Multiple Protein-protein Interactions Research Articles

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (SH2-S) for specifically binding and isolation of intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed for the identification of pTyr protein complexes by integrating ion exchange chromatography in conjunction with data independent acquisition mass spectrometry. Furthermore, we developed a novel complex-centric algorithm for quantifying protein complexes based on the protein complex elution curve. Utilizing this algorithm, we effectively quantified 216 putative protein complexes. We further screened 21 regulated pTyr protein complexes related to the epidermal growth factor signal. Our study engenders a comprehensive framework for the intricate examination of pTyr protein complexes and presents, for the foremost occasion, a quantitative landscape delineating the composition of pTyr protein complexes in HeLa cells.

Abstract P52: Combination of 5-fluorouracil and Menadione Exerts Synergistic Effect on Colorectal Cancer Cells by Targeting Wnt/β-catenin Signaling Pathway

Abstract Colorectal cancer is the third most common type of cancer and the second most in terms of mortality rate worldwide. 5-fluorouracil (5-FU), the fluoropyrimidine drug, that interferes with DNA replication and repair processes, is the first-line-of-treatment against colorectal cancer. However, cells gain resistance over time and relapses occur upon continuous 5-FU therapy. Interestingly, 5-FU promotes stemness in colorectal cancer via Wnt signaling pathway which is critical for its progression. Studies have pointed out the possibility of using a Wnt signaling inhibitor along with 5-FU as a better strategy to overcome the poor outcomes of current 5-FU based therapies. We have previously shown that menadione (vitamin K3) suppresses Wnt signaling pathway in colorectal cancer cells. In the present study, we attempted to evaluate if the combination of menadione and 5-FU can exert a synergistic effect in the human colorectal cancer cell lines HCT116 and SW620. Analysis of the cell viability assay results using CompuSyn software confirmed that the combination indeed acts synergistically. The combination efficiently downregulated the expression of the Wnt signal transducer protein, β-catenin, and the expression of other proteins in the pathway such as phospho-GSK3β in the cell lines HCT116 and SW620. Docking of the individual drugs and their combination on β-catenin using Autodock Vina showed that 5-FU and menadione in combination bind to key protein-protein interaction (PPI) sites of β-catenin whereby they may inhibit β-catenin-TCF interaction or activate the β-catenin destruction complex. Taken together, our data possibly explains that the synergistic action of 5-FU and menadione in colorectal cancer cells may be due to the simultaneous binding of the two drugs to multiple PPI sites thereby blocking the activity of β-catenin. Citation Format: Vidya P Warrier, Sankaran Venkatachalam, Sakthivel Ramasamy, M Michael Gromiha, Devarajan Karunagaran. Combination of 5-fluorouracil and Menadione Exerts Synergistic Effect on Colorectal Cancer Cells by Targeting Wnt/β-catenin Signaling Pathway [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P52.

Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

High photoreactivation activities of Rad2 and Rad14 in recovering insecticidal Beauveria bassiana from solar UV damage

Anti-ultraviolet (UV) roles of Rad2 and Rad14 depend on nucleotide excision repair (NER) of UV-induced DNA lesions in budding yeast but remain unexplored yet in filamentous fungi. Here, nucleus-specific Rad2 and Rad14 orthologs are shown to recover Beauveria bassiana, a main source of wide-spectrum mycoinsecticides, from solar UV damage through photorepair-depending photoreactivation. As a photorepair index, photoreactivation (germination) rates of lethal UVB dose-irradiated conidia via a 3- or 5-h light plus 9- or 7-h dark incubation at 25 °C were drastically reduced in the Δrad2 and Δrad14 mutants versus a wild-type strain. As an NER index, nighttime-mimicking 12-h dark reactivation rates of low UVB dose-impaired conidia decreased sharply compared to the corresponding photoreactivation rates in the presence or absence of either ortholog, indicating that its extant NER activity was limited to recovering light UVB damage in the field. The high photoreactivation activity of either Rad2 or Rad14 was derived from its tight link to a large protein complex formed by photolyase regulators and other anti-UV proteins through multiple protein-protein interactions revealed by yeast two-hybrid assays. Therefore, Rad2 and Rad14 recover B. bassiana from solar UV damage through photoreactiovation in vivo that depends primarily on photorepair, although they contribute little to the fungal lifecycle-related phenotypes. These findings unveil a novel scenario distinguished from the NER-depending anti-UV roles of Rad2 and Rad14 in the model yeast and broaden a biological basis crucial for rational application of fungal insecticides to improve pest control efficacy via feasible recovery of solar UV damage.

Proteomic investigations of adult polyglucosan body disease: insights into the pathobiology of a neurodegenerative disorder.

Inadequate glycogen branching enzyme 1 (GBE1) activity results in different forms of glycogen storage disease type IV, including adult polyglucosan body disorder (APBD). APBD is clinically characterized by adult-onset development of progressive spasticity, neuropathy, and neurogenic bladder and is histologically characterized by the accumulation of structurally abnormal glycogen (polyglucosan bodies) in multiple cell types. How insufficient GBE1 activity causes the disease phenotype of APBD is poorly understood. We hypothesized that proteomic analysis of tissue from GBE1-deficient individuals would provide insights into GBE1-mediated pathobiology. In this discovery study, we utilized label-free LC-MS/MS to quantify the proteomes of lymphoblasts from 3 persons with APBD and 15 age- and gender-matched controls, with validation of the findings by targeted MS. There were 531 differentially expressed proteins out of 3,427 detected between APBD subjects vs. controls, including pronounced deficiency of GBE1. Bioinformatic analyses indicated multiple canonical pathways and protein-protein interaction networks to be statistically markedly enriched in APBD subjects, including: RNA processing/transport/translation, cell cycle control/replication, mTOR signaling, protein ubiquitination, unfolded protein and endoplasmic reticulum stress responses, glycolysis and cell death/apoptosis. Dysregulation of these processes, therefore, are primary or secondary factors in APBD pathobiology in this model system. Our findings further suggest that proteomic analysis of GBE1 mutant lymphoblasts can be leveraged as part of the screening for pharmaceutical agents for the treatment of APBD.

Identifying Significantly Perturbed Subnetworks in Cancer Using Multiple Protein-Protein Interaction Networks.

The identification of cancer driver genes and key molecular pathways has been the focus of large-scale cancer genome studies. Network-based methods detect significantly perturbed subnetworks as putative cancer pathways by incorporating genomics data with the topological information of PPI networks. However, commonly used PPI networks have distinct topological structures, making the results of the same method vary widely when applied to different networks. Furthermore, emerging context-specific PPI networks often have incomplete topological structures, which pose serious challenges for existing subnetwork detection algorithms. In this paper, we propose a novel method, referred to as MultiFDRnet, to address the above issues. The basic idea is to model a set of PPI networks as a multiplex network to preserve the topological structure of individual networks, while introducing dependencies among them, and, then, to detect significantly perturbed subnetworks on the modeled multiplex network using all the structural information simultaneously. To illustrate the effectiveness of the proposed approach, an extensive benchmark analysis was conducted on both simulated and real cancer data. The experimental results showed that the proposed method is able to detect significantly perturbed subnetworks jointly supported by multiple PPI networks and to identify novel modular structures in context-specific PPI networks.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

The Impact of Human DNA Glycosylases on the Activity of DNA Polymerase β toward Various Base Excision Repair Intermediates.

Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligase. Coordination of BER is implemented by multiple protein-protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polβ's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polβ efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5'-dRP-mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polβ's activity toward the model DNA intermediates.

INKILN is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.

Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1β-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.

A partially shared joint clustering framework for detecting protein complexes from multiple state-specific signed interaction networks

Detecting protein complexes is critical for studying cellular organizations and functions. The accumulation of protein–protein interaction (PPI) data enables the identification of protein complexes computationally. Although a great number of computational methods have been proposed to identify protein complexes from PPI networks, most of them ignore the signs of PPIs that reflect the ways proteins interact (activation or inhibition). As not all PPIs imply co-complex relationships, taking into account the signs of PPIs can benefit the identification of protein complexes. Moreover, PPI networks are not static, but vary with the change of cell states or environments. However, existing methods are primarily designed for single-network clustering, and rarely consider joint clustering of multiple PPI networks. In this study, we propose a novel partially shared signed network clustering (PS-SNC) model for identifying protein complexes from multiple state-specific signed PPI networks jointly. PS-SNC can not only consider the signs of PPIs, but also identify the common and unique protein complexes in different states. Experimental results on synthetic and real datasets show that our PS-SNC model can achieve better performance than other state-of-the-art protein complex detection methods. Extensive analysis on real datasets demonstrate the effectiveness of PS-SNC in revealing novel insights about the underlying patterns of different cell lines.

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.

Abstract P2-11-35: A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer

Abstract A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer Han Gao1, Fiona Ruge1, Lin Ye1, Jane Lane1, Eleri Davies2, Wen G. Jiang1, Tracey A. Martin1 1Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK 2Wales Breast Centre, University Llandough Hospital, Cardiff CF64 2XX, UK Introduction. MUPP1 (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) or MPDZ is a scaffold protein with multiple PDZ domains, critical for multiple protein docking and protein-protein interactions in cells. MUPP1 is particularly interesting as it offers docking sites for a number of tight junction (TJ) proteins and proteins known to regulate cell junction and motility, including angiomotin (AMOT) and angiomotin-like proteins, the latter being important in regulating cell migration of endothelial cells and an important member in TJ protein complexes. The MUPP1 protein complex at cell junctions also includes CAR/CXADR (coxsackie adenovirus receptor) and members of the ERM (Moesin-Ezrin-Radixin) protein family. It was proposed that the junctional-related MUPP1 docking protein complex may collectively have a role in the development and progression of cancer including breast cancer. The present study examined the profile and integrated expression of these MUPP1 docking molecules in relationship with the outcome, clinical and pathological factors of breast cancer. Methods. Using the datasets of an existing breast cancer cohort at Cardiff, we visited the expression profile of the MUPP1/MPDZ docking molecules including MUPP1, four members of the ERM family, including ezrin, moesin, radixin and EHM2 (also known as NF2) and CAR/CXADR (coxsackie adenovirus receptor). The profile and integrated expression of the profile were analysed against clinical, pathological and outcome factors and also the hormone receptor status of breast cancers. Results. MUPP1 is a weakly linked to the disease progression, so as angiomotin, angiomotin-like1 and angiomotin-like2. Alone, only EHM2 showed significance in linking with the survival of the patient’s OS (p=0.38), neither of the remaining MUPP1 complex molecules, including MUPP1, AMOT, AMOTL1, AMOTL2, ezrin, moesin and radixin was able to offer a significant predictive value for the survival of the patients. However, it was found that MUPP1 has a significant correlation with AMOTs, EHM2 and ezrin. When these significantly correlated molecules were integrated in the analysis, the integrated expression pattern showed a highly significant value in evaluating the overall survival (p=0.003) and disease-free survival (p=0.007). Both of these predictive powers are independent in a multivariate analyses including the clinical and hormone receptors for OS (p=0.022, Hazard Ratio (HR)=1.352 95%CI (1.044-1.752)) and RFS (p=0.023, HR=1.241 (95% CI. 1.031-1.498)). It was interesting to note that the link between the expression pattern and OS was more prominent in ER negative (p=0.018) than in ER positive tumours (p=0.177). The same was observed with disease free survival for ER negative (p=0.011) compared with ER positive (p=0.462) tumours. Non-triple negative breast cancer had shown a similar sensitivity compared with triple negative breast cancer. Discussion. MUPP1, a PDZ domain containing docking protein closed linked with TJ structure, forms an interesting expression profile with its docking molecules including angiomotins and EHM2 that allows evaluation of disease progress and clinical outcome of the patients with breast cancer. Citation Format: Han Gao, Fiona Ruge, Lin Ye, Jane Lane, Eleri Davies, Wen G. Jiang, Tracey A. Martin. A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-35.

A Transformer-Based Ensemble Framework for the Prediction of Protein-Protein Interaction Sites.

The identification of protein-protein interaction (PPI) sites is essential in the research of protein function and the discovery of new drugs. So far, a variety of computational tools based on machine learning have been developed to accelerate the identification of PPI sites. However, existing methods suffer from the low predictive accuracy or the limited scope of application. Specifically, some methods learned only global or local sequential features, leading to low predictive accuracy, while others achieved improved performance by extracting residue interactions from structures but were limited in their application scope for the serious dependence on precise structure information. There is an urgent need to develop a method that integrates comprehensive information to realize proteome-wide accurate profiling of PPI sites. Herein, a novel ensemble framework for PPI sites prediction, EnsemPPIS, was therefore proposed based on transformer and gated convolutional networks. EnsemPPIS can effectively capture not only global and local patterns but also residue interactions. Specifically, EnsemPPIS was unique in (a) extracting residue interactions from protein sequences with transformer and (b) further integrating global and local sequential features with the ensemble learning strategy. Compared with various existing methods, EnsemPPIS exhibited either superior performance or broader applicability on multiple PPI sites prediction tasks. Moreover, pattern analysis based on the interpretability of EnsemPPIS demonstrated that EnsemPPIS was fully capable of learning residue interactions within the local structure of PPI sites using only sequence information. The web server of EnsemPPIS is freely available at http://idrblab.org/ensemppis.

Efficient Photoreactivation of Solar UV-Injured Metarhizium robertsii by Rad1 and Rad10 Linked to DNA Photorepair-Required Proteins.

Nucleotide excision repair (NER) of ultraviolet (UV)-induced DNA lesions known as cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts depends on the activities of multiple anti-UV radiation (RAD) proteins in budding yeast. However, NER remains poorly known in filamentous fungi, whose DNA lesions are photorepaired by one or two photolyases, namely CPD-specific Phr1 and/or 6-4PP-specific Phr2. Previously, the white collar proteins WC1 and WC2 were proven to regulate expressions of phr2 and phr1 and photorepair 6-4PP and CDP DNA lesions, respectively, in Metarhizium robertsii, a filamentous entomopathogenic-phytoendophytic fungus. We report here high activities of orthologous Rad1 and Rad10 in 5-h photoreactivation of UVB-injured or UVB-inactivated conidia but a severely compromised capability of their reactivating those conidia via 24-h dark incubation in M. robertsii. The null mutants of rad1 and rad10 were much more compromised in conidial UVB resistance and photoreactivation capability than the previous null mutants of phr1, phr2, wc1 and wc2. Multiple protein-protein (Rad1-Rad10, Rad1-WC2, Rad10-Phr1, WC1-Phr1/2 and WC2-Phr1/2) interactions detected suggest direct/indirect links of Rad1 and Rad10 to Phr1/2 and WC1/2 and an importance of the links for their photoreactivation activities. Conclusively, Rad1 and Rad10 photoreactivate UVB-impaired M. robertsii through their interactions with the DNA photorepair-required proteins.

Identification of Human Protein Subcellular Location with Multiple Networks

Background:Protein function is closely related to its location within the cell. Determination of protein subcellular location is helpful in uncovering its functions. However, traditional biological experiments to determine the subcellular location are of high cost and low efficiency, which cannot meet today’s needs. In recent years, many computational models have been set up to identify the subcellular location of proteins. Most models use features derived from protein sequences. Recently, features extracted from the protein-protein interaction (PPI) network have become popular in studying various protein-related problems.Objective:A novel model with features derived from multiple PPI networks was proposed to predict protein subcellular location.Methods:Protein features were obtained by a newly designed network embedding algorithm, Mnode2vec, which is a generalized version of the classic Node2vec algorithm. Two classic classification algorithms: support vector machine and random forest, were employed to build the model.Results:Such model provided good performance and was superior to the model with features extracted by Node2vec. Also, this model outperformed some classic models. Furthermore, Mnode2vec was found to produce powerful features when the path length was small.Conclusion:The proposed model can be a powerful tool to determine protein subcellular location, and Mnode2vec can efficiently extract informative features from multiple networks.

Integrating multi-omics data reveals function and therapeutic potential of deubiquitinating enzymes.

Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).

Evaluation of the Anti‐Migratory Activity of Small‐molecule Gankyrin Inhibitors in A549 and MDA‐MB‐231 Cells

IntroductionGankyrin is a 25 kDa oncoprotein that plays a crucial role in cellular growth, proliferation, and migration. It is highly conserved and has 7 ankyrin repeats which bind to the S6 ATPase subunit of the 26S proteasome. It has been shown that gankyrin exerts its oncogenic activities by interacting with numerous tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein (Rb), which in turn promotes their proteasomal degradation. While Rb plays an important role in cell adhesion, p53 inhibits Rac/CDC42 which is responsible for lamellipodia and filopodia formation. Gankyrin is also reported to increase the levels of interleukin‐6 (IL‐6) which activates the JAK/STAT3 pathway. The phosphorylation of STAT3 to p‐STAT3 and the subsequent nuclear translocation of p‐STAT3 results in transcription of migratory factors. The inhibition of these gankyrin‐based protein‐protein interactions (PPI) by a small molecule inhibitor, should result in an anti‐migratory effect. Cjoc42 is the first known binder of gankyrin, and since its discovery we have developed two derivatives with an enhanced ability to bind gankyrin, – AFM‐2 and JA‐38 In order to determine if next‐generation gankyrin inhibitors could affect cancer cell migration, we evaluated both compounds in gankyrin overexpressing cell lines A549 and MDA‐MB‐231. Their ability to disrupt multiple gankyrin‐based PPIs is hypothesized to be due to their ability to induce a conformational change in gankyrin upon binding. Various assays such as the scratch assay, Boyden chamber assay, western blotting, co‐immunoprecipitation (co‐IP) assay and fluorescence‐based assays were used to evaluate our hypotheses.MethodsWe evaluated the anti‐migratory potential of the molecules by scratch and Boyden chamber assay. The molecules’ ability to disrupt gankyrin’s interactions with p53, Rb and the S6 proteasome was evaluated by co‐IP assay. Using western blotting, we evaluated the changes in the levels of various TSPs and migratory proteins upon treatment with the molecules. To test our hypothesis of induction of conformational change in gankyrin upon treatment with the molecules, we utilized florescence‐based experiments such as intrinsic fluorescence evaluation and thermal shift.DiscussionThe cell‐based scratch and Boyden chamber assay showed significant decreases in cellular migration in both cell lines upon treatment with the molecules. Co‐IP experiments demonstrated that both molecules disrupt various gankyrin‐based PPIs. Disruption of gankyrin’s interactions with the TSPs would prevent their subsequent proteasomal degradation and subsequently affect the levels of downstream proteins such as IL‐6 and Rac/CDC42. Significant reduction in the migratory proteins and a significant increase in the TSPs was confirmed by western blotting. Lastly, using fluorescence‐based experiments, we believe that the binding of the two molecules caused a conformation change in gankyrin, which could be why gankyrin’s multiple PPIs are disrupted.ConclusionGankyrin plays a key role controlling cellular migratory pathways through its various PPIs. By inducing a conformational change in gankyrin, our molecules disrupt these PPI and exhibit significant anti‐migratory activity which could prevent cancer metastasis.

WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7.

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.

Crystal structure of the PDZ4 domain of MAGI2 in complex with PBM of ARMS reveals a canonical PDZ recognition mode

Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2) is a neuronal scaffold protein that plays critical roles at synaptic junctions by assembling neurotransmitter receptors and cell adhesion proteins through its multiple protein-protein interaction domains, including six PDZ domains, two phosphoserine-phosphothreonine binding WW domains, and a guanylate kinase GK domain. Previous studies showed that MAGI2 participates in formation of tetrameric complexes with PDZ-GEF1, TrkA receptor, and ankyrin repeat-rich membrane spanning (ARMS) protein at late endosomes and is crucial for neurite outgrowth. However, the molecular mechanism governing the assembly of these complexes remains unknown. Here, we characterize the direct interaction between MAGI2 and ARMS through multiple biochemical assays. Moreover, our solved crystal structure of the truncated PDZ4/PBM (PDZ binding motifs) complex of MAGI2 and ARMS proteins (MAGI2-PDZ4/ARMS-PBM) reveals that the binding interface lies between the αB/βB groove from the PDZ4 of MAGI2 and the C-terminal PBM from ARMS. The structure reveals high similarity to others in this protein family where canonical PDZ/PBM interactions are observed. However, the conserved “GLGF” motif in the PSD-95-PDZ3 changes to “GFGF” in the MAGI2-PDZ4/ARMS-PBM complex. We further validated our crystal structure through serial mutagenesis assays. Taken together, our study provides the biochemical details and binding mechanisms that underpin the stabilization of the MAGI2-PDZ4/ARMS-PBM complex, thereby offering a biochemical and structural basis for further understanding of the functional roles of MAGI2, ARMS, PDZ-GEF1, and TrkA in forming the tetrameric receptor complex in neuronal signaling.

The BEAF-32 Protein Directly Interacts with Z4/putzig and Chriz/Chromator Proteins in Drosophila melanogaster.

In Drosophila, the BEAF-32, Z4/putzig, and Chriz/Chromator proteins colocalize in the interbands of polytene chromosomes. It was assumed that these proteins can form a complex that affects the structure of chromatin. However, the mechanism of the formation of such a complex has not been studied. We have proved for the first time that the BEAF-32, Z4/putzig, and Chriz/Chromator proteins interact directly with each other and localized the protein domains that provide multiple protein-protein interactions. Based on the data obtained, we developed a model of the mechanism of the formation the BEAF/Z4/Chriz complex and its recruitment to chromatin.