Aberrant Protein-protein Interactions Research Articles

Selective inhibitors targeting Fis1/Mid51 protein-protein interactions protect against hypoxia-induced damage in cardiomyocytes.

Cardiovascular diseases (CVDs) are the most common non-communicable diseases globally. An estimated 17.9 million people died from CVDs in 2019, representing 32% of all global deaths. Mitochondria play critical roles in cellular metabolic homeostasis, cell survival, and cell death, as well as producing most of the cell's energy. Protein-protein interactions (PPIs) have a significant role in physiological and pathological processes, and aberrant PPIs are associated with various diseases, therefore they are potential drug targets for a broad range of therapeutic areas. Due to their ability to mimic natural interaction motifs and cover relatively larger interaction region, peptides are very promising as PPI inhibitors. To expedite drug discovery, computational approaches are widely used for screening potential lead compounds. Here, we developed peptides that inhibit mitochondrial fission 1 (Fis1)/mitochondrial dynamics 51kDa (Mid51) PPI to reduce the cellular damage that can lead to various human pathologies, such as CVDs. Based on a rational design approach we developed peptide inhibitors of the Fis1/Mid51 PPI. In silico and in vitro studies were done to evaluate the biological activity and molecular interactions of the peptides. Two peptides, CVP-241 and CVP-242 were identified based on low binding energy and molecular dynamics simulations. These peptides inhibit Fis1/Mid51 PPI (-1324.9kcal mol-1) in docking calculations (CVP-241, -741.3kcal mol-1, and CVP-242, -747.4kcal mol-1), as well as in vitro experimental studies Fis1/Mid51 PPI (KD 0.054µM) Fis1/Mid51 PPI + CVP-241 (KD 3.43µM), and Fis1/Mid51 PPI + CVP-242 (KD 44.58µM). Finally, these peptides have no toxicity to H9c2 cells, and they increase cell viability in cardiomyocytes (H9c2 cells). Consequently, the identified inhibitor peptides could serve as potent molecules in basic research and as leads for therapeutic development.

SMA-linked SMN mutants prevent phase separation properties and SMN interactions with FMRP family members.

Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of the SMN1 gene in most cases or mutations in rare cases. Interestingly, several SMN1 mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that in SMN1 mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMRFM). Importantly, SMA-linked SMNTUDOR mutant forms (SMNST) failed to interact with FMRFM In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.

The Emerging Roles of Protein Interactions with O-GlcNAc Cycling Enzymes in Cancer.

Simple SummaryDynamic O-GlcNAc modification regulates the functions of proteins in a broad range of cellular processes. Dysregulation of the sole enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), and the associated cellular O-GlcNAc profile is a common feature across nearly every cancer type. Here, we highlight recent studies on the structural features of OGT and OGA, as well as the emerging roles and molecular mechanisms of their aberrant protein–protein interactions (PPIs) in rewiring cancer networks, to help identify key protein contacts and functional modules that drive malignancies and to promote cancer therapeutic innovations.The dynamic O-GlcNAc modification of intracellular proteins is an important nutrient sensor for integrating metabolic signals into vast networks of highly coordinated cellular activities. Dysregulation of the sole enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), and the associated cellular O-GlcNAc profile is a common feature across nearly every cancer type. Many studies have investigated the effects of aberrant OGT/OGA expression on global O-GlcNAcylation activity in cancer cells. However, recent studies have begun to elucidate the roles of protein–protein interactions (PPIs), potentially through regions outside of the immediate catalytic site of OGT/OGA, that regulate greater protein networks to facilitate substrate-specific modification, protein translocalization, and the assembly of larger biomolecular complexes. Perturbation of OGT/OGA PPI networks makes profound changes in the cell and may directly contribute to cancer malignancies. Herein, we highlight recent studies on the structural features of OGT and OGA, as well as the emerging roles and molecular mechanisms of their aberrant PPIs in rewiring cancer networks. By integrating complementary approaches, the research in this area will aid in the identification of key protein contacts and functional modules derived from OGT/OGA that drive oncogenesis and will illuminate new directions for anti-cancer drug development.

Hotspot Identification and Drug Design of Protein-Protein Interaction Modulators Using the Fragment Molecular Orbital Method.

Protein-protein interactions (PPIs) are essential for the function of many proteins. Aberrant PPIs have the potential to lead to disease, making PPIs promising targets for drug discovery. There are over 64,000 PPIs in the human interactome reference database; however, to date, very few PPI modulators have been approved for clinical use. Further development of PPI-specific therapeutics is highly dependent on the availability of structural data and the existence of reliable computational tools to explore the interface between two interacting proteins. The fragment molecular orbital (FMO) quantum mechanics method offers comprehensive and computationally inexpensive means of identifying the strength (in kcal/mol) and the chemical nature (electrostatic or hydrophobic) of the molecular interactions taking place at the protein-protein interface. We have integrated FMO and PPI exploration (FMO-PPI) to identify the residues that are critical for protein-protein binding (hotspots). To validate this approach, we have applied FMO-PPI to a dataset of protein-protein complexes representing several different protein subfamilies and obtained FMO-PPI results that are in agreement with published mutagenesis data. We observed that critical PPIs can be divided into three major categories: interactions between residues of two proteins (intermolecular), interactions between residues within the same protein (intramolecular), and interactions between residues of two proteins that are mediated by water molecules (water bridges). We extended our findings by demonstrating how this information obtained by FMO-PPI can be utilized to support the structure-based drug design of PPI modulators (SBDD-PPI).

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools.

Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

Splicing Variants, Protein-Protein Interactions, and Drug Targeting in Hutchinson-Gilford Progeria Syndrome and Small Cell Lung Cancer.

Alternative splicing (AS) is a biological operation that enables a messenger RNA to encode protein variants (isoforms) that give one gene several functions or properties. This process provides one of the major sources of use for understanding the proteomic diversity of multicellular organisms. In combination with post-translational modifications, it contributes to generating a variety of protein–protein interactions (PPIs) that are essential to cellular homeostasis or proteostasis. However, cells exposed to many kinds of stresses (aging, genetic changes, carcinogens, etc.) sometimes derive cancer or disease onset from aberrant PPIs caused by DNA mutations. In this review, we summarize how splicing variants may form a neomorphic protein complex and cause diseases such as Hutchinson-Gilford progeria syndrome (HGPS) and small cell lung cancer (SCLC), and we discuss how protein–protein interfaces obtained from the variants may represent efficient therapeutic target sites to treat HGPS and SCLC.

Abstract P2-03-03: Profiling protein-protein interactions at single-molecule level as a novel biomarker-driven screening method for the detection of tumor recurrence in breast cancer patients

Abstract Background: Protein-protein interactions (PPIs) modulate the vast majority of cellular processes. Therefore, profiling PPIs occurred in a clinical specimen may facilitate to understand molecular mechanisms involved in disease progression such as cancer. In breast cancer diagnosis, molecular subtyping is an important step for decision-making clinically. However, the molecular subtype is determined simply according to the expression level of receptors (hormone receptors and HER2). Here, we present the use of single-molecule analysis to visualize PPIs in a clinical specimen. We further assess the potential use of PPI-profiling to screen a subgroup of breast cancer patients who show aberrant PPIs. Methods: In total, 138 fresh-frozen breast tumor tissues were profiled. Specimens were selected based on IHC scores with an average follow-up of 7.5 years; ≥ 7 for hormone receptors (ER/PR+) and ≤ 1 for HER2 (HER2-) determined by IHC-scoring guidelines. We analyzed expression levels and strengths of PPIs for HER2, HER3 and EGFR from breast cancer specimens by applying single-molecule pull-down and co-immunoprecipitation techniques - a novel technology based on single-molecule fluorescence imaging (Proteina co., Ltd.). Tumor proteins were extracted from fresh-frozen tumor tissue via cryogenic grinding followed by the addition of tissue lysis buffer. Single-molecule PPI and expression level analysis were performed by using EGFR-PPI-Dx kit and proto-type HER2/HER3-PPI-Dx kit, respectively. Single-molecule analysis for the prepared kit was carried out using ProViewTM, an automated single-molecule fluorescence imaging equipment. Disease-free survival between groups were compared using Kaplan-Meier survival curve analysis and log-rank test was performed to determine the difference of survival distributions between groups. Results: 72 (discovery set) and 66 (validation set) patients were analyzed, respectively. Single-molecule measurement for HER2 level demonstrated that clinical specimens showed significant differences in HER2 level even though they had the same IHC score (up to 65-fold from 117 patients with ‘IHC-0’ and 33-fold from 21 patients with ‘IHC 1’, respectively). By combining HER2 PPI and expression data, we screened 12 patients (17%) as a high HER2-signal group, which showed positive HER2-PPI and enhanced HER2-level from a discovery set. Interestingly, this high HER2-signal group showed the enhanced tumor recurrence rate compared to that of low HER2-signal (58% for high HER2-signal vs. 17% for low HER2-signal, log-rank p=0.001). Using the same cutoff, we screened 17 patients from validation set and this screened group showed higher tumor recurrence risk rate than the low HER2-signal/group-prevalence (18% vs. 12%/13.6%, log-rank p=0.033). Conclusions: Defining a cancer patient by individual molecular features is a cornerstone of precision medicine. A comprehensive understanding of the molecular landscape may help a physician deciding a biomarker-driven treatment patient-by-patient. Molecular profiling (PPI and expression level) at single-molecule level enables to determine the strength of PPIs and the quantity of proteins quantitatively and precisely. In this study, we describe a novel concept of patient-classification by single-molecule PPI analysis. Our PPI analysis reveals a subgroup of HR+/HER2- patients who show an enhanced HER2-initiated signaling, which was hidden by conventional techniques. We further identified that this subgroup exhibited higher recurrence rate than the prevalence of whole group we tested. Our findings propose that adjuvant HER2-targeted therapies along with hormone-therapy may be a new opportunity to patients screened by PPI profiling for reduced risk of tumor recurrence. Citation Format: Hong Won Lee, Hee Jun Choi, Hyunwoo Kim, Seul Lee, Sangmin Kim, Tae-Young Yoon, Jeong Eun Lee. Profiling protein-protein interactions at single-molecule level as a novel biomarker-driven screening method for the detection of tumor recurrence in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-03-03.

Protein-Protein Interactions of Huntingtin in the Hippocampus

Huntingtin (HTT) occurs in the neuronal cytoplasm and can interact with structural elements of synapses. Huntington's disease (HD) results from pathological expansion of a polyglutamine stretch in the HTT molecule, being probably associated with aberrant protein-protein interactions. The pathogenetic mechanism is still incompletely understood. Alterations of the synaptic structure and plasticity in the hippocampus are observed in early HD. The objective of the study was to theoretically evaluate the HTT contribution to changes in synaptic plasticity by integrating the available experimental data. HTT protein complexes are involved in maintaining the efficiency of synaptic transmission. A pathogenic HTT form (polyQ-HTT) probably disrupts the protein-protein interactions in distorts the dynamics of molecular processes in the synapsis. It was assumed that polyQ-HTT may compete with postsynaptic density proteins and proteins regulating cytoskeleton remodeling.

A familial ATP13A2 mutation enhances alpha-synuclein aggregation and promotes cell death.

Aberrant protein-protein interactions are a common pathological hallmark among neurodegenerative diseases, including Parkinson's disease (PD). Thus far, mutations in more than 20 genes have been associated with PD. These genes encode for proteins involved in distinct intracellular pathways, complicating our understanding of the precise molecular mechanisms underlying the disease. Recent reports suggested that the endolysosomal protein ATP13A2 can determine the fate of alpha-synuclein (α-Syn), although no consensus has yet been reached on the mechanisms underlying this effect. Here, we describe, for the first time, the deleterious effect arising from the interaction between the ATP13A2 familial mutant Dup22 with α-Syn. We show that this ATP13A2 mutant can enhance α-Syn oligomerization and aggregation in cell culture. Additionally, we report the accumulation of both proteins in abnormal endoplasmic reticulum membranous structures and the activation of the protein kinase RNA-like endoplasmic reticulum kinase pathway. Ultimately, our data bring new insight into the molecular mechanisms underlying the interplay of these two proteins, opening novel perspectives for therapeutic intervention.

Structural Re-engineering of the α-Helix Mimetic JY-1-106 into Small Molecules: Disruption of the Mcl-1-Bak-BH3 Protein-Protein Interaction with 2,6-Di-Substituted Nicotinates.

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: Ki =2.90 μm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D (1) H-(15) N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts.

Proximity Utilizing Biotinylation of Nuclear Proteins in vivo.

IntroductionThe human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular “proteome”). It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI) are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination) or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo.MethodsThe biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP) was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.ResultsUsing several experimental systems (BirA.A + BAP.B), we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1α (CBX5), HP1β (CBX1), HP1γ (CBX3), wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA), ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation) and is designed to purify and study the protein composition of chromatin in proximity to the nuclear protein of interest. Using the RAD18 protein as a model, we demonstrated that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2Az has a different pattern of H4 acetylation.ConclusionProgress in the last decade in cancer drug therapy has led us to the conclusion that the nucleus of eukaryotic cells is an active site for many cellular processes important to the development of cancer. These processes include changes in genetic and epigenetic landscape (e. g. methylation of DNA, modification of histones) and the expression levels of transcription factors, which regulates gene products (e.g. hypoxia-inducible factor 1α (HIF-1α) in chronic anemia, etc.) where protein-protein interactions play important role. Understanding the nature of protein-protein interactions may improve design strategies for small-molecule PPI modulators. PPI assay technologies that closely reflect physiological conditions hold the key to developing specific anti-cancer drugs.

Abstract LB-197: YK-4-279 is effective in treating EWS-FLI1 induced myeloid/erythroid leukemia in a transgenic mouse model

Abstract Ewing sarcoma is an aggressive neoplasm affecting bone and soft tissue occurring almost exclusively in children and young adults. It is characterized by a distinct chromosomal translocation involving EWS and FLI1 genes making targeted therapy feasible since the resulting oncoprotein, EWS-FLI1, is exclusively expressed in the cancerous cells. The fusion protein has been shown to confer its tumorigenic phenotype through aberrant transcriptional activity, splicing, and protein-protein interactions. Previous work from our lab has identified a small molecule inhibitor, YK-4-279 that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing Sarcoma cell lines. Uncertainty about the cell of origin for Ewing sarcoma contributes to the lack of in vivo models for the disease, which has hindered preclinical study efforts. Torchia et al. (Mol. and Cell. Bio. 2007. 27(22): 7918-7934) has generated a transgenic mouse model where expression of EWS-FLI1 was targeted to the bone marrow, spleen, and liver by crossing Rosa26-loxP-stop-loxP-EWS-FLI1 mice with mice that express cre recombinase under Mx1-promoter, which responds to pIpC. Expression of EWS-FLI1 in these tissues induce a rapid expansion of primitive myeloid progenitors leading to rapid leukemia development. We used this mouse model to assess the efficacy of YK-4-279 in disrupting oncogenic activity of EWS-FLI1 in vivo. The mice were injected with 1mg of pIpC at an age of one month to induce leukemia. Once an increase in white blood cell count is observed following pIpC injection, the mice were randomly assigned to a treatment or control groups. Treatment of the transgenic mice daily, five times a week, intraperitoneally with 75mg/kg YK-4-279 vs. vehicle led to improved overall survival. Median survival for control group was 10.5 days while for treatment group was 24 days (p=0.045). Mice treated with 75mg/kg YK-4-279 showed significant reduction of disease burden within two weeks as monitored by weekly White Blood Cell count (DMSO vs. 75mg/kg YK Week 1: p=0.023 and Week 2: p=0.004). Mice treated with 150mg/kg YK-4-279 showed even more reduction of disease burden where one week after treatment WBC for DMSO group increased on average by 6495 cells/ul of blood while for YK-4-279 group it decreased by 1278 cells/ul of blood. Two weeks after the treatment started, the WBC for DMSO group increased even more by an average of 17,500 cells/ul of blood while the disease was stabilized for 150mg/kg YK-4-279 treated group (DMSO vs. 150mg/kg YK-4-279 Week 1: p-value<0.0001 and Week 2: p-value<0.002). Additionally, we observed reduced proliferation of blast cells in the peripheral blood by differential counting in YK-4-279 treated group vs. those treated with vehicle alone. Furthermore, we show that following two weeks treatment, YK-4-279 inhibits genes that are up-regulated in response to EWS-FLI1 activation in EF/+ Mx1cre+ mice. In conclusion, findings from this in vivo study highlight the effect of YK-4-279 in treating EWS-FLI1 induced leukemia and suggest its potential for early clinical trials in humans. Citation Format: Tsion Z. Minas, Jenny Han, Sung-Hyeok Hong, Jeffrey Toretsky, Aykut Uren. YK-4-279 is effective in treating EWS-FLI1 induced myeloid/erythroid leukemia in a transgenic mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2014-LB-197

Approaching the Increasing Complexity of Non-small Cell Lung Cancer Taxonomy

The advent of molecular targeted agents is changing the treatment of solid tumors. In non-small-cell lung cancer, compounds directed against oncogenic proteins offer novel therapeutic opportunities for a fraction of patients whose tumors harbor specific genetic defects. With the increased level of resolution achieved by high-throughput technologies, the taxonomy of lung cancer is rapidly changing. For instance, by cataloguing genetic abnormalities in squamous cell lung cancer the Cancer Genome Atlas Network revealed the existence of multiple molecular entities, each one characterized by specific molecular abnormalities, and by a different spectrum of activated/ inactivated molecular networks. Although this increased complexity could be perceived as a further drawback in effective anticancer therapy, on the other hand the combined interrogation of genomic and proteomic data is expected to provide the whole molecular map of each tumor, and to determine the information flow in the explored biological system. In particular, novel genetic and proteomic approaches are offering the opportunity for matching specific genetic defects and aberrant protein-protein interactions with active pathway-targeted inhibitors. Moreover, the isolation and characterization of a cellular pool endowed with stem-like traits, and able to recapitulate the parental disease in animals, is enabling investigators to recreate the individual patient tumor in the laboratory. In this article, we discuss how novel technologies and cellular and animal models, applied to lung cancer research, hold the potential to foster a new wave of biomarker-driven clinical trials.

Predicting Druggable Sites in Protein-Protein Interfaces using FindBindSite

Significance: Aberrant protein-protein interactions are a hallmark of disease and many cancers. Disrupting these interactions is a current therapeutic strategy. However, developing inhibitors for protein-protein interfaces (PPI) remains challenging due to large surface area over which these interactions occur. Computational methods can greatly aid in identifying druggable sites on the PPI enabling rational inhibitor design for PPI.Approach: We have developed a computational method termed FindBindSite (FBS), to identify druggable sites in the PPI starting from free monomer structures. Our method virtually screens a small database of compounds or dipeptides over the entire protein surface and identifies regions with high docked ligand atom density. Densely populated regions are then clustered and scored based on cluster size. The clustering allows us to identify binding surfaces in the interface regions.Results: FBS was validated 41 protein-protein structures crystallized in complex form. Structures were selected giving preference to free, protein-inhibitor, and then protein-protein complex when structures were not available. We predicted binding sites in interface regions of 71% with a high confidence and 90% with a low confidence using our test set. We tested the performance of FBS on homology models of free monomers achieving a hit rate of 68% when using templates with sequence identity between 20-97%. Applying a 60% sequence identity cutoff we achieved a hit rate of 86%. Using a library of dipeptides we were able to achieve 85% hit rate. We demonstrate that FBS is a useful computational method to predict binding sites in protein-protein interfaces because it uses the probe molecule diversity to span beyond well formed pockets and identify regions where one could likely disrupt any PPIs are likely to occur.

Recapitulating the α-Helix: Nonpeptidic, Low-Molecular-Weight Ligands for the Modulation of Helix-Mediated Protein–Protein Interactions

Protein-protein interactions play critical roles in a wide variety of biological processes, and their dysregulations contribute to the pathogenesis of several diseases, including cancer. Chemical entities that can abrogate aberrant protein-protein interactions may provide novel therapeutic agents. A large number of protein-protein interactions are mediated by protein secondary structure, the most commonly encountered form of which is the α-helix. Accordingly, over the last decade, there has been a flood of nonpeptidic small molecules that recapitulate the projection and chemical nature of key side chains of the canonical α-helix as a strategy to disrupt helix-mediated protein-protein interactions. In this review, we discuss recent advances (post 2006) in the design of synthetic α-helix mimetics, which include single-faced and two-faced/amphipathic structures, for the modulation of protein-protein interactions.

A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

Intrinsically unstructured proteins and neurodegenerative diseases: Conformational promiscuity at its best

Neurodegenerative diseases are complex, multifactorial disorders where misfolding of proteins cause aberrant protein-protein interactions. They are not usually characterized by specific mutations especially for nonfamilial disease types. Most of the causative proteins, however, are intrinsically unstructured (IUP), loss of whose fine balance could play pivotal role in these processes. Very fast conformational switch of these IUPs between different functional forms, so as to choose different interaction partners and different functional niches within the cell, is the basic premise on which these proteins maintain their interaction network. We are working on the hypothesis that even small perturbations in conformation leads to disruption of the network and to the disease phenotype. Based on a comprehensive data search, the evidence was obtained for the role of IUPs in neurodegenerative disorders, and their mode of action through conformational promiscuity is elaborated through three case studies.

Abstract 4873: Protein complexes in cancer: A novel strategy to identify protein complexes through reduction of false discovery rate

Abstract Most cellular processes are controlled by protein-protein interactions, and aberrant protein-protein interactions can lead to human diseases such as cancer. Hence a comprehensive understanding of a protein interaction network and its dynamics response to different physiological conditions is essential for understanding a protein's function and may reveal novel therapeutic opportunities for fighting cancer. Affinity purification coupled with mass spectrometry has become the method of choice to study in vivo protein interactions and formation of protein complexes. However, the methodology has some limitations that reduce it application such as, under sampling of low abundant proteins and high background leading to an elevated false discovery rate (FDR). Herein we describe a strategy utilizing differential expression of the target protein followed by analysis using quantitative proteomics. By performing affinity purification on cells expressing increasing amounts of the polycomb complex protein Bmi-1, we show that known interacting proteins could be distinguished very efficiently from the background proteins based on their abundance profile. Applying this method to the data set significantly reduced the FDR and allowed us to identify potentially new interacting proteins for Bmi-1 that otherwise would have been considered non-specifically bound proteins. The method was also applied to characterize the interactome for Snail I, II and III a family of Zn-finger proteins thought to be involved in transcriptional repression. These proteins have been implicated in diverse biological function such as regulation of epithelial to mesenchymal transition, formation and maintenance of embryonic mesoderm, generation and migration of neural crest cells and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4873. doi:10.1158/1538-7445.AM2011-4873

The structure of the mammalian RNase H2 complex provides insight into RNA:DNA hybrid processing to prevent immune dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA:DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi‐Goutieres syndrome (AGS). We have determined the crystal structure of the three protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein‐protein interaction motifs that could allow for efficient participation in multiple cellular functions. Further structural and biochemical analyses show that some RNase H2 disease‐causing mutations likely result in aberrant protein‐protein interactions while the RNase H2A subunit‐G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification. Funding: NIH‐R01‐GM069962, Alliance for Lupus Research, ACS.

The Structure of the Mammalian RNase H2 Complex Provides Insight into RNA·DNA Hybrid Processing to Prevent Immune Dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.