Catalyzing the release of ADP-ribose from poly(ADP-ribose) or O-acetyl-ADP-ribose by contacting with an isolated ARH3 polypeptide; modifying DNA repair or chromatin structure by modifying ARH3 polypeptide activity; drug screening; antiinflammatory, antiischemic agents; aging resistance
Inventors
Moss, Joel • Oka, Shunya • KATO, Jiro • Zhu, Jianfeng • Kasamatsu, Atsushi
Assignees
US Department of Health and Human Services
Publication Number
US-7670806-B2
Publication Date
2010-03-02
Expiration Date
2026-09-12
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Abstract
This disclosure provides methods for catalyzing the release of ADP-ribose from poly(ADP-ribose) or O-acetyl-ADP-ribose. Also provided are methods for modifying DNA repair or chromatin structure by introducing into the cell an agent that modifies the activity of an ARH3 polypeptide, or variant or fragment thereof. Further provided are methods for screening molecules involved in the poly(ADP-ribosyl)ation of proteins or O-acetyl-ADP-ribose content, and method for treating disorders by altering activity of an ARH3 protein.
Core Innovation
This invention relates to methods for catalyzing the release of ADP-ribose from poly(ADP-ribose) or O-acetyl-ADP-ribose by contacting with an isolated ARH3 polypeptide, or a variant or fragment thereof. It further provides methods for modifying DNA repair or chromatin structure by introducing into a mammalian cell an agent that modifies the activity of an ARH3 polypeptide. The invention also includes methods for screening molecules that alter ARH3 hydrolysis activity or binding to ADP-ribose, and methods for treating disorders by altering ARH3 activity.
The background identifies the problem of regulation of ADP-ribose metabolism, which is involved in diverse critical cellular processes including DNA repair, carcinogenesis, cellular differentiation, and chromatin structure modulation. While poly(ADP-ribose) polymerases (PARPs) synthesize ADP-ribose polymers, only one poly(ADP-ribose) glycohydrolase (PARG) was known to degrade these polymers. Specific enzymes responsible for degrading signaling molecule O-acetyl-ADP-ribose were unclear beyond Nudix pyrophosphatases. Hence, there was a need for identifying additional enzymes capable of hydrolyzing poly(ADP-ribose) polymers and O-acetyl-ADP-ribose to regulate their biological effects and modulate associated processes such as DNA repair and chromatin structure.
The summary reveals that ARH3 protein has been discovered to possess poly(ADP-ribose) glycohydrolase activity and O-acetyl-ADP-ribose hydrolase enzymatic activity, catalyzing the release of ADP-ribose from both substrates. This activity allows ARH3 to regulate poly(ADP-ribose) levels and the Sir2-signaling molecule O-acetyl-ADP-ribose in cells. The invention includes producing ARH3 polypeptides with such hydrolase activity and using agents that modify ARH3 activity to alter biological processes including DNA repair, chromatin structure, apoptosis, and cellular differentiation. The methods have applications in treating cancer, disorders associated with excessive DNA damage, inflammation, and aging. Screening methods for molecules modulating ARH3 hydrolysis or ADP-ribose binding are also disclosed.
Claims Coverage
The claims include one independent claim covering methods of catalyzing ADP-ribose release using specified ARH3 polypeptides. Several dependent claims specify details about the polypeptide sequences and assay conditions. The main inventive features involve the use of ARH3 polypeptides with poly(ADP-ribose) glycohydrolase or O-acetyl-ADP-ribose hydrolase activity to catalyze ADP-ribose release.
Use of ARH3 polypeptides to catalyze ADP-ribose release
A method comprising contacting poly(ADP-ribose) or O-acetyl-ADP-ribose with an isolated ARH3 polypeptide that catalyzes the release of ADP-ribose, where the ARH3 polypeptide comprises specific amino acid sequences including SEQ ID NO: 2, 3, 4, or 6, or variants with at least 95% identity having hydrolase activity.
Inclusion of magnesium to enhance hydrolysis
The method further comprises adding magnesium to the poly(ADP-ribose) or O-acetyl-ADP-ribose to enhance ARH3 enzymatic activity.
Polypeptides with defined sequence identity and mutations
Claims specify ARH3 polypeptides consisting of or comprising amino acid sequences at least 95%, 98%, or 99% identical to SEQ ID NO: 2, including variants with glutamic acid to glutamine substitutions at positions 261 and 262 or 238 and 239, maintaining hydrolase activity.
Assaying enzymatic activity and ADP-ribose release
The methods include assaying poly(ADP-ribose) glycohydrolase activity or measuring the release of ADP-ribose to confirm enzymatic function of ARH3 polypeptides.
The claims cover the inventive use of isolated ARH3 polypeptides with specific or closely related amino acid sequences possessing poly(ADP-ribose) glycohydrolase or O-acetyl-ADP-ribose hydrolase activity to catalyze ADP-ribose release from corresponding substrates, optionally with magnesium addition, and include assay methods for activity confirmation.
Stated Advantages
ARH3 regulates poly(ADP-ribose) and O-acetyl-ADP-ribose levels affecting critical biological processes such as DNA repair, apoptosis, chromatin structure, and cellular differentiation.
Activation or inhibition of ARH3 provides therapeutic potential in treating cancer, disorders associated with excessive DNA damage, inflammation, graft-versus-host disease, transplant rejection, allergy, inflammatory arthritis, and atherosclerosis.
The methods enable screening molecules that modulate ARH3 hydrolysis activity or ADP-ribose binding, facilitating identification of new therapeutic agents.
ARH3 activity modulates Sir2-dependent signaling pathways and can influence aging and longevity.
Documented Applications
Treating cancer and radiosensitizing tumor cells by modulation of ARH3 activity.
Modifying DNA repair, chromatin structure, apoptosis, and cellular differentiation in mammalian cells by altering ARH3 activity.
Treating inflammation-related disorders including graft-versus-host disease, transplant rejection, allergy, inflammatory arthritis, and atherosclerosis by increasing ARH3 activity.
Screening molecules for use in altering cellular processes involving poly(ADP-ribosyl)ation and Sir2-related pathways, useful in cancer and DNA damage disorder therapies.
Enhancing aging resistance by modulating ARH3 activity as it degrades O-acetyl-ADP-ribose, a Sir2 reaction product.
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