RESUMO
There is a growing interest in the use of probiotic bacteria as biosensors for the detection of disease. However, there is a lack of bacterial receptors developed for specific disease biomarkers. Here, we have investigated the use of the peptide-regulated transcription factor ComR from Streptococcus spp. for specific peptide biomarker detection. ComR exhibits a number of attractive features that are potentially exploitable to create a biomolecular switch for engineered biosensor circuitry within the probiotic organism Lactiplantibacillus plantarum WCFS1. Through iterative design-build-test cycles, we developed a genomically integrated, ComR-based biosensor circuit that allowed WCFS1 to detect low nanomolar concentrations of ComR's cognate peptide XIP. By screening a library of ComR proteins with mutant residues substituted at the K100 position, we identified mutations that increased the specificity of ComR toward an amidated version of its cognate peptide, demonstrating the potential for ComR to detect this important class of biomarker.IMPORTANCEUsing bacteria to detect disease is an exciting possibility under active study. Detecting extracellular peptides with specific amino acid sequences would be particularly useful as these are important markers of health and disease (biomarkers). In this work, we show that a probiotic bacteria (Lactiplantibacillus plantarum) can be genetically engineered to detect specific extracellular peptides using the protein ComR from Streptococcus bacteria. In its natural form, ComR allowed the probiotic bacteria to detect a specific peptide, XIP. We then modified XIP to be more like the peptide biomarkers found in humans and engineered ComR so that it activated with this modified XIP and not the original XIP. This newly engineered ComR also worked in the probiotic bacteria, as expected. This suggests that with additional engineering, ComR might be able to activate with human peptide biomarkers and be used by genetically engineered probiotic bacteria to better detect disease.
Assuntos
Proteínas de Bactérias , Peptídeos , Fatores de Transcrição , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peptídeos/metabolismo , Peptídeos/genética , Probióticos/metabolismo , Mutação , Técnicas Biossensoriais , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptococcus/genética , Streptococcus/metabolismoRESUMO
Tumor protein p63 (TP63) is a member of the TP53 protein family that are important for development and in tumor suppression. Unlike TP53, TP63 is rarely mutated in cancer, but instead different TP63 isoforms regulate its activity. TA isoforms (TAp63) act as tumor suppressors, whereas ΔN isoforms are strong drivers of squamous or squamous-like cancers. Many of these tumors become addicted to ΔN isoforms and removal of ΔN isoforms result in cancer cell death. Furthermore, some TP53 conformational mutants (TP53CM) gain the ability to interact with TAp63 isoforms and inhibit their antitumorigenic function, while indirectly promoting tumorigenic function of ΔN isoforms, but the exact mechanism of TP63-TP53CM interaction is unclear. The changes in the balance of TP63 isoform activity are crucial to understanding the transition between normal and tumor cells. Here, we modeled TP63-TP53CM complex using computational approaches. We then used our models to design peptides to disrupt the TP63-TP53CM interaction and restore antitumorigenic TAp63 function. In addition, we studied ΔN isoform oligomerization and designed peptides to inhibit its oligomerization and reduce their tumorigenic activity. We show that some of our peptides promoted cell death in a TP63 highly expressed cancer cell line, but not in a TP63 lowly expressed cancer cell line. Furthermore, we performed kinetic-binding assays to validate binding of our peptides to their targets. Our computational and experimental analyses present a detailed model for the TP63-TP53CM interaction and provide a framework for potential therapeutic peptides for the elimination of TP53CM cancer cells.
Assuntos
Carcinoma de Células Escamosas , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Isoformas de Proteínas/metabolismo , Linhagem Celular Tumoral , Carcinogênese , Carcinoma de Células Escamosas/metabolismo , Simulação por Computador , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting spike (S) protein cleavage by several proteases. We developed a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.
Assuntos
COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Peptídeo Hidrolases , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do VírusRESUMO
Formation of Ras multimers, including dimers and nanoclusters, has emerged as an exciting, new front of research in the 'old' field of Ras biomedicine. With significant advances made in the past few years, we are beginning to understand the structure of Ras multimers and, albeit preliminary, mechanisms that regulate their formation in vitro and in cells. Here we aim to synthesize the knowledge accrued thus far on Ras multimers, particularly the presence of multiple globular (G-) domain interfaces, and discuss how membrane nanodomain composition and structure would influence Ras multimer formation. We end with some general thoughts on the potential implications of Ras multimers in basic and translational biology.
Assuntos
Transdução de Sinais , Proteínas ras , Membrana Celular/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.
Assuntos
Antineoplásicos/química , Proteínas de Ciclo Celular/química , Desenho de Fármacos , Fatores de Transcrição Forkhead/química , Modelos Moleculares , Peptídeos/química , Senoterapia/química , Proteína Supressora de Tumor p53/química , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Melanoma , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/farmacologia , Conformação Proteica , Senoterapia/farmacologia , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) ß-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and ß-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While ß-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the ß-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and ß-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sequência de Aminoácidos , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Simulação de Dinâmica Molecular , Mutação/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Drug repurposing is a creative and resourceful approach to increase the number of therapies by exploiting available and approved drugs. However, identifying new protein targets for previously approved drugs is challenging. Although new strategies have been developed for drug repurposing, there is broad agreement that there is room for further improvements. In this chapter, we review protein-protein interaction (PPI) interface-targeting strategies for drug repurposing applications. We discuss certain features, such as hot spot residue and hot region prediction and their importance in drug repurposing, and illustrate common methods used in PPI networks to identify drug off-targets. We also collect available online resources for hot spot prediction, binding pocket identification, and interface clustering which are effective resources in polypharmacology. Finally, we provide case studies showing the significance of protein interfaces and hot spots in drug repurposing.
Assuntos
Biologia Computacional , Reposicionamento de Medicamentos , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Biologia Computacional/métodos , Bases de Dados de Proteínas , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Ligantes , Modelos Moleculares , Conformação Molecular , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas/química , Relação Estrutura-AtividadeRESUMO
Proteins are dynamic, and this holds especially for their surfaces. They display ensembles of conformations, which allows them to interact with diverse partners, often via the same patch of surface, and execute their distinct functions. Binding a specific partner can stimulate - or suppress - a distinct signaling pathway. This diversity poses a challenge: how to reliably model a specific protein-protein interaction (PPI)? This problem is compounded in protein assemblies, which are typically large, involving multiple protein-protein interfaces. Integrative modeling (IM), which combines diverse data, has emerged as the most promising strategy; however, modeling dynamical interfaces, often at the detailed level, which are at the heart of reliable predictions of assemblies, still poses a challenge. Here we review hurdles and advances in integrative modeling of dynamical interfaces; while some could have been predicted or expected, others transformed modeling in unanticipated ways. We further comment on what we believe could be possible future advances.
Assuntos
Modelos Moleculares , Proteínas/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas/químicaRESUMO
Motivation: Single amino acid variations (SAVs) in protein-protein interaction (PPI) sites play critical roles in diseases. PPI sites (interfaces) have a small subset of residues called hot spots that contribute significantly to the binding energy, and they may form clusters called hot regions. Singlet hot spots are the single amino acid hot spots outside of the hot regions. The distribution of SAVs on the interface residues may be related to their disease association. Results: We performed statistical and structural analyses of SAVs with literature curated experimental thermodynamics data, and demonstrated that SAVs which destabilize PPIs are more likely to be found in singlet hot spots rather than hot regions and energetically less important interface residues. In contrast, non-hot spot residues are significantly enriched in neutral SAVs, which do not affect PPI stability. Surprisingly, we observed that singlet hot spots tend to be enriched in disease-causing SAVs, while benign SAVs significantly occur in non-hot spot residues. Our work demonstrates that SAVs in singlet hot spot residues have significant effect on protein stability and function. Availability and implementation: The dataset used in this paper is available as Supplementary Material. The data can be found at http://prism.ccbb.ku.edu.tr/data/sav/ as well. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Aminoácidos/química , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Humanos , Ligação Proteica , Conformação Proteica , Software , TermodinâmicaRESUMO
Proper localization of Ras proteins at the plasma membrane (PM) is crucial for their functions. To get to the PM, KRas4B and some other Ras family proteins bind to the PDEδ shuttling protein through their farnesylated hypervariable regions (HVRs). The docking of their farnesyl (and to a lesser extent geranylgeranyl) in the hydrophobic pocket of PDEδ's stabilizes the interaction. At the PM, guanosine 5'-triphosphate (GTP)-bound Arf-like protein 2 (Arl2) assists in the release of Ras from the PDEδ. However, exactly how is still unclear. Using all-atom molecular dynamics simulations, we unraveled the detailed mechanism of Arl2-mediated release of KRas4B, the most abundant oncogenic Ras isoform, from PDEδ. We simulated ternary Arl2-PDEδ-KRas4B HVR complexes and observed that Arl2 binding weakens the PDEδ-farnesylated HVR interaction. Our detailed analysis showed that allosteric changes (involving ß6 of PDEδ and additional PDEδ residues) compress the hydrophobic PDEδ pocket and push the HVR out. Mutating PDEδ residues that mediate allosteric changes in PDEδ terminates the release process. Mutant Ras proteins are enriched in human cancers, with currently no drugs in the clinics. This mechanistic account may inspire efforts to develop drugs suppressing oncogenic KRas4B release.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Proteínas de Ligação ao GTP/química , Proteínas Proto-Oncogênicas p21(ras)/química , Regulação Alostérica , Membrana Celular/química , Membrana Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Prenilação de Proteína , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.
Assuntos
Modelos Moleculares , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Regulação Alostérica , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Intrinsic fluctuations of a protein enable it to sample a large repertoire of conformers including the open and closed forms. These distinct forms of the protein called conformational substates pre-exist together in equilibrium as an ensemble independent from its ligands. The role of ligand might be simply to alter the equilibrium toward the most appropriate form for binding. Normal mode analysis is proved to be useful in identifying the directions of conformational changes between substates. In this study, we demonstrate that the ratios of normalized weights of a few normal modes driving the protein between its substates can give insights about the ratios of kinetic conversion rates of the substates, although a direct relation between the eigenvalues and kinetic conversion rates or populations of each substate could not be observed. The correlation between the normalized mode weight ratios and the kinetic rate ratios is around 83% on a set of 11 non-enzyme proteins and around 59% on a set of 17 enzymes. The results are suggestive that mode motions carry intrinsic relations with thermodynamics and kinetics of the proteins.
Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Cinética , Conformação Proteica , TermodinâmicaRESUMO
Here we report the synthesis of nanoparticles based on a conjugated oligomer which is synthesized through Heck-coupling of divinylfluorene and dibromobenzothiodiazole monomers. These water dispersible nanoparticles emit in the region of red tailing to the near-infrared region of the spectrum with high fluorescent quantum yield and brightness. The nanoparticles were found to be stable in water for a prolonged time without forming any aggregates and could carry camptothecin, an anticancer drug with high loading efficiency. MTT cell viability studies performed with breast cancer cell lines showed that half-maximal inhibitory concentration (IC50) values of nanoparticles for MCF7 and MDA-MB-231 were 44.7 µM and 24.8 µM, respectively. In order to further decrease the cytotoxicity and increase the stability of nanoparticles, amine groups were disguised by capping with cucurbit[7]uril (CB7). Drug release studies showed that drugs were released at low pH (at 5.0) faster than physiological pH (7.4) confirming the pH-responsive nature of the nanoparticles. On the other hand, CB7-capped drug-loaded nanoparticles regulated the release rate by providing slower release at pH 7.4 than the nanoparticles in the absence of CB7s. IC50 values for camptothecin in the presence of nanoparticles with or without CB7 were significantly reduced in MCF7 and MDA-MB-231 cells.