79 results about "Phosphorylation sites" patented technology

The invention discloses two newly-discovered Flt3 phosphorylation sites, tyrosine 589 (Tyr589) and tyrosine 591 (Tyr591) in the intracellular domain, and provides antibodies, both polyclonal and monoclonal, that selectively bind to Flt3 when phosphorylated at these novel sites. Also provided are assays utilizing these reagents, including methods for determining the phosphorylation of Flt3 in a biological sample, selecting a patient suitable for Flt3 inhibitor therapy, profiling Flt3 activation in a test tissue, and identifying a compound that modulates phosphorylation of Flt3 in a test tissue, by using a detectable reagent, such as the disclosed antibodies, that binds to Flt3 when phosphorylated at Tyr589 or Tyr591. The sample or test tissue may be taken from a subject suspected of having cancer, such as acute myelogenous leukemia (AML).

Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine / threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR / ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.

Modified proteins, modified interferons alpha 's and beta 's, phosphorylated modified proteins and DNA sequences encoding the above, applications and uses thereof. Modified phosphorylated Hu-IFN- alpha -like proteins are provided which carry an identifiable label such as a radio-label. Corresponding phosphorylatable Hu-IFN- alpha -like proteins which contain a putative phosphorylation site. DNA sequences which encode a Hu-IFN- alpha -like protein and contain a sequence encoding a putative phosphorylatable site. Appropriate expression vectors are used to transform compatible host cells of various microorganisms, such as E. coli. Numerous uses for the phosphorylated proteins are disclosed.

The invention discloses a prediction algorithm for recognizing tyrosine posttranslational modification sites. The algorithm comprises the steps of data collection, data processing, feature coding, feature optimization and model training and evaluation. The invention furthermore discloses application of the prediction algorithm. According to the algorithm, features of the tyrosine posttranslational modification sites are extracted comprehensively from the perspectives of protein sequence information, evolutional information and physical and chemical properties, Elastic Net is used as an optimization means to automatically select variables to screen multidimensional features, redundant information is removed, a prediction model of tyrosine nitration, sulfuration and phosphorylation sites is constructed in combination with an SVM, the prediction capability of the prediction model is improved, and the prediction quality of the tyrosine posttranslational modification sites is remarkably improved. Through a developed prediction software platform TyrPred, predictive analysis of nitration modification sites, sulfuration modification sites and phosphorylation modification sites of tyrosine on intact protein is realized, and a convenient, economical and rapid research tool and important reference are provided for research of tyrosine posttranslational modification.

There are provided a novel compound which captures a multisite phosphorylated peptide or protein specifically to a phosphorylation site and a method for detecting a multisite phosphorylated peptide or protein using the same. In particular, there are provided a compound which specifically detects an excessively phosphorylated tau protein observed in the brain affected by Alzheimer's disease and a method for diagnosing Alzheimer's disease in vitro or in vivo using the compound. By bringing a metal complex compound having two dipicolylamine (Dpa) moieties and a spacer including a chromogenic or luminescent functional group or atom group into contact with a multisite phosphorylated peptide or protein, the compound recognizes the distance between phosphate groups and specifically binds to the peptide or the protein, and a multisite phosphorylated peptide or protein or a kinase activity is optically detected by measuring the change, or a multisite phosphorylated peptide or protein or kinase activity is imaged by an optical imaging method applying the change in the luminescence.

The invention involves a method for measuring phosphorylation of proteins at specific sites and, as such, is an indicator of the protein kinase activity of enzymes capable of phosphorylating those sites. The method involves the in vitro or in vivo phosphorylation of a target protein at a specific serine, threonine or tyrosine residue, subjecting that protein (non-phosphorylated) to reaction mixture containing all reagents, including phosphokinase which allow the creation of a phosphorylated form of protein. The phosphorylated protein is measured by contacting it with an antibody specific for the phosphorylation site(s). The invention includes antibodies useful in practicing the methods of the invention. The invention particularly relates to all proteins modified by phosphorylation and dephosphorylation as illustrated by Tau, Rb and EGFR proteins and antibodies specific for the site of phosphorylation of the Tau, Rb or EGFR proteins.

The present invention provides methods for measuring the phosphorylation activity of Cdc7-ASK kinase complex by using as an indicator the level of phosphorylation at a phosphorylation site of MCM, which is a substrate of Cdc7-ASK kinase complex. The effects of test compounds on the phosphorylation activity of Cdc7-ASK kinase complex can also be evaluated based on these measurement methods. Compounds that inhibit this phosphorylation activity are useful as anti-cancer agents having superior specificity for cancer.

The invention discloses a magnetic metal-organic framework nanosphere with multiple affinity sites as well as a preparation method and application thereof. The magnetic metal-organic framework nanosphere consists of a Fe3O4 magnetic sphere, a high-molecular polymer intermediate layer coating the surface of the magnetic sphere, a metal-organic framework growing on the high-molecular polymer intermediate layer and arginine grafted on the metal-organic framework. The magnetic metal-organic framework nanosphere disclosed by the invention takes the Fe3O4 magnetic sphere as a kernel and has good magnetic response performance; the metal-organic framework is introduced onto the surface of the magnetic sphere, and the organic ligand surface is modified through the arginine; as metal ions forming the metal-organic framework, a guanidyl on the arginine and the like can be taken as the multiple affinity sites for enriching phosphorylated polypeptides, the enrichment of the phosphorylated polypeptides for single-phosphorylation sites and multi-phosphorylation sites is achieved; the magnetic metal-organic framework nanosphere has high enrichment efficiency and has very important significance in studying the phosphorylation process of physiological behavioral proteins.

This invention relates to novel phosphorylation sites in the desmin protein that are associated with the onset of heart failure. The phosphorylation sites, i.e., Ser-27 and Ser-31, can be used as biomarkers for (i) identifying subjects at risk for the development of heart failure, (ii) treating subjects having a higher than normal level of the biomarker, and (iii) monitoring therapy of a subject at risk for the development of heart failure. Also described are antibodies, reagents, and kits for carrying out a method of the present invention.

The invention involves a method for measuring phosphorylation of proteins at specific sites and, as such, is an indicator of the protein kinase activity of enzymes capable of phosphorylating those sites. The method involves the in vitro or in vivo phosphorylation of a target protein at a specific serine, threonine or tyrosine residue, subjecting that protein (non-phosphorylated) to reaction mixture containing all reagents, including phosphokinase which allow the creation of a phosphorylated form of protein. The phosphorylated protein is measured by contacting it with an antibody specific for the phosphorylation site(s). The invention includes antibodies useful in practicing the methods of the invention. The invention particularly relates to all proteins modified by phosphorylation and dephosphorylation as illustrated by Tau, Rb and EGFR proteins and antibodies specific for the site of phosphorylation of the Tau, Rb or EGFR proteins.

Modified proteins, modified interferons alpha's and beta's, phosphorylated modified proteins and DNA sequences encoding the above, applications and uses thereof. Modified phosphorylated Hu-IFN-alpha-like proteins are provided which carry an identifiable label such as a radio-label. Corresponding phosphorylatable Hu-IFN-alpha-like proteins which contain a putative phosphorylation site. DNA sequences which encode a Hu-IFN-alpha-like protein and contain a sequence encoding a putative phosphorylatable site. Appropriate expression vectors are used to transform compatible host cells of various microorganisms, such as E. coli. Numerous uses for the phosphorylated proteins are disclosed.

The invention discloses 347 novel phosphorylation sites identified in carcinoma, peptides (including AQUA peptides) comprising a phosphorylation site of the invention, antibodies specifically bind to a novel phosphorylation site of the invention, and diagnostic and therapeutic uses of the above.

The present invention relates to modified G-protein coupled receptors (GPCRs). The modified GPCRs of the present invention include GPCRs that have been modified to have carboxyl terminal tails comprising one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites. The modified GPCRs of the present invention may comprise a retained portion of a carboxyl-terminus region from a first GPCR fused to a polypeptide, wherein the polypeptide comprises the one or more clusters of phosphorylation. The present invention also relates to methods of screening compounds and sample solutions for GPCR activity using the modified GPCRs.

The invention discloses a protein phosphorylation site identification method, system and device and a storage medium. The method comprises the following steps: acquiring an amino acid sequence fragment of a protein phosphorylation site to be identified; performing logic operation on binary codes of amino acids in the amino acid sequence fragment to obtain logic binary feature vectors of the aminoacid sequence fragment; performing kernel principal component analysis on the logic binary feature vector according to a preset kernel function to obtain a kernel principal component logic binary feature vector; and inputting the kernel principal component logic binary feature vector into a random forest model for processing to obtain an identification result of the protein phosphorylation site. Based on theoretical calculation of the random forest model, the method can achieve the quick and accurate recognition of the information of a large number of protein phosphorylation sites, is low in cost, facilitates the development of phosphorylation mechanism and phosphorylation and disease relation research, and is widely applied to the field of protein phosphorylation site recognition.

Sensitive assays for candidate compounds affecting GPCR activity are provided using a cell containing fusion proteins comprising a first fusion protein comprising (a) a target GPCR fused to a small fragment of β-galactosidase through a linker comprising a phosphorylation site or (b) a GPCR or a protein of interest, where the GPCR and protein of interest form a complex and one of them is fused to the small fragment of β-galactosidase; and a second fusion protein comprising arrestin fused to a large fragment of β-galactosidase. In (a), the affinity of the small and large fragments is optimized based on the background to signal ratio and the absolute signal observed. The assay is performed using a β-galactosidase substrate that provides a detectable optical signal.

The invention provides an isotope label-free method for relative quantitative analysis of a protein phosphorylation modification level. The method comprises: conducting enzymolysis on protein to make it into peptide fragments, making use of a data-independent MS / MS technique to determine peptide fragments with phosphorylation sites, and utilizing a label-free quantitation technique to carry out relative quantitative analysis on the peptide fragments with phosphorylation sites. Compared with the existing methods combining data-dependent MS / MS analysis and stable isotope-labeling technologies, the method does not need an isotope labeling reagent, thus having the advantages of reducing the experiment cost and operation complexity.

The invention discloses 214 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human carcinoma, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites / proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: Adaptor / Scaffold proteins, Cytoskeleton proteins, GTP Signaling proteins, Kinases, Metabolism proteins, Phosphatases / Phospho-diesterases / Proteases, Receptor proteins, RNA Processing proteins, Transcription proteins, Translation proteins, Transporter proteins, and Ubitquitin proteins, as well as other protein types.

The invention relates to the technical field of protein modification site identification, in particular to a protein kinase specificity prediction method and device based on a nearest neighbor algorithm. According to the prediction method, amino acid information on the upstream and the downstream of a phosphorylation site is fully utilized, and the accuracy of prediction is increased. According to the protein kinase specificity prediction method, an amino acid permutation matrix is used for scoring the similarity of a phosphorylation site peptide fragment to be detected and a known phosphorylation site peptide fragment, the phosphorylation site peptide fragment to be detected is marked to be the highest scored known phosphorylation site peptide fragment, and the sensitivity and the specificity of prediction are improved.

The invention discloses saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate. A regulatory factor G1n3p in saccharomyces cerevisiae is transformed through engineering bacteria. The concrete strategy is that a phosphorylation site on a G1n3p nuclear localization sequence is subjected to mutation to form three phosphorylation sites on the G1n3p nuclear localization sequence; the three phosphorylation sites respectively are the 344th, 347th and 355th serine. In order to obtain a better technical effect, a nuclear localization regulatory region of combining G1n3p with an upstream regulatory factor can be further removed; the G1n3p of a regulatory sequence at the tail end of C is removed by truncated expression, wherein an expression sequence is 1-653. The EC yield of the engineering bacteria disclosed by the invention is reduced by 62% by detection of a yellow rice wine simulation system. In addition, the content of main ingredients is not significantly changed, and the fermentation characteristics and the growth characteristics of a bacterial strain are not affected after the content of other ingredients in a fermentation liquor after fermentation is detected.

Diacylglycerol (DAG) plays a central role in both the synthesis of complex lipids and in intracellular signaling; diacylglycerol kinase (DGK) catalyzes the phosphorylation of DAG, which yields phosphatidic acid. A family of DGKs has been identified in multicellular organisms over the past few years, but the physiological function(s) of this diversity is not clear. One clue has come from the Drosophila DGK2, rdgA, since mutations in this gene cause retinal degeneration. The present invention relates to a novel DGK, designated DGKι, which was isolated from human retina and brain libraries. DGKι contains two cysteine-rich repeats, a region similar to the phosphorylation site domain of MARCKS, a conserved catalytic domain, and four ankyrin repeats at its C-terminus. By primary structure, DGKι is most similar to human DGKζ and Drosophila rdgA. A>12 kb mRNA for DGKι was detected only in brain and retina among the tissues examined. In cells transfected with the DGKι cDNA, an approximately 130 kDa protein was detected by immunoassay, and activity assays demonstrated that it encodes a functional DAG kinase. The protein was found to be in both the cytoplasm and nucleus, with this localization controlled by PKC isoforms alpha and gamma. The gene encoding DGKι was localized to human chromosome 7q32.3-33, which is known to be a locus for an inherited form of retinitis pigmentosa. These results have defined a novel isoform of DAG kinase, which may have important cellular functions in the retina and brain.

The invention discloses 94 novel phosphorylation sites identified in carcinoma and leukemia, peptides (including AQUA peptides) comprising a phosphorylation site of the invention, antibodies that specifically bind to a novel phosphorylation site of the invention, and diagnostic and therapeutic uses of the above.

The present invention relates to isolated phosphorylated NF45 peptides and isolated phosphorylation site-specific antibody or antigen-binding portion thereof that specifically binds a phosphorylated NF45 protein. The present invention also relates to methods of utilizing these antibodies to determine the therapeutic efficacy of a candidate compound and methods for screening for candidate compounds that increase the phosphorylation of NF45 protein in a cell.

The invention discloses 211 novel phosphorylation sites identified in signal transduction proteins and pathways underlying Anaplastic Large Cell Lymphoma (ALCL) involving the ALK-NPM translocation / fusion, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites / proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: Protein Kinases (including Receptor Tyrosine Kinases), Adaptor / Scaffold Proteins, Cellular Metabolism or Miscellaneous Enzymes, Oxidoreductases, Transcription Factors, Cytoskeletal Proteins, Translation Initiation Complexes, RNA Binding Proteins, Proteases, Acetyltransferases, G protein regulators / GTPases, Helicases, Apoptosis / Cell Cycle Regulation proteins, and Hydrolases.

The disclosure relates to a cytoplasmic protein complex comprising: (a) a first recombinant fusion protein comprising a kinase, fused to a first interaction polypeptide; and (b) a second recombinant fusion protein comprising a domain comprising a reporter phosphorylation site, whereby the domain is fused to a second interaction polypeptide. The disclosure relates further to a method to detect compound-compound-interaction using the cytoplasmic protein complex, and to cells comprising such cytoplasmic protein complex.

The invention belongs to the field of microbial genetics and molecular biology and discloses a method with a function of reducing accumulation of ethyl carbamate in rice wine fermentation. Genetically engineered bacteria modified by nitrogen catabolite repression regulation factors are adopted for rice wine fermentation, and engineered saccharomyces cerevisiae eliminates nuclear localization regulatory sequences of the regulation factors Gln3p and Gat1p and mutates phosphorylation sites of the nuclear localization sequences. In a simulated rice wine fermentation system, compared with rice wine with wild strains for fermentation, rice wine fermented with the genetically engineered bacteria has the advantages that contents of carbamide and ethyl carbamate in the rice wine are decreased by 63% and 72% respectively, the content of the ethyl carbamate in the rice wine is decreased to about 55.53 microgram / L, and contents of major nutrient substances and characteristic flavor substances are less in difference. Therefore, the method has a huge potential of application to rice wine production.

The invention provides a method for producing fucoxanthin by using a mutant strain for expressing exogenous pyruvate phosphate dikinase in phaeodactylum tricornutum, and particularly relates to algae preference codon optimization on an original CyPPDK gene to obtain a new CyPPDK gene with a nucleotide sequence as shown in SEQ ID NO.7; the constructed shuttle expression vector is named as pPha-fcp-PPDK; and the obtained new gene is introduced into a host algae cell phaeodactylum tricornutum cell in an electric transformation manner, and a transformant is screened to obtain a phaeodactylum tricornutum transgenic algae strain for overexpression of pyruvate phosphate dikinase. Compared with an unoptimized transformant, the optimized transformant for phosphorylation sites of the CyPPDK gene has the advantages that the biomass accumulation is improved by 33.3%, the content of EPA in grease is improved by 11.5%, and the fucoxanthin yield is improved by 21.2%.

The invention discloses novel phosphorylation sites identified in signal transduction proteins and pathways, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites / proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: adaptor / scaffold proteins, adhesion / extracellular matrix protein, apoptosis proteins, calcium binding proteins, cell cycle regulation proteins, chaperone proteins, chromatin, DNA binding / repair / replication proteins, cytoskeletal proteins, endoplasmic reticulum or golgi proteins, enzyme proteins, G / regulator proteins, inhibitor proteins, motor / contractile proteins, phosphatase, protease, Ser / Thr protein kinases, Protein kinase (Tyr)s, receptor / channel / cell surface proteins, RNA binding proteins, transcriptional regulators, tumor suppressor proteins, ubiquitan conjugating system proteins and proteins of unknown function.