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Structural and functional assessment of APOBEC3G macromolecular complexes
Bogdan Polevoda1, William M McDougall1, Ryan P Bennett2
1Department of Biochemistry and Biophysics, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Methods (San Diego, Calif.)
|March 19, 2016
Summary
The human APOBEC protein family, crucial for cellular functions, forms complex structures through interactions with DNA and RNA. Understanding these macromolecular interactions using cross-linking methods is key to their diverse roles.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The human APOBEC (Apolipoprotein B mRNA Editing Enzyme, Catalytic Polypeptide-like) protein family comprises eleven evolutionarily related members with diverse functions.
- These proteins, originating from gene duplication events, exhibit varied enzymatic and non-enzymatic roles within cells.
- APOBEC protein functionality is intrinsically linked to their ability to form higher-order structures via protein-protein and protein-nucleic acid interactions.
Purpose of the Study:
- To review and exemplify the application of cross-linking methods for characterizing macromolecular interactions of APOBEC proteins.
- To elucidate the functional implications of these interactions, using APOBEC3G as a specific research example.
- To propose the general applicability of these methods for studying other DNA and RNA editing/modifying proteins.
Main Methods:
- Utilized protein-protein and protein-nucleic acid cross-linking techniques.
- Integrated mass spectrometry, electrophoretic mobility shift assays (EMSA), glycerol gradient sedimentation, and fluorescence anisotropy.
- Employed APOBEC deaminase assays to quantify functional outcomes of identified interactions.
Main Results:
- Demonstrated the utility of cross-linking coupled with various biochemical assays in mapping critical interacting surfaces.
- Provided insights into how macromolecular interactions govern APOBEC subcellular localization, complex formation, and activity modulation.
- Successfully characterized interactions essential for processes including gene targeting, antiviral activity, and protein degradation.
Conclusions:
- Macromolecular interactions are fundamental drivers of APOBEC protein function, including subcellular compartmentalization and holoenzyme formation.
- Cross-linking methodologies offer powerful tools for dissecting these complex interactions and their functional consequences.
- The described methods are broadly applicable to the study of other nucleic acid editing and modifying enzymes within the APOBEC family and beyond.
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