Essential Protein-protein Interactions Research Articles

Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.

Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation to resolve condensates from the dilute phase. Condensate-specific lifetimes were used to track protein-protein interactions by measuring FLIM-Förster resonance energy transfer (FRET). We used condensate FLIM-FRET to evaluate whether mRNA decapping complex subunits can form decapping-competent interactions within P-bodies. Condensate FLIM-FRET revealed the presence of core subunit interactions within P-bodies under basal conditions and the disruption of interactions between the decapping enzyme (Dcp2) and a critical cofactor (Dcp1A) during oxidative stress. Our results show a context-dependent plasticity of the P-body interaction network, which can be rewired within minutes in response to stimuli. Together, our FLIM-based approaches provide investigators with an automated and rigorous method to uncover and track essential protein-protein interaction dynamics within RNP condensates in live cells.

Inhibiting KSHV replication by targeting the essential activities of KSHV processivity protein, PF-8.

Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are no cures for KSHV infection. KSHV establishes biphasic lifelong infections. During the lytic phase, new genomes are replicated by seven viral DNA replication proteins. The processivity factor's (PF-8) functions to tether DNA polymerase to DNA, so new viral genomes are efficiently synthesized. PF-8 self-associates, interacts with KSHV DNA replication proteins and the viral DNA. Inhibition of viral DNA replication would diminish the infection within a host and reduce transmission to new individuals. In this review we summarize PF-8 molecular and structural studies, detail the essential protein-protein and nucleic acid interactions needed for efficient lytic DNA replication, identify future areas for investigation and propose PF-8 as a promising antiviral target. Additionally, we discuss similarities that the processivity factor from Epstein-Barr virus shares with PF-8, which could promote a pan-herpesvirus antiviral therapeutic targeting strategy.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Abstract 419: Elevated Akt/mTOR signaling drives therapy resistance in ALM

Abstract Metastatic acral lentiginous melanoma (ALM) is the most lethal subtype of cutaneous melanoma and effective therapies for patients have not been established. Studies revealed a link between the dysregulation of CDK4/6 and ALM mortality, however CDK4/6 inhibitors (CDK4i/6i) have proved ineffective in patients with advanced ALM. The tumor suppressor Rb was recently shown to have a novel suppressive role towards mTORC2. Here, we elucidate the role of a non-canonical tumor suppressor function of Rb in negative regulation of mTOR in CDK4i/6i-resistance. We hypothesize that the loss of Rb induced by CDK4i/6i triggers phosphorylation of Akt and downstream mTORC1 signaling pathways. We characterized baseline activity of the PI3K/AKT/mTOR pathway in a panel of ALM cell lines, finding heterogeneity across signaling. We immunoblotted for key proteins involved in the signal reprogramming hypothesized: pRb, pAkt, pS6 to define activation status and found the loss of Rb phosphorylation preceded the enrichment of pAKT, supporting the non-canonical role of Rb tumor suppression and the Rb-mTORC2 signaling axis as intrinsic CDK4i/6i mechanism. In parallel, we assessed the essential protein-protein interactions required for mechanistic mTORC1 activation—mTOR and Rheb GTPase. The enrichment of these interactions shortly after introducing CDK4i/6i supported our hypothesized mechanism. Translational utility of CDK4i/6i and mTORi dual inhibition was determined via long-term Colony Formation Assays (CFAs) using Palbociclib, rapamycin and sapanisertib (TAK228), a catalytic mTOR inhibitor. We generated Palbociclib (acquired)-resistant ALM cell lines and investigated the pharmacological inhibition of PI3K signaling pathways through immunoblot and colony-formation assays. Concurrent mTORi and CDK4i/6i inhibition showed effective reduction in pRb, pS6, Cyclin D and increases in Cleaved PARP protein, suggesting apoptotic cell death was elevated with combined CDK4i/6i+mTORi treatment. Similarly, CFAs revealed dual inhibition elicited superior antiproliferative effect over the monotherapy treatments. CFAs with acquired-resistance cell lines also showed that combination inhibition decreased colony formation, showing the potential utility of concurrent CDK4i/6i and mTORi as an approach against acquired resistance. Our results show the efficacy of combined inhibition as an anti-tumor strategy. Citation Format: Jeremy A. Bravo Narula, Vito W. Rebecca. Elevated Akt/mTOR signaling drives therapy resistance in ALM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 419.

Functional analysis of PsbS transmembrane domains through base editing in Physcomitrium patens.

Plants exposed to light fluctuations are protected from photodamage by non-photochemical quenching (NPQ), a reversible mechanism that enables dissipation of excess absorbed energy as heat, which is essential for plant fitness and crop productivity. In plants NPQ requires the presence of the membrane protein PsbS, which upon activation interacts with antenna proteins, inducing their dissipative conformation. Here, we exploited base editing (BE) in the moss Physcomitrium patens to introduce specific amino acid changes invivo and assess their impact on PsbS activity, targeting transmembrane regions to investigate their role in essential protein-protein interactions. This approach enabled the recognition of residues essential for protein stability and the identification of a hydrophobic cluster of amino acids impacting PsbS activity. This work provides new information on the molecular mechanism of PsbS while also demonstrating the potential of BE approaches for in planta gene function analysis.

Midgut membrane protein BmSUH facilitates Bombyx mori nucleopolyhedrovirus oral infection.

Baculoviruses are virulent pathogens that infect a wide range of insects. They initiate infections via specific interactions between the structural proteins on the envelopes of occlusion-derived virions (ODVs) and the midgut cell surface receptors in hosts. However, host factors that are hijacked by baculoviruses for efficient infection remain largely unknown. In this study, we identified a membrane-associated protein sucrose hydrolase (BmSUH) as an ODV binding factor during Bombyx mori nucleopolyhedrovirus (BmNPV) primary infection. BmSUH was specifically expressed in the midgut microvilli where the ODV-midgut fusion happened. Knockout of BmSUH by CRISPR/Cas9 resulted in a significantly higher survival rate after BmNPV orally infection. Liquid chromatography-tandem mass spectrometry analysis and co-immunoprecipitation analysis demonstrated that PIF protein complex required for ODV binding could interact with BmSUH. Furthermore, fluorescence dequenching assay showed that the amount of ODV binding and fusion to the midgut decreased in BmSUH mutants compared to wild-type silkworm, suggesting the role of BmSUH as an ODV binding factor that mediates the ODV entry process. Based on a multilevel survey, the data showed that BmSUH acted as a host factor that facilitates BmNPV oral infection. More generally, this study indicated that disrupting essential protein-protein interactions required for baculovirus efficient entry may be broadly applicable to against viral infection.

Signaling Pathways and Protein-Protein Interaction of Vimentin in Invasive and Migration Cells: A Review.

The vimentin (encoded by VIM) is one of the 70 human intermediate filaments (IFs), building highly dynamic and cell-type-specific web networks in the cytoplasm. Vim-/- mice exhibit process defects associated with cell differentiation, which can have implications for understanding cancer and disease. This review showed recent reports from studies that unveiled vimentin intermediate filaments (VIFs) as an essential component of the cytoskeleton, followed by a description of vimentin's physiological functions and process reports in VIF signaling pathway and gene network studies. The main focus of the discussion is on vital signaling pathways associated with how VIF coordinates invasion cells and migration. The current research will open up multiple processes to research the function of VIF and other IF proteins in cellular and molecular biology, and they will lead to essential insights into different VIF levels for the invasive metastatic cancer cells. Enrich GO databases used Gene Ontology and Pathway Enrichment Analysis. Estimation with STRING online was to predict the functional and molecular interactions of proteins-protein with Cytoscape analysis to search and select the master genes. Using Cytoscape and STRING analysis, we presented eight genes, RhoA, Smad3, Akt1, Cdk2, Rock1, Rock2, Mapk1, and Mapk8, as the essential protein-protein interaction with vimentin involved in the invasion.

Purification and functional analysis of the ferrous iron transport protein B (FeoB) incorporated into SMA‐copolymer nanodiscs

The acquisition and transport of ferrous iron (Fe2+) is essential for the survival and the virulence of many infectious prokaryotes. While bacteria possess several methods to acquire Fe2+, the ferrous iron transport (Feo) system is the most important Fe2+ transport complex with strong ties to bacterial virulence. Despite this significance, an understanding of the structure‐function relationship of the transporter FeoB is limited due to its intrinsically insoluble nature that renders it generally intractable outside of a lipid bilayer. Traditionally, detergents have been used to solubilize membrane proteins such as FeoB; however, detergent micelles can disrupt protein folding, lead to protein instability, and even inhibit important and essential protein‐protein interactions. In lieu of traditional amphipathic detergents, styrene‐maleic acid (SMA) copolymers have been demonstrated to solubilize membrane proteins into a nanodisc while encapsulating native lipids, thereby preventing protein misfolding and preserving protein‐protein interaction sites. In this work, we describe the first successful solubilization and purification of Escherichia coli FeoB (EcFeoB) in SMA nanodiscs. This approach has afforded our ability to obtain highly pure, monomeric EcFeoB‐SMA nanodiscs. In addition, we compare the NTPase activity of detergent‐solubilized EcFeoB to that of SMA‐solubilized EcFeoB nanodiscs to probe nucleotide‐hydrolysis rates in the presence of an artificial micelle compared to a lipid bilayer. This approach represents a promising first step in understanding the structure and the mechanism of the FeoB transporter in its native environment.

The Human STAT2 Coiled-Coil Domain Contains a Degron for Zika Virus Interferon Evasion.

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers, including the type I and type III interferon (IFN) antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN-stimulated gene transcription factor ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil domain contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response and identify new targets for antiviral therapeutic approaches. IMPORTANCE Zika virus infection can cause mild fever, rash, and muscle pain and in rare cases can lead to brain or nervous system diseases, including Guillain-Barré syndrome. Infections in pregnant women can increase the risk of miscarriage or serious birth defects, including brain anomalies and microcephaly. There are no drugs or vaccines for Zika disease. Zika virus is known to break down the host antiviral immune response, and this research project reveals how the virus suppresses interferon signaling, and may reveal therapeutic vulnerabilities.

Abstract 1817: C/EBPβ antagonist peptide, ST101, attenuates oncogenic gene transactivation in cancer cells to drive antitumor activity

Abstract Transcription factor dysregulation is common in cancer, resulting in aberrant gene expression that drives oncogenesis. Antagonism of oncogenic transcription factor activity, by disrupting essential protein-protein interactions needed for activation of downstream effector molecules or association with DNA, represents a powerful approach to target this previously ‘undruggable' class of proteins. CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is a transcription factor overexpressed in many cancers that regulates expression of factors that promote tumor survival, proliferation and inhibit differentiation. Here, we decribe the anti-tumor activity of ST101, a cell-penetrating peptide antagonist of C/EBPβ. To demonstrate ST101 disruption of the interaction of C/EBPβ with co-factor activating transcription factor 5 (ATF5), a competition ELISA assay was performed. Recombinant ATF5 associated with plate-bound C/EBPβ with a binding affinity of ∼1 nM. Addition of ST101 inhibited this interaction in dose-dependent manner, resulting in an IC50 of 25 nM. To demonstrate ST101 disruption of C/EBPβ phosphorylation and gene transactivation in cancer cells, western blot analysis and quantitative polymerase chain reaction (qPCR) were performed on U251 glioblastoma, MCF7 breast adenocarcinoma and A549 lung adenocarcinoma cells. Administration of ST101 resulted in a dose-dependent decrease in C/EBPβ activation, as evidenced by a decrease in Thr189 phosphorylation. Anatgonism of C/EBPβ activity resulted in a dose-dependent decrease in mRNA expression of genes involved in survival (BCL2 and the baculoviral inhibitor of apoptosis factors BIRC3, BIRC5), inhibition of differentiation (Inhibitor of DNA binding proteins ID1, ID2 and ID3) and proliferation (cyclins CCNB1 and CCNA2 and cyclin-dependent kinases CDK1 and CDK2). Finally, in a mouse xenograft model, 25mg/kg ST101 administered three times per week for three weeks resulted in significant and sustained tumor growth inhibition in U251 subcutaneous tumors, both when ST101 administration was initiated early (day 2, 200 mm3 tumors, p<0.05) or late (day 16, >500mm3 tumors, p<0.05). These data demonstrate the therapeutic potential of systemic administration of ST101 and support clinical development of ST101 as a potent peptide therapeutic for a variety of solid tumor malignancies. Citation Format: Jim A. Rotolo, Rick Ramirez, Mark Koester, Erin Gallagher, Siok Leong, Lila Ghamsari, Gene Merutka, Barry J. Kappel. C/EBPβ antagonist peptide, ST101, attenuates oncogenic gene transactivation in cancer cells to drive antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1817.

Cell-penetrating peptide inhibits retromer-mediated human papillomavirus trafficking during virus entry

Virus replication requires critical interactions between viral proteins and cellular proteins that mediate many aspects of infection, including the transport of viral genomes to the site of replication. In human papillomavirus (HPV) infection, the cellular protein complex known as retromer binds to the L2 capsid protein and sorts incoming virions into the retrograde transport pathway for trafficking to the nucleus. Here, we show that short synthetic peptides containing the HPV16 L2 retromer-binding site and a cell-penetrating sequence enter cells, sequester retromer from the incoming HPV pseudovirus, and inhibit HPV exit from the endosome, resulting in loss of viral components from cells and in a profound, dose-dependent block to infection. The peptide also inhibits cervicovaginal HPV16 pseudovirus infection in a mouse model. These results confirm the retromer-mediated model of retrograde HPV entry and validate intracellular virus trafficking as an antiviral target. More generally, inhibiting virus replication with agents that can enter cells and disrupt essential protein-protein interactions may be applicable in broad outline to many viruses.

A CURE Biochemistry Laboratory Module to Study Protein-Protein Interactions by NMR Spectroscopy.

Design of undergraduate laboratory courses that provide meaningful research-based experiences enhance undergraduate curricula and prepare future graduate students for research careers. In this article, a Course-based Undergraduate Research Experience (CURE) laboratory module was designed for upper-division undergraduate biochemistry and chemistry students. The laboratory module enabled students to build upon recently published data in the literature to decipher atomistic insight for an essential protein-protein interaction in human biology through the use of biomolecular NMR spectroscopy. Students compared their results with published data with the goal of identifying specific regions of the protein-protein interaction responsible for triggering an allosteric conformational change. The laboratory module introduced students to basic and advance laboratory techniques, including protein purification, NMR spectroscopy, and analysis of protein structure using molecular visualization software.

The Wnt-Driven Mll1 Epigenome Regulates Salivary Gland and Head and Neck Cancer

We identified a regulatory system that acts downstream of Wnt/β-catenin signaling in salivary gland and head and neck carcinomas. We show in a mouse tumor model of K14-Cre-induced Wnt/β-catenin gain-of-function and Bmpr1a loss-of-function mutations that tumor-propagating cells exhibit increased Mll1 activity and genome-wide increased H3K4 tri-methylation at promoters. Null mutations of Mll1 in tumor mice and in xenotransplanted human head and neck tumors resulted in loss of self-renewal of tumor-propagating cells and in block of tumor formation but did not alter normal tissue homeostasis. CRISPR/Cas9 mutagenesis and pharmacological interference of Mll1 at sequences that inhibit essential protein-protein interactions or the SET enzyme active site also blocked the self-renewal of mouse and human tumor-propagating cells. Our work provides strong genetic evidence for a crucial role of Mll1 in solid tumors. Moreover, inhibitors targeting specific Mll1 interactions might offer additional directions for therapies to treat these aggressive tumors.

Rational Design of a 310 -Helical PIP-Box Mimetic Targeting PCNA, the Human Sliding Clamp.

The human sliding clamp (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 310 -helical structure required for PCNA binding. NMR studies confirmed that while the unmodified p21 peptide had little defined structure in solution, mimetic ACR2 pre-organized into 310 -helical structure prior to interaction with PCNA. ACR2 displayed higher affinity binding than most known PIP-box peptides, and retains the native PCNA binding mode, as observed in the co-crystal structure of ACR2 bound to PCNA. This study offers a promising new strategy for PCNA inhibitor design for use as anti-cancer therapeutics.

The Transactions of NS3 and NS5 in Flaviviral RNA Replication.

Dengue virus (DENV) replication occurs in virus-induced vesicles that contain the replication complex (RC) where viral RNA, viral proteins and host proteins participate in RNA-RNA, RNA-protein and protein-protein interactions to ensure viral genome synthesis. However, the details of the multitude of interactions involved in the biogenesis of the infectious virion are not fully understood. In this review, we will focus on the interaction between non-structural (NS) proteins NS3 and NS5, as well as their interactions with viral RNA and briefly also the interaction of NS5 with the host nuclear transport receptor protein importin-α. The multifunctional NS3 protease/helicase and NS5 methyltransferase (MTase)/RNA-dependent RNA polymerase (RdRp) contain all the enzymatic activities required to synthesize the viral RNA genome. The success stories of drug discovery and development with Hepatitis C virus (HCV), a member of the Flaviviridae family, has led to the view that DENV NS3 and NS5 may be attractive antiviral drug targets. However, more than 10years of intensive research effort by Novatis has revealed that they are not "low hanging fruits" and therefore, the search for potent directly acting antivirals (DAAs) remains a pipeline goal for several medium to large drug discovery enterprises. The effort to discover DAAs for DENV has been boosted by the epidemic outbreak of the closely related flavivirus member- Zika virus (ZIKV). Because the viral RNA replication occurs within a molecular machine that is composed several viral and host proteins, much interest has turned to characterising functionally essential protein-protein interactions in order to identify potential allosteric inhibitor binding sites within the RC.

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging.

The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.

Abstract PR15: Predicting the essentialities of protein-protein interactions in cancer

Abstract Many protein-protein interactions (PPIs) are essential to cellular proliferation and survival, and thus represent potential targets for novel compounds with high specificity and therapeutic potential in the treatment of cancer. Despite their therapeutic significance, there are currently no high-throughput experimental platforms to interrogate the essentiality of individual protein-protein interactions. The ability to computationally predict or infer PPI essentiality would help prioritize the development of therapeutic targets and advance understanding of cancer biology, and remains a significant barrier. We have developed a computational method to predict PPI essentiality by combining shRNA studies with network models of protein interaction pathways in an analytic framework. High-throughput single-gene shRNA silencing is a well-established experimental approach to study protein essentiality in genome-wide screens. The silencing of a single gene in an shRNA screen effectively disrupts multiple PPIs, masking their individual contributions to the observed protein essentiality. Our method uses a network model to infer cliques of PPIs that are disrupted as each gene is silenced, then uses these sets to formulate an additive model of how the unknown PPI essentialities combine to form the observed protein essentialities. This process is then deconvoluted to recover the unknown essentialities of each PPI using a regularized solver. We performed this analysis in 108 cell lines characterized for protein essentiality in Project Achilles [1, 2]. A superpathway of 2186 proteins and 11488 protein interactions was constructed from prior knowledge databases [3] and protein interaction screenings. We demonstrate the validity of our approach via prediction of essential PPIs such as TP53-MDM4 in cell lines sensitive to nutlin and CDK4-RB1 interactions in cell lines sensitive to CDK4 inhibitors. [1] Cowley, Weir & Vazquez, et al. Nature Scientific Data 1, Article number: 140035. September 30, 2014 [2] Cheung HW, Cowley GS, Weir BA, et al. Proc Natl Acad Sci. 2011 Jul 26; 108(30):12372-7. [3] Schaefer CF, Anthony K, Krupa S, et al. Nucleic Acids Research. Jan 2009. 37:674-9 Citation Format: Lee AD Cooper, Josue D. Moran, Zenggang Li, Yuhong Du, Sahar Harati, Andrey A. Ivanov, Phillip Webber, Jonathan J. Havel, Margaret A. Johns, Haian Fu, Carlos S. Moreno. Predicting the essentialities of protein-protein interactions in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR15.

Mad2 Inhibitor-1 (M2I-1): A Small Molecule Protein-Protein Interaction Inhibitor Targeting the Mitotic Spindle Assembly Checkpoint.

The genetic integrity of each organism depends on the faithful segregation of its genome during mitosis. To meet this challenge, a cellular surveillance mechanism, termed the spindle assembly checkpoint (SAC), evolved that monitors the correct attachment of chromosomes and blocks progression through mitosis if corrections are needed. While the central role of the SAC for genome integrity is well established, its functional dissection has been hampered by the limited availability of appropriate small molecule inhibitors. Using a fluorescence polarization-based screen, we identify Mad2 inhibitor-1 (M2I-1), the first small molecule inhibitor targeting the binding of Mad2 to Cdc20, an essential protein-protein interaction (PPI) within the SAC. Based on computational and biochemical analyses, we propose that M2I-1 disturbs conformational dynamics of Mad2 critical for complex formation with Cdc20. Cellular studies revealed that M2I-1 weakens the SAC response, indicating that the compound might be active in cells. Thus, our study identifies the SAC specific complex formation between Mad2 and Cdc20 as a protein-protein interaction that can be targeted by small molecules.

Influence of macrocyclization on allosteric, juxtamembrane-derived, stapled peptide inhibitors of the epidermal growth factor receptor (EGFR).

The hydrocarbon-stapled peptide E1S allosterically inhibits the kinase activity of the epidermal growth factor receptor (EGFR) by blocking a distant but essential protein–protein interaction: a coiled coil formed from the juxtamembrane segment (JM) of each member of the dimeric partnership.1 Macrocyclization is not required for activity: the analogous unstapled (but alkene-bearing) peptide is equipotent in cell viability, immunoblot, and bipartite display experiments to detect coiled coil formation on the cell surface.

Identifying hierarchical and overlapping protein complexes based on essential protein-protein interactions and "seed-expanding" method.

Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.