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Regulation of autophagy pathway phosphorylation and uses thereof

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Regulation of autophagy pathway phosphorylation and uses thereof


The invention relates to polypeptides and proteins known to function in the autophagy pathway that have novel phosphorylation sites. The invention also relates to antibodies specific to these polypeptides and proteins that are phosphorylated or not phosphorylated at novel phosphorylated sites. The invention also relates to methods of producing these antibodies and use of these antibodies in the treatment of diseases related to autophagocytosis.

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Inventors: Chun Wu, John A. Mountzouris, Bingren Hu, Chunli Liu
USPTO Applicaton #: #20120258550 - Class: 436501 (USPTO) - 10/11/12 - Class 436 
Chemistry: Analytical And Immunological Testing > Biospecific Ligand Binding Assay



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The Patent Description & Claims data below is from USPTO Patent Application 20120258550, Regulation of autophagy pathway phosphorylation and uses thereof.

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/505,281, now U.S. Pat. No. 8,148,088, which 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 disclosures of which are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

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 Name Date of Creation Size (bytes) 549132000401Seqlist.txt May 30, 2012 1,032,007 bytes

TECHNICAL FIELD

The claimed compositions, methods, and kits are directed to antibodies to detect autophagy proteins in either the phosphorylated or nonphosphorylated forms.

BACKGROUND ART

Autophagy is a process whereby cells convert proteins and organelles into amino acids as a source of food. Many cells in the human body rely on autophagy to maintain homeostasis, especially when insulin levels are low. Autophagocytosis may play a role in human disease and aging. In eukayrotic cells autophagy occur constitutively at low levels in all cells to perform housekeeping functions such as destruction of dysfunctional organelles. Dramatic upregulation occurs (e.g., cytoplasmic and organelle turnover) in the presence of external stressors (starvation, hormonal imbalance, oxidation, extreme temperature, and infection), and internal needs (generation of source materials for architectural remodeling, removal of protein aggregates). Autophagy is highly regulated through the coordinated action of various kinases, phosphatases, and guanosine triphosphatases (GTPases).

At least three different autophagy mechanisms are known, all of which result in targeting of cytosolic proteins and organelles to the lysosome in order to provide amino acids and energy in the form of catabolites. These types are macroautophagy, microautophagy, and chaperone-mediated autophagy.

Macroautophagy is a major inducible pathway for the general turnover of cytoplasmic constituents in eukaryotic cells and also plays a significant role in the degradation of active cytoplasmic enzymes and organelles during nutrient starvation. Macroautophagy involves the formation of double-membrane bound autophagosomes which enclose the cytoplasmic constituent targeted for degradation in a membrane bound structure, which then fuse with the lysosome (or vacuole) releasing a single-membrane bound autophagic bodies which are then degraded within the lysosome (or vacuole). MAP1A and MAP1B are microtubule-associated proteins which mediate the physical interactions between microtubules and components of the cytoskeleton. These proteins are involved in formation of autophagosomal vacuoles (autophagosomes). MAP1A and MAP1B each consist of a heavy chain subunit and multiple light chain subunits. Apg8a is one of the light chain subunits and can associate with either MAP1A or MAP1B. The precursor molecule is cleaved by APG4B/ATG4B to form the cytosolic form, Apg8a-I. This is activated by APG7L/ATG7, transferred to ATG3 and conjugated to phospholipid to form the membrane-bound form, Apg8a-II.

Microautophagy circumvents the autophagosomic step of macrophagy, and begins with the direct uptake of cytosolic material via invaginations and pinching off of the lysosomal membrane. The internalized cytosolic components are digested by lysosomal enzymes released when the vacuolar membrane disintegrates, as in macroautophagy.

In chaperone-mediated autophagy, specific chaperone proteins bind to target proteins containing a KFERQ (SEQ ID NO: 1) sequence and channel these proteins to the surface of the lysosome. These proteins bind to Lamp2a and are then transported across the lysosomal membrane with the assistance of lysosomal chaperones, after which they are degraded by vacuolar proteases.

Mizushima, et al., describes autophagy as promoting both cell survival and cell death. By maintaining homeostasis during times of cellular stress, autophagy generally promotes survival when it is controlled. See Mizushima, N., et al., “Autophagy fights disease through cellular self-digestion” Nature (2008) 451:1069-1075.

However, dramatic upregulation of autophagy via Beclin 1 overexpression brings about cell death. Mizushima, et al., describe how the autophagy and apoptosis pathways share many common regulatory factors, with the likelihood of significant cross-talk between these pathways in the cell. Since apoptosis is known to be implicated in human disease, autophagy also is likely an important phenomenon to target in order to treat disease.

Proteins that regulate autophagy in cancer cells make attractive therapeutic and diagnostic targets. Cancer cells rely on autophagy in order to evade anti-cancer treatments designed to reduce nutrient supply and enhance the stress on rapidly dividing cells. A compound that downregulates autophagy may be a useful additional drug in cancer treatment. Mizushima, et al., state that since autophagy may help prevent cancer, there is a potential need to target autophagy in a context-specific manner. The targeting of specific autophagy regulatory proteins rather than a targeting of autophagy in general may be critical in developing a treatment of cancer as well as new modes of diagnosing cancer.

Alterations in the autophagy degradation pathway have been described in normal brain aging and in age-related neurodegenerative diseases including Alzheimer's and Parkinson's diseases. See Nixon, R., “Autophagy in neurodegenerative disease: friend, foe, or turncoat?” Trends in Neurosciences (2006) 29(9):528-535. An improper clearance of proteins in these diseases may result either from a compromise in the autophagy degradation pathway or induced alterations in this pathway, and may result in neuron dysfunction and neuron loss. The targeting of specific autophagy regulatory proteins, rather than a targeting of autophagy in general, may be critical in developing a treatment of neurodegenerative diseases as well as new modes of diagnosing neurodegenerative diseases. Therefore, there exists a need to develop an assay to monitor the activity of autophagy proteins that does not rely exclusively on protein localization.

DISCLOSURE OF THE INVENTION

Several of the following aspects provide peptides comprising amino acids that are novel phosphorylation sites on various autophagy proteins. Several of the following aspects also provide antibodies that react specifically to the phosphorylated forms of these proteins, and antibodies that react specifically to the non-phosphorylated forms of these proteins.

In one aspect, the present disclosure provides an isolated autophagy peptide that comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 1, wherein the x residue is nonphosphorylated or phosphorylated serine, threonine, or tyrosine, and with the proviso that the peptide is not a full-length autophagy protein comprising an amino acid sequence set forth in Table 2. The peptides of this embodiment are referred to as “autophagy peptides”.

In some embodiments, an isolated peptide of the present disclosure comprises an amino sequence found in Table 3. The peptides of this embodiment are a subset of possible autophagy peptides.

In another embodiment, isolated peptides of the present disclosure comprise an amino acid sequence found in Table 4. In some embodiments, the x residue may be phosphorylated. In some embodiments, the x residue may be nonphosphorylated.

In another aspect, the disclosure provides for an immunogen, which comprises an autophagy peptide and immune response potentiator.

In another aspect, the disclosure provides for a multiple antigenic peptide (MAP), which comprises a branched oligolysine core conjugated with a plurality of isolated autophagy peptides.

In another aspect, the disclosure provides for a method for producing an antibody to an autophagy polypeptide. The method comprises introducing an isolated autophagy peptide comprising a sequence set forth in Table 1 to a mammal in an amount sufficient to produce an antibody to the autophagy peptide; and recovering the antibody from the mammal.

In another aspect, the disclosure provides for a kit for producing an antibody to an autophagy polypeptide. The kit comprises an isolated autophagy peptide comprising a sequence set forth in Table 1, a means for introducing the isolated autophagy peptide to a mammal in an amount sufficient to produce an antibody to the autophagy peptide, and a means for recovering the antibody from the mammal.

In another aspect, the disclosure provides for a method for producing an antibody to an autophagy polypeptide. The method comprises introducing an autophagy protein to a mammal in an amount sufficient to produce an antibody to the autophagy protein, recovering the antibody from the mammal, and affinity purifying an autophagy antibody that specifically binds to an epitope of the sequence in Table 3. This method is referred to below as the “method for producing an affinity-purified autophagy antibody” In one embodiment, the disclosure provides for an antibody to an autophagy polypeptide produced by this method.

In another aspect, the disclosure provides for a kit for producing an antibody to an autophagy polypeptide. The kit comprises an autophagy protein, a means for introducing the autophagy protein to a mammal in an amount sufficient to produce an antibody to the autophagy polypeptide, a means for recovering the antibody from the mammal, and an isolated autophagy peptide comprising a sequence from Table 1.

In another aspect, the disclosure provides for an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x (also referred to herein as the “X residue”) in an amino acid sequence set forth in Table 3, wherein the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine. In one embodiment, the epitope comprises the amino acid residue x in one amino acid sequence set forth in Table 3 and an amino acid residue of autophagy protein that is outside the same amino acid sequence set forth in Table 3. In another embodiment, the epitope comprises the amino acid residue x in one amino acid sequence set forth in Table 3 and amino acid residues of an autophagy protein that are outside the same amino acid sequence set forth in Table 3.

In another aspect, the disclosure provides for a method for detecting an autophagy protein or fragment comprising an amino acid sequence set forth in Table 4, wherein the x residue is either phosphorylated or nonphosphorylated serine, threonine, or tyrosine, in a sample. The method comprises the following steps. First, a sample containing or suspected of containing an autophagy protein or fragment comprising the amino acid sequence set forth in Table 4, wherein the x residue is either phosphorylated or nonphosphorylated, is contacted with an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein x is either phosphorylated or nonphosphorylated. The next step is assessing a complex formed between the autophagy protein or fragment, if present in the sample, and the antibody, to determine the presence, absence and/or amount of the autophagy protein or fragment in the sample.

In another aspect, the disclosure provides for a kit for detecting an autophagy protein or fragment comprising amino acid sequence set forth in Table 4 wherein x is serine or phosphoserine in a sample, which kit comprises, in a container, an isolated antibody that specifically binds to an epitope that comprises the amino acid residue x in the amino acid sequence set forth in Table 4, wherein x is serine or phosphoserine.

In another aspect, the disclosure provides for a method for treating a disease or disorder associated with abnormal phosphorylation status of an autophagy protein or fragment comprising amino acid sequence set forth in Table 4 wherein the x residue is either phosphorylated or nonphosphorylated, which method comprises administering, to a subject when such a treatment is needed or desired, a sufficient amount of an isolated antibody that specifically binds to an epitope that comprises the x residue in the amino acid sequence set forth in Table 4, wherein x is either phosphorylated or nonphosphorylated.

Another aspect is a method for identifying a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing autophagy polypeptide comprising an amino acid sequence listed in Table 1, wherein x is nonphosphorylated serine, threonine, or tyrosine; (2) contacting the autophagy polypeptide with a test protein and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide; and (3) assessing the phosphorylation status of the autophagy polypeptide to determine whether the test protein is a kinase for the autophagy protein on the x residue.

Another aspect is a method for identifying a modulator of a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence selected from a sequence listed in Table 1, wherein the x residue is not phosphorylated; (2) contacting the autophagy polypeptide with a kinase that phosphorylates the protein on the residue indicated by x and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide in the presence or absence of a test substance; and assessing and comparing phosphorylation status of the autophagy polypeptide by the kinase to determine whether the test substance modulates the kinase.

Another aspect is a method for identifying a phosphatase that dephosphorylates an autophagy protein on a phosphorylated x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence selected from the group consisting of a sequence listed in Table 1, wherein x is phosphorylated; (2) contacting the autophagy polypeptide with a test protein and H2O under conditions suitable for the dephosphorylation of the phosphoserine residue of the autophagy polypeptide; and (3) assessing phosphorylation status of the autophagy polypeptide to determine whether the test protein is a phosphatase for the autophagy polypeptide on the x residue.

Another aspect is a method for identifying a modulator of a kinase that phosphorylates an autophagy protein on the x residue. The method comprises the steps of: (1) providing an autophagy polypeptide comprising an amino acid sequence listed in Table 1, wherein x is nonphosphorylated serine, threonine, or tyrosine; (2) contacting the autophagy polypeptide with a kinase that phosphorylates an autophagy polypeptide on the x residue and ATP under conditions suitable for the phosphorylation of the x residue of the autophagy polypeptide in the presence or absence of a test substance; and (3) assessing and comparing the phosphorylation status of the autophagy polypeptide by the kinase to determine whether the test substance modulates the kinase.

Another aspect is an isolated nucleic acid fragment which is comprised of a sequence of nucleotides encoding an autophagy peptide comprising an amino acid sequence selected from the group consisting of sequences set forth in Table 1, wherein the x residue is nonphosphorylated serine, threonine, or tyrosine. The autophagy peptide is not a full-length autophagy protein comprising an amino acid sequence set forth in Table 2. The nucleic acid may be DNA. The nucleic acid may also be RNA.

Other objects, features, and technical advantages of the present invention will become more apparent from a consideration of the detailed description herein and from the accompanying drawings.

All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a dot blot of an antibody that specifically binds to APG3L that is phosphorylated at tyrosine-18.

FIG. 2 shows a dot blot of an antibody that specifically binds to APG4A that is phosphorylated at serine-100.

FIG. 3 shows a dot blot of an antibody that specifically binds to APG4C that is phosphorylated at serine-166.

FIG. 4 shows a dot blot of an antibody that specifically binds to APG4C that is phosphorylated at serine-177.

FIG. 5 shows a dot blot of an antibody that specifically binds to APG4C that is phosphorylated at serine-398.

FIG. 6 shows a dot blot of an antibody that specifically binds to APG4C that is phosphorylated at serine-451.

FIG. 7 shows a dot blot of an antibody that specifically binds to APG4D that is phosphorylated at serine-15.

FIG. 8 shows a dot blot of an antibody that specifically binds to APG4D that is phosphorylated at serine-341.

FIG. 9 shows a dot blot of an antibody that specifically binds to APG4D that is phosphorylated at serine-467.

FIG. 10 shows a dot blot of an antibody that specifically binds to APG7L that is phosphorylated at serine-95.

FIG. 11 shows a dot blot of an antibody that specifically binds to APG9L1 that is phosphorylated at serine-735.

FIG. 12 shows a dot blot of an antibody that specifically binds to APG16L that is phosphorylated at serine-213.

FIG. 13 shows a dot blot of an antibody that specifically binds to APG16L that is phosphorylated at serine-287.



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stats Patent Info
Application #
US 20120258550 A1
Publish Date
10/11/2012
Document #
13438770
File Date
04/03/2012
USPTO Class
436501
Other USPTO Classes
530330, 5303879
International Class
/
Drawings
13


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