Regulation of apg8 phosphorylation and uses thereof

a technology of apg8 and phosphorylation, applied in the direction of antibody medical ingredients, peptide/protein ingredients, immunological disorders, etc., can solve the problems of cell death, neuron dysfunction and neuron loss, and potentially unreliable markers

Inactive Publication Date: 2010-04-15
ABGENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, dramatic upregulation of autophagy via Beclin 1 overexpression brings about cell death.
However since APG8A:GFP may aggregate independently of autophagocytosis, this is a potentially unreliable marker.
An improper clearance of proteins in these diseases may result either from a compromise in the autophagy degradation pathway or induce alterations in this pathway, and may result in neuron dysfunction and neuron loss.

Method used

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  • Regulation of apg8 phosphorylation and uses thereof
  • Regulation of apg8 phosphorylation and uses thereof
  • Regulation of apg8 phosphorylation and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of an APG8a Phosphospecific Polyclonal Antibody

[0252]A 16 amino acid phospho-peptide antigen, MPSDRPFKQRRS*FADRC (SEQ ID NO:156) (where S*=phosphoserine), corresponding to residues 1-16 of human APG8a (SEQ ID NO:1) plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin / Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, pages 75 76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264 283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962)).

[0253]This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per...

example 2

Western Blot Assay Using APG8a Antibody

[0255]The antibody described in Example 1 is tested for phosphospecificity in detection of cellular protein using Western blot assay. Jurkat and 293 cells are collected, washed with PBS and directly lysed in cell lysis buffer. The loading buffer is added into the respective cell lysate and the mixture is boiled at 100° C. for 5 minutes. 10-20 μg of lysate is added onto 7.5% SDS-PAGE gel. A standard Western blot is performed according to the Immunoblotting Protocol set out in the Abgent 2006-2008 Catalog and Technical Reference, p. 262. APG8a phosphospecific polyclonal antibody dilutions are optimized between 1:60 and 1:1000 from a stock concentration of 0.25 mg / mL. For each lysate, sample is incubated as follows: Lane 1, negative sera; Lane 2, phosphospecific APG8a antibody; Lane 3, phosphospecific APG8a antibody that is pre-incubated with phosphorylated peptide MPSDRPFKQRRS*FADRC (SEQ ID NO:156) (optimized in the range of 1:4 to 1:50 stoichiom...

example 3

Production of an APG8c Phosphospecific Polyclonal Antibody

[0256]A 16 amino acid phospho-peptide antigen, QKIPS*VRPFKQRC (SEQ ID NO:158) (where S*=phosphoserine), corresponding to residues 5-16 of human APG8c (SEQ ID NO:8) plus cysteine on the C-terminal for coupling, was constructed according to standard synthesis techniques using a Rainin / Protein Technologies, Inc., Symphony peptide synthesizer. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75 76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264 283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21 49 (1962)).

[0257]This peptide was coupled to KLH, and rabbits were injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (200 μg antigen per rabbit). The rabbits were boosted with same antigen in incomplete Freund adjuvant (100 μg antigen per rabbit) weekly for eight. Coincident with the dates of the third through eighth boosts, 15-20 mL bleed per rabbit...

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Abstract

The invention relates to proteins and peptides of the alpha isoform of light chain 3 (APG8) and antibodies to the APG8 protein, particularly antibodies that specifically bind to the alpha isoform of APG8 when either phosphorylated or not phosphorylated at serine-12 and antibodies that bind to the gamma isoform of APG8 when either phosphorylated or not phosphorylated at serine-9. The invention also relates to methods of producing these antibodies and use of these antibodies in the treatment of diseases related to autophagocytosis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61 / 082,174 filed Jul. 18, 2008 and U.S. Provisional Application Ser. No. 61 / 082,179 filed Jul. 18, 2008, the entire contents of which are herein incorporated by reference.REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB[0002]The entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:File NameDate of CreationSize (bytes)549132000500Seqlist.txtNov. 6, 200954,365 bytesTECHNICAL FIELD[0003]The claimed compositions, methods, and kits are directed to an antibody to detect the alpha and gamma isoforms of APG8 in either phosphorylated or nonphosphorylated form at an N-terminal serine resid...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K5/10C07K5/103C07K7/06C07K7/08C07K14/00C07K16/00A61P43/00
CPCC07K5/1008C07K5/101C07K5/1013C07K5/1016C07K16/44C07K5/1021C07K5/1024C07K5/1027C07K5/1019A61P37/02A61P43/00
Inventor WU, CHUNMOUNTZOURIS, JOHN A.HU, BINGRENLIU, CHUNLI
Owner ABGENT
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