Interacting Protein Research Articles

PHIP variants associated with Chung-Jansen syndrome disrupt replication fork stability and genome integrity.

Chung–Jansen syndrome (CJS) is a rare, autosomal dominant disorder characterized by developmental delay, intellectual disability/cognitive impairment, behavioral challenges, obesity, and dysmorphic features. CJS is associated with heterozygous variants in PHIP (Pleckstrin-Homology Interacting Protein), a gene that encodes one of several substrate receptors for Cullin4-RING (CRL4) E3 ubiquitin ligase complex. Full-length PHIP, also called DCAF14, was recently identified to function as a replication stress response protein. Herein, we report the identification of two PHIP missense variants identified by exome sequencing in unrelated individuals with CJS. The variants p.D488V and p.E963G occur in different functional elements of DCAF14-WD40 repeat domain and pleckstrin homology-binding region (PBR), respectively. Using DNA fiber assays, we reveal that cells expressing either variant exhibit defective replication fork progression in conditions of replication stress. Furthermore, unlike wild-type DCAF14, both variants fail to accomplish DNA replication after exposure to genotoxic stress indicating a critical role of DCAF14 in protecting stalled replication forks. Thus, we have identified replication defects associated with CJS variants and predict replication-associated genome instability with CJS syndrome.

Integrative study of gene expression datasets in retinal samples of Diabetic Retinopathy

Diabetic Retinopathy is prevalent among patients with uncontrolled hyperglycemia resulting in vision loss. Despite numerous challenges to create a link among these conditions, the characterization of pathological neovascularization causing retinal damage due to the prognosis of early non-proliferative diabetic retinopathy to late proliferative diabetic retinopathy needs deep understanding. In this study, meta-analysis-based integration of gene expression datasets for the fibrovascular membrane of PDR and neural retina of NPDR were compared, to investigate the differentially expressed genes involved in retinal angiogenesis. Human samples with gene expression profiling of the same experiment type and platform with sufficient information for analysis were included in the study. The studies from cell lines and non-human studies, human samples that include serum, cornea, lens, and/or other ocular tissues or fluids, and studies that lack basic information for analysis were excluded. The microarray datasets available in the Gene Expression Omnibus database of the early and late stages in DR were screened to find common gene expression profiles. Using the INMEX bioinformatics tool, significantly upregulated and downregulated genes in the neural retina of Non-Proliferative Diabetic Retinopathy and fibrovascular membrane of Proliferative Diabetic Retinopathy were compared and studied by the combine effect size method. Using the STRING database PPI network, 50 upregulated and 50 downregulated genes were used to find the key candidate genes involved in retinal disease/degeneration in eye/retinal tissues. In the extensive gene expression meta-analysis performed using INMEX bioinformatics tool, overall, 7935 differentially expressed genes were identified and the respective heatmap was created by using the visualization tools of INVEX. STRING database PPI network identified Retinol Binding Protein 3, Neural Retina Leucine Zipper, S-Antigen Visual Arrestin, Peripherin 2, and Aryl Hydrocarbon Receptor Interacting Protein Like-1 to be the most highly ranked hub genes. The newly discovered potential genes related to retinal angiogenesis causing FVM formation in DR may provide insight into the cellular pathogenesis of NPDR to PDR.

Glutamate Receptor Interacting Protein 1 in the Dorsal CA1 Drives Alpha-amino-3-hydroxy-5-methyl-4-Isoxazolepropionic Acid Receptor Endocytosis and Exocytosis Bidirectionally and Mediates Forgetting, Exploratory, and Anxiety-like Behavior

Endocytosis of GluA2-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in CA1 of the hippocampus regulates forgetting; deficits in forgetting nociceptive memory can induce serious stress disorders. As a transporter of GluA2-containing AMPAR, the functions of glutamate receptor interacting protein 1 (GRIP1) in forgetting and possible stress responses remain unclear. Lentivirus-mediated interference of GRIP1 expression or function in the dorsal CA1 of the hippocampus in mice indicated that GRIP1 overexpression enhanced spatial memory, impaired forgetting in a Barnes maze, and induced anxiety-like behavior in the open field and elevated plus-maze test. In contrast, GRIP1 knockdown impaired learning capacity. Furthermore, inhibition of the PDZ2 and PDZ4/5 domains of GRIP1 and GluA2-dn enhanced learning capacity, whereas GluA2-dn impaired spatial memory; inhibition of the PDZ2 and PDZ4/5 domains of GRIP1 also decreased forgetting capacity to some extent. Importantly, inhibition of both the PDZ2 and PDZ4/5 domains of GRIP1 induced anxiety-like behavior but not GluA2-dn. Furthermore, optogenetic control of both GluA1 and GluA2 insertion into the postsynaptic membrane impaired memory and induced anxiety-like behavior. In vitro experiments showed that GRIP1-ov and -dn, inhibition of PDZ2 and PDZ4/5 domains of GRIP1, and GluA2-dn decreased glycine-induced GluA1 and GluA2 exocytosis; meanwhile, GRIP1-ov and -dn, and interference of PDZ2 and PDZ4/5 domains of GRIP1 blocked AMPA- and NMDA-induced GluA1 and GluA2 endocytosis. Overall, these results suggest that GRIP1 drives AMPA receptor endocytosis and exocytosis bidirectionally; furthermore, GRIP1-induced stabilization of anchoring postsynaptic GluA1 and GluA2 impairs forgetting and induces anxiety-like behavior. GRIP1 may provide a potential therapeutic target in posttraumatic syndromes and anxiety disorders.

Investigation on the cellular mechanism of Prunetin evidenced through next generation sequencing and bioinformatic approaches against gastric cancer

Gastric cancer is the common type of malignancy positioned at second in mortality rate causing burden worldwide with increasing treatment options. More accurate and reliable diagnostic methods/biomarkers are urgently needed. The application of transcriptomics technologies possesses the high efficiency of identifying key metabolic pathways and functional genes in cancer research. In this study, we performed a transcriptome analysis on Prunetin treated AGS cells. A total of 1,118 differentially expressed (DE) genes on Prunetin treated AGS cancer cells, among which 463 were up-regulated and 655 were down-regulated. Notably, around 40 genes were found to be related with necroptosis, among which 16 genes were found to be in close association with Receptor Interacting Protein Kinase (RIPK) family. Validation of the RIPK genes through GEPIA identified 8 genes (NRP1, MNX1, SSRP1, PRDX2, PLRG1, LGALS4, SNX5 and FXYD3) which are highly expressed in stomach cancer were significantly down-regulated in PRU treated samples. In conclusion, the sequencing data explores the expression of RIPK mediated genes through necroptosis signaling network in treating gastric cancer. The futuristic validations on the 8 genes as candidate biomarkers will offer a treatment approach against gastric cancer using PRU.

The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

PDZK1 Interacting Protein 1 Promotes the Progression of Papillary Thyroid Cancer.

The incidence of papillary thyroid cancer (PTC) has increased rapidly in recent decades, and tumor progression events are common in PTC. The purpose of our study is to identify the differentially expressed genes (DEGs) correlated with PTC progression and investigate the function of PDZK1IP1 (PDZK1 interacting protein 1) in PTC. We first analyzed DEGs associated with PTC progression between paired PTC and normal thyroid tissues in 3 Gene Expression Omnibus data sets (GSE29265, GSE33630, and GSE60542) and The Cancer Genome Atlas (TCGA) database. Data from the TCGA database and our institution were utilized to explore the relationship between PDZK1IP1 expression and clinicopathological characteristics of PTC. The CCK8 cell proliferation assay, clone formation assay, flow cytometry assay, and the xenograft model were used to investigate the function of PDZK1IP1 in PTC. Thirty-nine DEGs associated with PTC progression were identified, in which only PDZK1IP1 was upregulated in PTC tissue at both messenger RNA and protein levels. In addition, we found that high expression of PDZK1IP1 in the TCGA database was associated with poor progression-free survival, extrathyroidal extension, high stage, tall cell variant, and BRAFV600E mutation of the PTC (P < 0.001). In our collected samples, high expression of PDZK1IP1 was only related to lymph node metastasis (P < 0.05). Overexpression of PDZK1IP1 significantly promoted proliferation and inhibited apoptosis of PTC cells. Knockdown of PDZK1IP1 significantly inhibited proliferation, promoted apoptosis, and prevented xenograft formation of PTC cells. PDZK1IP1 is an oncogene for tumorigenesis and development of PTC and might be a potential therapeutic target.

Identification and Expression Analysis of an Interacting Protein (LvFABP) that Mediates Vibrio parahaemolyticus AHPND Toxin Action.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus causing AHPND (VPAHPND) is the most serious disease affecting shrimp farming. The PirAvp and PirBvp toxins of VPAHPND are known virulence factors. However, the corresponding target protein in shrimp that mediates their action has not been identified. By screening yeast two-hybrid cDNA libraries from intestine, stomach, and hepatopancreas of Litopenaeus vannamei, the protein with the largest increase in gene expression in shrimp hepatopancreas in response to VPAHPND challenge was identified and designated LvFABP. Analysis revealed high sequence homology of the LvFABP gene and a lipocalin/cytosolic fatty acid binding gene. Yeast two-hybrid pairwise analysis, GST-pull down assay, and far-western blot assay were performed to determine the interaction between LvFABP and PirBvp. LvFABP was able to directly bind to PirBvp. The expression of LvFABP in the hepatopancreas was significantly higher at P23 and P27 developmental stages of L. vannamei. RNA interference (RNAi) of LvFABP reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the intestine, stomach, and hepatopancreas after VPAHPND challenge. We concluded that the LvFABP was involved in AHPND pathogenesis and acted as a VPAHPND toxin interacting protein. This is the first identification of VPAHPND toxin interacting protein from the shrimp digestive system by yeast two-hybrid library screening and were confirmed by in vitro protein interaction verification and in vivo challenge experiments. This study provides novel insight into the contributions of LvFABP towards AHPND pathogenesis in shrimp. The findings could inform AHPND preventative measures in shrimp farming.

Coilin as a regulator of NF-kB mediated inflammation in preeclampsia.

ABSTRACTThe nuclear factor-Kappa B (NF-κB) pathway is a crucial mediator of inflammatory signaling. Aberrant activation of NF-κB is associated with several disorders including preeclampsia (PE). Many regulators of the NF-κB pathway have been identified, including microRNAs (miRNAs). Specifically, miR-517-3p targets mRNA encoding TNFAIP3 Interacting Protein 1 (TNIP1), an inhibitor of NF-κB signaling. Activation of NF-κB increases production of the cytokine TNF superfamily member 15 (TNFSF15), leading to the upregulation of anti-angiogenic soluble vascular endothelial growth factor receptor 1 (sFlt-1). We have previously observed that Cajal bodies (CBs), subnuclear domains, are associated with the chromosome 19 miRNA gene cluster (C19MC), which encodes miR-517-3p. We have also found that coilin, the CB marker protein, is a positive regulator of miRNA biogenesis. Here we report that coilin is a regulator of miR-517-3p, sFlt-1, TNIP1, TNFSF15 and NF-κB activation, and this regulation is influenced by hypoxia. We also report that coilin and CBs are induced in the reduced uterine perfusion pressure (RUPP) rat model of PE. Collectively, the data presented here implicate coilin as a novel regulator of NF-κB activation and sFlt-1 upregulation.

Physiological and Pathophysiological Roles of Thioredoxin Interacting Protein: A Perspective on Redox Inflammation and Metabolism.

Significance: Thioredoxin interacting protein (TXNIP) is a member of the arrestin fold superfamily with important cellular functions, including cellular transport, mitochondrial energy generation, and protein cycling. It is the only arrestin-domain protein known to covalently bind to thioredoxin and plays roles in glucose metabolism, inflammation, apoptosis, and cancer. Recent Advances: The crystal structure of the TXNIP-thioredoxin complex provided details about this fascinating interaction. Recent studies showed that TXNIP is induced by endoplasmic reticulum (ER) stress, activates NLR family pyrin domain containing 3 (NLRP3) inflammasomes, and can regulate glucose transport into cells. The tumor suppressor role of TXNIP in various cancer types and the role of TXNIP in fructose absorption are now described. Critical Issues: The influence of TXNIP on redox state is more complex than its interaction with thioredoxin. Future Directions: It is incompletely understood which functions of TXNIP are thioredoxin-dependent. It is also unclear whether TXNIP binding can inhibit glucose transporters without endocytosis. TXNIP-regulated control of ER stress should also be investigated further. Antioxid. Redox Signal. 38, 442-460.

Distinct interactors define the p63 transcriptional signature in epithelial development or cancer.

The TP63 is an indispensable transcription factor for development and homeostasis of epithelia and its derived glandular tissue. It is also involved in female germline cell quality control, muscle and thymus development. It is expressed as multiple isoforms transcribed by two independent promoters, in addition to alternative splicing occurring at the mRNA 3′-UTR. Expression of the TP63 gene, specifically the amino-deleted p63 isoform, ΔNp63, is required to regulate numerous biological activities, including lineage specification, self-renewal capacity of epithelial stem cells, proliferation/expansion of basal keratinocytes, differentiation of stratified epithelia. In cancer, ΔNp63 is implicated in squamous cancers pathogenesis of different origin including skin, head and neck and lung and in sustaining self-renewal of cancer stem cells. How this transcription factor can control such a diverse set of biological pathways is central to the understanding of the molecular mechanisms through which p63 acquires oncogenic activity, profoundly changing its down-stream transcriptional signature. Here, we highlight how different proteins interacting with p63 allow it to regulate the transcription of several central genes. The interacting proteins include transcription factors/regulators, epigenetic modifiers, and post-transcriptional modifiers. Moreover, as p63 depends on its interactome, we discuss the hypothesis to target the protein interactors to directly affect p63 oncogenic activities and p63-related diseases.

P417: A SINGLE AMINO ACID AT THE PNT DOMAIN OF ERG MEDIATES ITS LEUKEMOGENIC ACTIVITY THROUGH INTERACTION WITH THE NCOR-HDAC3 CO-REPRESSOR COMPLEX.

Background: The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized. Aims: Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity. Methods: A genomic and proteomic analysis has been used to study the role of P199 in the leukemogenic pathway of ERG. Results: We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC). As P199 is located within the PNT domain (protein interaction domain), we attempted to identify key protein interactions associated with leukemia progression induced by ERG. To this end, we used proximity ligation-mass spectrometry (BioID). In HEK293 cells, proximity maps of WT-ERG and a mutated form of ERG at position 199 (P199L-ERG) were compared. A total of 240 putative protein interactors with a significance analysis of interactome were identified. The hits were highly enriched with chromatin modifiers. Most significantly, the mutation severely impaired ERG’s interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with wild-type ERG. ChIP sequencing for histone markers conducted on ER-Hoxb8 cells (transformed murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells. Next, we addressed the role of HDAC3 in ERG’s transcriptional activity. RNA sequencing of human ER-Hoxb8 cells treated with BRD3308 (an HDAC3 inhibitor) for 48 hours was performed. Interestingly, leukemia-associated pathways (Hoxa9-Meis1, CBFA2T3 and repression of myeloid differentiation genes) were ranked at the leading edge of gene sets that were significantly perturbed after HDAC3 inhibition. Moreover, we tested the significance of the ERG/NCoR-HDAC3 interaction in models of human leukemia. We demonstrated that ERG-dependent human AML cells as SKNO1 (AML1-ETO) and TF1 (CBFA2T3-ABDH12) were more sensitive to HDAC3 inhibition (RGFP966) as compared to ERG independent cells. Furthermore, RGFP966 exhibited a significant in-vivo antileukemic effect as measured by the burden of disease in the bone marrow of NSG mice transplanted with SKNO1 cells. By using the CRISPR-dCAS9 system we were able to show that leukemia development is abrogated in mice transplanted with SKNO1 cells expressing a reduced level of HDAC3. Remarkably, this effect was translated into a significant survival advantage. Summary/Conclusion: Taken together, our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression.

TRAF3IP3 Is Cleaved by EV71 3C Protease and Exhibits Antiviral Activity.

Enterovirus 71 (EV71) is one of the major pathogens of hand, foot, and mouth disease, which poses a major risk to public health and infant safety. 3C protease (3Cpro), a non-structural protein of EV71, promotes viral protein maturation by cleaving polyprotein precursors and facilitates viral immune escape by cleaving host proteins. In this study, we screened for human proteins that could interact with EV71 3Cpro using a yeast two-hybrid assay. Immune-associated protein TRAF3 Interacting Protein 3 (TRAF3IP3) was selected for further study. The results of co-immunoprecipitation and immunofluorescence demonstrated the interaction between TRAF3IP3 and EV71 3Cpro. A cleavage band was detected, indicating that both transfected 3Cpro and EV71 infection could cleave TRAF3IP3. 87Q-88G was identified as the only 3Cpro cleavage site in TRAF3IP3. In Jurkat and rhabdomyosarcoma (RD) cells, TRAF3IP3 inhibited EV71 replication, and 3Cpro cleavage partially resisted TRAF3IP3-induced inhibition. Additionally, the nuclear localization signal (NLS) and nuclear export signal (NES) of TRAF3IP3 were identified. The NES contributed to TRAF3IP3 alteration of 3Cpro localization and inhibition of EV71 replication. Together, these results indicate that TRAF3IP3 inhibits EV71 replication and 3Cpro resists such inhibition via proteolytic cleavage, providing a new example of virus-host interaction.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by Inhibitor of Receptor Interacting Protein Kinase 3 Alleviates Necroptosis in Glycation End Products-Induced Cardiomyocytes Injury.

Necroptosisis a regulatory programmed form of necrosis. Receptor interacting protein kinase 3 (RIPK3) is a robust indicator of necroptosis. RIPK3 mediates myocardial necroptosis through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) in cardiac ischemia-reperfusion (I/R) injury and heart failure. However, the exact mechanism of RIPK3 in advanced glycation end products (AGEs)-induced cardiomyocytes necroptosis is not clear. In this study, cardiomyocytes were subjected to AGEs stimulation for 24 h. RIPK3 expression, CaMKII expression, and necroptosis were determined in cardiomyocytes after AGEs stimulation. Then, cardiomyocytes were transfected with RIPK3 siRNA to downregulate RIPK3 followed by AGEs stimulation for 24 h. CaMKIIδ alternative splicing, CaMKII activity, oxidative stress, necroptosis, and cell damage were detected again. Next, cardiomyocytes were pretreated with GSK′872, a specific RIPK3 inhibitor to assess whether it could protect cardiomyocytes against AGEs stimulation. We found that AGEs increased the expression of RIPK3, aggravated the disorder of CaMKII δ alternative splicing, promoted CaMKII activation, enhanced oxidative stress, induced necroptosis, and damaged cardiomyocytes. RIPK3 downregulation or RIPK3 inhibitor GSK′872 corrected CaMKIIδ alternative splicing disorder, inhibited CaMKII activation, reduced oxidative stress, attenuated necroptosis, and improved cell damage in cardiomyocytes.

NOD/RIPK2 signalling pathway contributes to osteoarthritis susceptibility

ObjectivesHow inflammatory signalling contributes to osteoarthritis (OA) susceptibility is undetermined. An allele encoding a hyperactive form of the Receptor Interacting Protein Kinase 2 (RIPK2) proinflammatory signalling intermediate has been associated...

In-silico Analysis of 2-cysteine peroxiredoxin Genes in Arabidopsis thaliana with Possible Role in Carbon Dioxide Fixation through Carbonic Anhydrase regulation

Targeting first neighbours and adding interactive protein to investigate the direct and indirect influence of neighbour genes is an important novel technique used in cancer studies. Weighing neighbour genes on the basis of topological analysis methods for essential proteins identification and multi-omics data is useful in predicting significant interactions in such investigations. A similar approach can also be adopted to help target specific genes in photosynthetic research, especially under stress conditions like low CO2 and the transformation of plants from C3 to C4 photosynthesis. In this study, a computational approach has been used to identify two Genes i.e. 2-cys peroxiredoxin A, (2CPA; AT3G11630/BAS1) and 2-cysteine peroxiredoxin B (2CPB: AT5G06290) that may regulate Carbonic anhydrase during CO2 fixation process during photosynthesis in Arabidopsis thaliana. We have retrieved almost one hundred genes in the Protein-Protein Interaction (PPI) network of Carbonic anhydrase and identified the hub proteins in the network using Cyto-Hubba and AraNet gene prioritization tools to show significant interactions of essential proteins with CA. In the top thirty-four (34) essential proteins, the GeneMania web tool predicted direct interactions among six proteins i.e. Photosystem I P subunit (CURT1B: AT2G46820), Photosystem I subunit l (PSAL: AT4G12800), Ferredoxin-NADP[+]-oxidoreductase 1 (FNR1:AT5G66190), ATPase (F0 complex subunit B/B' (PDE334:AT4G32260), Glucose-6-phosphate/phosphate translocator-like protein (TPT; AT5G46110) and Photosystem I subunit H2 (PSAH2; AT1G52230) that were participating in the same biological process and are found in the chloroplast. Upon addition of interactor protein, BAS1 and 2CPB were also found to have significant interactions with CA on the basis of multi-omics data. GO Biological process enrichment analysis confirmed that six genes are involved in photosynthesis except for BAS1 and 2CPB. These genes may interact with CA and possibly regulate it under high temperature and low CO2 conditions.

Abstract 5320: BRG399, a small molecule modulator of UBE2K demonstrated dose-dependent anti-cancer efficacy in an in vivo model for gastric cancer

Abstract Gastric cancer is difficult to treat and exhibits poor survival with current therapies (five-year survival for all stages is 32% and 5% for metastatic gastric cancer) demonstrating a clear unmet clinical need. UBE2K was identified as a drug target from the BERG Interrogative Biology® Platform, from an Artificial Intelligence derived in vitro pan-cancer Bayesian network. UBE2K (also known as Huntington Interacting Protein 2 and E2-25K) is one of the ~40 E2 enzymes from the Ubiquitin Proteasome System and belongs to the Class II of E2 enzymes that preferentially catalyze the synthesis of Lys48-linked changes on monoubiquitinated substrates. Recently, high expression of UBE2K in tumors of gastric cancer patients has been shown to associate with poor prognosis. BERG has developed BRG399 as a candidate molecule to modulate UBE2K activity for use in oncology. Here, we evaluated the anti-cancer effect of BRG399 using Hs 746T, a human xenograft in vivo model for gastric cancer. In vitro evaluation of BRG399 potency revealed an IC50 of 44.3nM in Hs 746T cells. For determination of BRG399 potency in vivo, nude mice were inoculated subcutaneously with Hs 746T cell line and tumors were allowed to reach 120mm3. Animals were then randomized into four groups to receive oral administration of vehicle, 75, 100, or 150mg/kg of BRG399 twice per day for 15 days. The result revealed that BRG399 significantly decreased the growth of Hs 746T tumors in a dose-dependent manner. Statistical analysis using a mixed effect linear model of the tumor volumes on the last day of study demonstrated a tumor growth inhibition of 60.3, 71.2 and 85.3% at the three doses, respectively, without any significant effect on body weight. These results are well correlated with the dose-dependent increase of BRG399 in both plasma and tumor tissues. Anti-tumor effect of BRG399 appears to be the result of G2/M arrest, as indicated by a dose-dependent elevation of two cell cycle-dependent biomarkers, CNNB1 and pHH3, in tumor tissue. This is consistent with the role of UBE2K in regulating several components of cell cycle for dictating the precise timing of cell division. Mixed effect multivariable regression modeling was employed to identify the relationship between these two biomarkers and response. This analysis indicated that the level of plasma concentration of BRG399, together with the tumor levels of CNNB1 and pHH3, are significantly and negatively associated with tumor volume, allowing an inference that these biomarkers are indicative and/or involved in the tumor response of BRG399. In summary, these results signify the anti-cancer efficacy of BRG399 and support its potential as a drug candidate for treatment of gastric cancer. Citation Format: Shiva Kazerounian, Mollie O'Hara, Arcan Guven, Kashni Grover, Nicole Pellegrino, Kayleigh Gray, Can Bruce, Gregory M. Miller, Maria-Dorothea Nastke, Michael A. Kiebish, Eric M. Grund, Rangaprasad Sarangarajan, Niven R. Narain, Stephane Gesta, Vivek K. Vishnudas. BRG399, a small molecule modulator of UBE2K demonstrated dose-dependent anti-cancer efficacy in an in vivo model for gastric 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 5320.

Circ-RANGAP1/MicroRNA-542-3p/Myosin Regulatory Light Chain Interacting Protein Axis Modulates the Osteosarcoma Cell Progression.

Objective This study is aimed at exploring the influence of circular RNA- (circRNA-) RANGAP1 targeting microRNA- (miR-) 542-3p/myosin regulatory light chain interacting protein (MYLIP) on the biological function of osteosarcoma (OS) cells. Methods Tumor tissues and normal tissues were collected from OS patients and circ-RANGAP1, miR-542-3p, and MYLIP expression was tested by RT-qPCR. The correlation between the clinicopathology/prognosis of patients with OS and circ-RANGAP1 expression was observed. Human OS cell line MG-63 was screened to determine the influences of circ-RANGAP1 and miR-542-3p on OS cell progression. The targeting relation of circ-RANGAP1, miR-542-3p, and MYLIP was probed. Results circ-RANGAP1 expression was elevated in tumor tissues from OS patients, which was correlated to the poor clinicopathology. circ-RANGAP1 expression was augmented in males or patients younger than 20 years old or patients with advanced OS. Higher circ-RANGAP1 expression indicated a poor prognosis in OS patients. After silencing circ-RANGAP1 or elevating miR-542-3p in MG63 cells, cell progression was limited. miR-542-3p downregulation reduced the therapeutic efficacy of silenced circ-RANGAP1. circ-RANGAP1 bound with miR-542-3p to target MYLIP. Conclusion Silenced circ-RANGAP1 boosts MYLIP expression via competitive binding of miR-542-3p to facilitate OS cell progression.

KNIME workflow for retrieving causal drug and protein interactions, building networks, and performing topological enrichment analysis demonstrated by a DILI case study

As an alternative to one drug-one target approaches, systems biology methods can provide a deeper insight into the holistic effects of drugs. Network-based approaches are tools of systems biology, that can represent valuable methods for visualizing and analysing drug-protein and protein–protein interactions. In this study, a KNIME workflow is presented which connects drugs to causal target proteins and target proteins to their causal protein interactors. With the collected data, networks can be constructed for visualizing and interpreting the connections. The last part of the workflow provides a topological enrichment test for identifying relevant pathways and processes connected to the submitted data. The workflow is based on openly available databases and their web services. As a case study, compounds of DILIRank were analysed. DILIRank is the benchmark dataset for Drug-Induced Liver Injury by the FDA, where compounds are categorized by their likeliness of causing DILI. The study includes the drugs that are most likely to cause DILI (“mostDILI”) and the ones that are not likely to cause DILI (“noDILI”). After selecting the compounds of interest, down- and upregulated proteins connected to the mostDILI group were identified; furthermore, a liver-specific subset of those was created. The downregulated sub-list had considerably more entries, therefore, network and causal interactome were constructed and topological pathway enrichment analysis was performed with this list. The workflow identified proteins such as Prostaglandin G7H synthase 1 and UDP-glucuronosyltransferase 1A9 as key participants in the potential toxic events disclosing the possible mode of action. The topological network analysis resulted in pathways such as recycling of bile acids and salts and glucuronidation, indicating their involvement in DILI. The KNIME pipeline was built to support target and network-based approaches to analyse any sets of drug data and identify their target proteins, mode of actions and processes they are involved in. The fragments of the pipeline can be used separately or can be combined as required.

TXNIP inhibition in the treatment of diabetes. Verapamil as a novel therapeutic modality in diabetic patients.

Loss of pancreatic β–cell is a critical factor in the pathogenesis of type 1 diabetes and it also occurs in type 2. TXNIP (thioredoxin – interacting protein), also known as vitamin D3-upregulated protein 1, or thioredoxin-binding-protein-2, regulates this process and modulates cellular redox balance. TXNIP is localized primarily in the nucleus, but under oxidative stress it moves to mitochondria, where it interacts with mitochondrial thioredoxin 2. Overexpression of TXNIP induced by hyperglycaemia is typical for diabetes and insulin resistance and leads to apoptosis of pancreatic β–cell, cardiomyopathy, metabolic disorders and multiple harmful effects. It activates NLRP3 inflamasomme and IL–1β, a cytokine involved in type 2 diabetes and insulin resistance. TXNIP influences peroxisome proliferator-activated receptor alpha transcriptional activity, expression of glucose transporter–1, nitric oxide production in endothelium and insulin production in β–cells. TXNIP overexpression leads to diabetic retinopathy, nephropathy, atherosclerosis, it occurs in cancers and autoimmune diseases, while its deficiency protects β cells. Reduction of TXNIP is an important target in diabetes treatment. In this mechanism insulin, metformin and inhibitors of dipeptydylopeptydase IV are involved. It has been observed that calcium channel blockers (CCB) used in hypertension also inhibit TXNIP expression in cardiomyocytes. L–type channels identification in pancreatic β-cells revealed that CCB inhibit TXNIP expression also in β–cells. For the first time, verapamil was distinguished as an agent that not only inhibits TXNIP expression in pancreatic β-cells, but also enhances β cell survival and function, and possibly prevents diabetes.

200-OR: IER3IP1 Is Critical for Maintaining Glucose Homeostasis through Regulating the Endoplasmic Reticulum Function and Survival of ß-Cells

Recessive mutations in IER3IP1 (Immediate Early Response 3 Interacting Protein 1) cause a triad of microcephaly, epilepsy, and permanent neonatal diabetes syndrome (MEDS) . IER3IP1 encodes an endoplasmic reticulum (ER) membrane protein, which is crucial for brain development, however the role of IER3IP1 in β cell function and glucose homeostasis remains unknown. Herein, we established a new β cell-specific IER3IP1 knockout (IER3IP1-βKO) mouse model and found that IER3IP1 deficiency in β cells causes severe early onset insulin deficient diabetes. Functional studies revealed a markedly dilated ER along with significantly increased proinsulin misfolding and elevated expression of the ER chaperones including PDI, ERO1, BiP, and P58IPK. Transcriptome analysis confirmed by qRT-PCR revealed a markedly decreased expression of genes associated with β cell maturation, cell cycle, and anti-apoptosis, whereas there was a significantly increased expression of anti-proliferation genes. Indeed, multiple independent approaches further demonstrated that IER3IP1-βKO impaired β cell maturation and proliferation, and promoted cell death. These data not only uncover a critical role of IER3IP1 in normal β cell function and survival, but they also provide insight into the mechanism (s) by which loss-of-function mutations of IER3IP1 lead to diabetes. Disclosure J.Yang: None. J.Sun: None. N.Li: None. T.Liu: None. S.Wang: None. X.Zhang: None. P.Arvan: n/a. M.Liu: None. J.Zhen: None. W.Feng: None. Z.Fan: None. Y.Huang: None. H.Shu: None. X.Li: None. J.Qiao: None. Y.Fan: None. Funding National Natural Science Foundation of China (81830025, 81620108004, 81870533, 81900720, 81800733, 82100865, 82070805, and 81870535)