Palbociclib (PD 0332991) Interaction with Kinases. Theoretical and Comparative Molecular Docking Study

The optimized geometry of palbociclib, (PD 0332991) (8-cyclopentyl-6-ethanoyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one), electrostatic potential map, molecular orbitals were calculated using the density functional theory. The geometry was used in a molecular docking study of palbociclib-kinase complexes, results could be explained by the charge of the nitrogen and oxygen atoms within the palbociclib. Energy gap of HOMO-LUMO surfaces, could help to explain the reactivity of the ligand and the hydrogen bonding with three different kinases, two of CDK6 and one of CDK4 type. Docking results are similar and complementary with literature reports using molecular dynamics, were hydrogen bonding was obtained and analyzed. The promiscuity of three kinases with palbociclib was detected by the docking results, thus, palbociclib could be used in other types of cancer besides myeloid leukemia. Some similarities are found with CDK4/CDK6 kinases which allow us to determine that palbociclib could be used to control other resistant inhibitor types of cancer.     

Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements

Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET) measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET) measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM), and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro₇ and the second polypeptide was a flexible molecule consisting of (Gly-Ser)₇ repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.     

Predicting the Binding Patterns of Hub Proteins: A Study Using Yeast Protein Interaction Networks

Background

Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH) with one or two binding sites, or multiple-interface hubs (MIH) with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations) or party hubs (i.e., simultaneously interact with multiple partners).

Methodology

Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB) protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques.

Conclusions

Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions.

Availability

We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.     

利用FRET技术研究Hepcidin 和Fpn 相互作用

铁是生命必需的微量元素,ferroportin(Fpn)是小肠吸收细胞铁释放的重要蛋白。新近发现肝脏分泌的抗菌多肽 hepcidin 具有调节肠铁吸收的重要作用,但目前尚缺少Fpn和hepcidin发生作用的实验依据。应用荧光共振能量转移技术(fluorescence resonance energy transfer ,FRET)对hepcidin和Fpn之间的作用关系进行了深入研究。首先进行了hepcidin-CF P融合蛋白表达载体的构建及表达鉴定;然后对含YFP,Fpn-YFP基因动物细胞表达载体的构建、表达和FRET检测。实验结果证实hepcidin和Fpn之间存在直接的相互作用,并发现两种蛋白发生相互作用后hepcidin也在细胞质中有分布。为临床治疗铁代谢紊乱性疾病提供了新的治疗策略和重要理论依据。     

利用荧光共振能量转移技术研究活细胞TLR4与MD-2作用结构域

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用．研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义．运用基于强度的三通道荧光共振能量转移技术(FRET)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域．结果表明：N端Glu²⁴～Met⁴¹缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu²⁴～Met⁴¹的TLR4不能聚合．上述结果提示,TLR4的Glu²⁴～Met⁴¹不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用．     

Study of the Interaction of the Myelin Monomeric GTP-Binding Proteins with Other Brain Proteins

Abstract: Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.     

FRET Reveals Novel Protein-Receptor Interaction of Bone Morphogenetic Proteins Receptors and Adaptor Protein 2 at the Cell Surface

Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 β-isoform and caveolae of the caveolin-1 α/β-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

Binding Site Identification and Flexible Docking of Single Stranded RNA to Proteins Using a Fragment-Based Approach

Protein-RNA docking is hampered by the high flexibility of RNA, and particularly single-stranded RNA (ssRNA). Yet, ssRNA regions typically carry the specificity of protein recognition. The lack of methodology for modeling such regions limits the accuracy of current protein-RNA docking methods. We developed a fragment-based approach to model protein-bound ssRNA, based on the structure of the protein and the sequence of the RNA, without any prior knowledge of the RNA binding site or the RNA structure. The conformational diversity of each fragment is sampled by an exhaustive RNA fragment library that was created from all the existing experimental structures of protein-ssRNA complexes. A systematic and detailed analysis of fragment-based ssRNA docking was performed which constitutes a proof-of-principle for the fragment-based approach. The method was tested on two 8-homo-nucleotide ssRNA-protein complexes and was able to identify the binding site on the protein within 10 Å. Moreover, a structure of each bound ssRNA could be generated in close agreement with the crystal structure with a mean deviation of ~1.5 Å except for a terminal nucleotide. This is the first time a bound ssRNA could be modeled from sequence with high precision.     

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular‐Docking Method

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular‐docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three‐dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular‐docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH₃ in R₁ had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8‐anilino‐1‐naphthalenesulfonic acid, was changed with norgestrel addition.     

采用酵母双杂交系统筛选GmDREB5的互作蛋白

以无自激活活性的GmDREB5蛋白73～226位氨基酸区段为诱饵,采用酵母双杂交系统筛选干旱处理5 h大豆cDNA文库.结果发现:一个互作蛋白含有保守的TPR(Tetratricopeptide repeat)结构域,与拟南芥的TPR蛋白仅有14%的相似性,说明其可能是一类新的大豆TPR蛋白,将其定名为GmTPR1;表达特性分析表明,GmTPR1基因受干旱、低温、高盐、ABA的诱导;证明GmTPR1不仅参与植物对非生物胁迫的响应,同时参与对GmDREB5蛋白水平的调控.     

Triazinylamidophosphate Oligonucleotides: Synthesis and Study of Their Interaction with Cells and DNA-Binding Proteins

Russian Journal of Bioorganic Chemistry - In this work, representatives of the class of triazinylamidophosphate oligonucleotide derivatives were obtained for the first time. A scheme for the...     

A Comparative Analysis of the Synergistic Interaction Between N'-Phenylsulfanilamides and Benzylpyrimidines Using Qsar Techniques

Abstract

Growth inhibition of E. coli cell culture has been determined for a series of 4-substituted-N'-phenylsulfonamides tested in the presence and absence of synergistic concentrations of trimethoprim. Quantitative structure-activity relationships, established by regression analysis, exhibit an identical dependence of bacterial growth inhibition on sulfonamide pK_a irrespective of the presence or absence of trimethoprim. Examination of a small series of benzylpyrimidines in the presence or absence of 4-dimethylamino-N¹-phenylsulfanilamide gave similar results. Since the presence of a synergistic agent affords no change in structure-activity relationships, it is concluded that no direct interaction between sulfonamides and benzylpyrimidines occurs and that the synergism observed is solely the result of the kinetic consequences of sequential blockade of the folate biosynthetic pathway.     

PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。     

A Matrix Protein Silences Transposons and Repeats through Interaction with Retinoblastoma-Associated Proteins

1. Download : Download high-res image (133KB)
2. Download : Download full-size image

Highlights? The Arabidopsis matrix protein TEK silences transposons and repeat-containing genes ? Binding of TEK on targets affects chromatin conformation and histone modifications ? TEK protein associates with FVE/MSI5-containing histone deacetylation complex ? TEK directs repressive modification as a key structural component in gene silencing     

Structural Model for p75–TrkA Intracellular Domain Interaction: A Combined FRET and Bioinformatics Study

Nerve growth factor (NGF) is a member of the neurotrophins, which are important regulators of embryonic development and adult function in the vertebrate nervous systems. The signaling elicited by NGF regulates diverse activities, including survival, axon growth, and synaptic plasticity. NGF action is mediated by engagement with two structurally unrelated transmembrane receptors, p75^NTR and TrkA, which are co-expressed in a variety of cells. The functional interactions of these receptors have been widely demonstrated and include complex formation, convergence of signaling pathways, and indirect interaction through adaptor proteins. Each domain of the receptors was shown to be important for the formation of TrkA and p75^NTR complexes, but only the intramembrane and transmembrane domains seemed to be crucial for the creation of high-affinity binding sites. However, whether these occur through a physical association of the receptors is unclear. In the present work, we demonstrate by Förster resonance energy transfer that p75^NTR and TrkA are physically associated through their intracellular (IC) domains and that this interaction occurs predominantly at the cell membrane and prior to NGF stimulation. Our data suggest that there is a pool of receptors dimerized before NGF stimulus, which could contribute to the high-affinity binding sites. We modeled the three-dimensional structure of the TrkA IC domain by homology modeling, and with this and the NMR-resolved structure of p75^NTR, we modeled the heterodimerization of TrkA and p75^NTR by docking methods and molecular dynamics. These models, together with the results obtained by Förster resonance energy transfer, provide structural insights into the receptors' physical association.