36 results about "Autophagosome" patented technology

A composition, comprising: an enriched population of autophagosomes derived from a non-small cell lung carcinoma cell line, and wherein the enriched population of autophagosomes includes: one or more toll-like receptor agonists; one or more tumor antigens; and one or more damage-associated molecular pattern molecules. In this way, an off-the-shelf vaccine may be available to be administered in order to stimulate a targeted immune response in patients bearing different tumor types.

The drug action mechanisms and core techniques of the present invention are summarized in figure 1. Specifically, the invention provides malignant denatured proteins, such as mutant Huntingtin proteinor alpha-synuclein, stick together to grow into oligomer aggregates (1, 2), fibrillar aggregates (3), and ultimately inclusion bodies (4). Young neuronal cells produce a large quantity of Nt-Arg through N-terminal arginylation (5) of endoplasmic reticulum chaperones, such as BiP, and thereafter, arginylated BiP (R-BiP) comes into the cytoplasm and binds with denatured proteins (6). Nt-Arg of R-BiP, as a ligand, binds with the ZZ domain of p62 (7) to induce the structural activation of p62 (8) while the ordinarily closed inactive form of p62 is changed with an open form thereof, and thus PB1 and LC3-binding domains are exposed. On the basis of oligomerization (9) by the PB1 domain, p62 binds with the denatured protein aggregates to be concentrated to autophagically degradable aggregates, that is, p62 bodies (10). Thereafter, p62 completes autophagy targeting (11) and lysosomal proteolysis through binding with LC3 protruding on the autophagosomal membranes. In young neuronal cells, theautophagic proteolysis occurring through steps 5-11 is strong, and thus the cytotoxic protein aggregates (1-5) do not accumulate, but in aged neuronal cells, the autophagic proteolysis occurring through steps 5-11 is weakened, and thus the protein aggregates (1-5) accumulate, resulting in a vicious cycle. The present invention attempts to effectively remove Huntingtin and alpha-synuclein protein aggregates and the like by artificially activating p62 using low-mass ligands of the p62 ZZ domain (12, 13). Specifically, p62 binding the ligands through step 12 promotes p62-R-BiP-denatured protein oligomerization (9) and autophagy aggregate formation (10). In addition, the ligand-62 conjugates step 13 act as autophagy activators (14), to promote LC3 synthesis, the conversion of LC3-I into LC3-II, and the like, thereby promoting the formation of autophagosomes (15).

The invention discloses application of H22 liver cancer cell autophagosome DRibbles to the preparation of a liver cancer therapeutic vaccine. Liver cancer is one of the most common malignant tumors in China and can be effectively prevented by developing a liver cancer vaccine; and years of research results prove that tumor cell autophagosome is one of effective vectors for tumor antigen cross-presentation. The application comprises a core technology for extracting the autophagosome DRibbles from human liver cancer cells and proves that the autophagosome can effectively induce anti-tumor immune response of organisms and has a good biotherapy prospect.

The invention discloses an electrochemical immunosensor for measuring secretory autophagosomes, a preparation method and application thereof, and belongs to the technical field of biomedical detection. The base electrode was modified by GH‑MB solution, and the autophagosome surface marker LC3B primary antibody solution was covalently bound to the modified base electrode to prepare an electrochemical immunosensor. The present invention also provides a method for detecting secreted autophagosomes in human peripheral blood and mouse peripheral blood based on an electrochemical immunosensor for GH-MB signal amplification. Secreted autophagosomes are composed of Fe 3 o 4 ‑The Au-modified LC3B secondary antibody specifically binds to the LC3B primary antibody after capture. The detection method has high sensitivity, good specificity, wide linear range, low detection limit and low cost.

The invention discloses an autophagy monitoring method for fat cells. Due to the adoption of a special structure, mature fat cells are full of plenty of lipid droplets, so that organelle and nucleus are displaced towards the periphery, and a typical autophagosome is hard to be seen under an electron microscope. Proved by a plurality of experiments, the method disclosed by the invention finally and successfully observes autophagy structures in various autophagy stages in mature 3T3-L1 fat cells by continuously improving the experiment condition. Besides, mature fat has a characteristic of being not easy to be transfected, so common plasmid transfection is hard to play an expectant effect. Therefore, the method disclosed by the invention utilizes a GFP-LC3 lentivirus method to infect 3T3-L1 fat cells, and the infection efficiency is as high as 80% or greater by observation under a fluorescence microscope. At nutrition and hunger condition, by the method disclosed by the invention, GFP-LC3 point-like aggregation phenomenon is successfully observed. The establishment of the autophagy monitoring method for mature fat cells lays the foundation of research on the relationship between autophagy and fat metabolism for us.

The pharmacokinetics and key technologies of the present invention are summarized in FIG. 1. Particularly, malignant misfolded proteins such as mutant huntingtin and alpha-synuclein are coagulated and grow into oligomeric coagulum (①, ②, fibrillar coagulum (③) and eventually inclusion body (④). Young neurons produce a large amount of Nt-Arg through N-terminal arginylation (⑤) of vesicle chaperones such as BiP secreted into the cytoplasm, and then arginylated BiP (R-BiP) is secreted binds to the misfolded proteins (⑥). As a ligand, the Nt-Arg of R-BiP binds to the p62 ZZ domain (⑦), and the normally inactivated closed form of p62 is changed to an open form, leading to structural activation (⑧). As a result, PB1 and LC3-binding domains are exposed. The PB1 domain induces oligomerization (⑨), leading to the concentration as a p62 body (⑩) that is a coagulum capable of being degraded by autophagy. Then, p62 binds to LC3, which is protruding from the autopagosomal membranes, leading to the completion of autophagy targeting (⑪) and lysosomal proteolysis. Since autophagy proteolysis including steps (⑤)-(⑪) is strong in young neurons, cytotoxic protein coagulums (①-⑤) do not accumulate. However in aged neurons, autophagy proteolysis including steps ⑤-⑪ is weakened, and protein coagulums (①-⑤) accumulate and become cytotoxic. In this invention, p62 is intentionally activated (⑫, ⑬) by using low mass ligands of the p62 ZZ domain to effectively remove huntingtin and alpha-synuclein protein coagulums. Particularly, in step {circle around (12)}, p62 ligated with a ligand accelerates the oligomerization of p62-R-BiP-misfolded protein (⑨) and the formation of autophagy coagulum (⑩). In step (⑬), the ligand-p62 conjugate acts as an autophagy activator (⑭) to induce the synthesis of LC3 and the conversion of LC3-I into LC3-II in order to accelerate the formation of autophagosomes (⑮).

The invention discloses a chimeric molecule that mediates the degradation of Tau protein based on an autophagy mechanism, and its amino acid sequence is shown in SEQ ID NO:5 or 7. Also disclosed is a nucleic acid molecule encoding the chimeric molecule that mediates Tau protein degradation based on an autophagy mechanism; an expression vector comprising the nucleic acid molecule. Also disclosed are the applications of the chimeric molecules, nucleic acid molecules and expression vectors that mediate Tau protein degradation based on autophagy mechanism in degrading Tau protein. The invention develops a novel targeting chimeric molecule, which is combined on the surface of the aggregation protein Tau, thereby recruiting the Tau protein into the cell autophagosome and promoting its degradation through the cell autophagy pathway. The new protein targeted degradation technology provided by the present invention is expected to apply this new technology to different target proteins, thereby overcoming the inability of the current protein targeted degradation technology based on the ubiquitin-proteasome pathway to efficiently degrade intracellular macromolecules point defects.