvana Leit, David L. Laughton, Tom Baker, Suzanne L. Jacques, Beth Browning, Angela V. Toms, Samantha Garside, Simon D'Archivio, Katarzyna Kopycka, Xiaohua Zhu, Allan M. Jordan, Stuart Thomson, Christine Loh, Peter Tummino, David N. Ciccone. Discovery of NTX-001, a potent Cbl-b inhibitor with antitumor activity in syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 589.

DUSP3 regulates phosphorylation-mediated degradation of occludin and is required for maintaining epithelial tight junction

BackgroundTight junctions (TJ) are multi-protein complexes that hold epithelial cells together and form structural and functional barriers for maintaining proper biological activities. Dual specificity phosphatase 3 (DUSP3), a suppressor of multiple protein tyrosine (Tyr) kinases, is decreased in lung cancer tissues. Here we demonstrated the role of DUSP3 in regulation of epithelial TJ.MethodsBarrier functions of TJ were examined in wild-type or DUSP3-deficient lung epithelial cells. Animal and clinical data were analyzed for the association between DUSP3 deficiency and lung cancer progression. Proximity ligation assay, immunoblotting, and phosphatase assay were performed to study the effect of DUSP3 on the TJ protein occludin (OCLN). Mutations of Tyr residues on OCLN showed the role of Tyr phosphorylation in regulating OCLN.ResultsCompared to those of the DUSP3-expressing cells, we found the expression and distribution of ZO-1, a TJ-anchoring molecule, were abnormal in DUSP3-deficient cells. OCLN had an increased phosphorylation level in DUSP3-deficient cells. We identified that OCLN is a direct substrate of DUSP3. DUSP3 regulated OCLN ubiquitination and degradation through decreasing OCLN tyrosine phosphorylation directly or through suppressing focal adhesion kinase, the OCLN kinase.ConclusionOur study revealed that DUSP3 is an important TJ regulatory protein and its decrease may be involved in progression of epithelial cancers.

Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.

191-OR: Hepatic Expression of the Branched-Chain Keto Acid Dehydrogenase Kinase Is Sufficient to Cause Glucose Intolerance in Lean Mice

Perturbed branched-chain amino acid homeostasis is a feature of obesity, insulin-resistance and type 2 diabetes. Induction of the branched-chain keto acid dehydrogenase kinase (BDK) in liver is one mechanism that is thought to contribute to elevated BCAA levels in these conditions. Whether higher levels of BDK in liver are sufficient to disturb systemic BCAA homeostasis and promote glucose intolerance in the absence of obesity or insulin resistance has never been directly tested. We treated 12 week-old C57BL6/J mice with adeno-associated virus, serotype 8 (AAV8) -BDK or control AAV8-GFP to promote long term expression of BDK specifically in liver of lean mice. Mice were then maintained on a standard low-fat chow diet for 8 weeks (n= 9 AAV8-GFP and n=AAV8-BDK) . AAV8-BDK significantly raised BDK protein and mRNA expression in liver but not adipose, skeletal muscle, or cardiac tissue compared to AAV8-GFP control mice. The higher expression of BDK in liver was associated with robust inhibitory phosphorylation of the branched-chain keto acid dehydrogenase (BCKDH) on ser293. AAV8-mediated induction of BDK expression in liver in our study was sufficient to promote significant elevations in circulating levels of the direct substrate of BCKDH, the branched chain α-keto acids as well as their cognate BCAA. There was no effect of AAV8-BDK to alter food intake, weight gain or adiposity. However, glucose tolerance measured by a 1g/kg intraperitoneal glucose tolerance test at week 7 was significantly impaired in mice receiving AAV8-BDK compared to AAV8-GFP control. These data demonstrate for the first time that induction of BDK in liver, as occurs with obesity, is sufficient to disturb systemic BCAA and glucose homeostasis in lean mice. Disclosure J.Walejko: None. R.W.Mcgarrah: None. P.J.White: None. Funding American Diabetes Association (1-16-INI-17)