Membrane-less structures are prevalent in cells and executing unique functions (e.g., DNA repair foci and nucleoli for making ribosomal subunits). The mechanisms by which their formation, size, number, and dynamics are regulated, however, remain unclear. Emergent studies revealed that their formation can be partly explained by a biophysical phenomenon known as liquid-liquid phase separation, whereby the nucleoplasm and cytoplasm are considered complex fluids that stably segregate like oil and water. Phase separation is often triggered when proteins bind to a common scaffold such as the nucleic acids DNA and RNA, resulting in the condensation of proteins to form higher-order structures. We previously discovered that an under-studied nucleic acid called poly(ADP-ribose) (PAR) is critical for the formation of a class of membrane-less organelles implicated in cancer, virus infection and neurodegeneration called stress granules. Stress granules are cytoplasmic RNA-protein assemblies formed in different sizes in response to stressors such as hypoxia, oxidative stress and heat shock. Most granule components dynamically exchange with the surrounding cytoplasm, and individual granules grow in size over time through fusion. Notably, stress granules in models of neuropathological diseases, such as amyotrophic lateral sclerosis (ALS), have slower exchange dynamics and are less able to fuse. However, the molecular factors that control the stress granule dynamics and fusion (which affects size and number of granules) remain poorly understood. In this proposal, we will (1) determine how PAR regulates phase separation in stress granules using innovative techniques to define critical parameters of ADP- ribosylation for stress granule formation in cells and in vitro, and (2) determine whether PAR- protein interactions regulate stress granule fusion using mutagenesis, live-cell imaging, biophysical methods and proteomics. Projected Impact: Besides its role in stress granules, PAR is also critical for the formation of time- and location-specific membrane-less structures, including DNA repair complexes and nucleoli. This proposal will thus advance the field by defining critical parameters for physiologically relevant PAR-mediated phase separation and by identifying ADP-ribosylated proteins required for these phenomena. Given that PARPs are druggable and actively targeted by pharmaceutical companies, next-generation inhibitors may be designed to modulate the formation and dynamics of physiological and pathological membrane-less structures in neurological or other diseases.

Public Health Relevance

s This proposal focuses on the regulation of a class of membrane-less structures implicated in cancer, virus infection and neurodegeneration called stress granules by an understudied, but therapeutically important, poly(ADP-ribose). Given that the enzyme family that controls poly(ADP-ribose) formation is druggable and actively targeted by pharmaceutical companies, next-generation inhibitors may be designed to modulate the formation and dynamics of physiological and pathological membrane-less structures in a range of diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Sakalian, Michael
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Johns Hopkins University
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McPherson, Robert Lyle; Ong, Shao-En; Leung, Anthony K L (2018) Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry. Methods Mol Biol 1813:271-283
Abraham, Rachy; Hauer, Debra; McPherson, Robert Lyle et al. (2018) ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication. Proc Natl Acad Sci U S A 115:E10457-E10466
Abraham, Rachy; McPherson, Robert Lyle; Sreekumar, Easwaran et al. (2018) Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation. Methods Mol Biol 1813:297-316
Leung, Anthony K L (2017) PARPs. Curr Biol 27:R1256-R1258
Leung, Anthony K L (2017) SERious Surprises for ADP-Ribosylation Specificity: HPF1 Switches PARP1 Specificity to Ser Residues. Mol Cell 65:777-778
Fischer, Joseph W; Leung, Anthony K L (2017) CircRNAs: a regulator of cellular stress. Crit Rev Biochem Mol Biol 52:220-233
Vivelo, Christina A; Wat, Ricky; Agrawal, Charul et al. (2017) ADPriboDB: The database of ADP-ribosylated proteins. Nucleic Acids Res 45:D204-D209
McPherson, Robert Lyle; Abraham, Rachy; Sreekumar, Easwaran et al. (2017) ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence. Proc Natl Acad Sci U S A 114:1666-1671
Daniels, Casey M; Ong, Shao-En; Leung, Anthony K L (2017) ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method. Methods Mol Biol 1608:79-93
McPherson, Robert Lyle; Leung, Anthony K L (2016) ADPr-ChAP: Mapping ADP-Ribosylation onto the Genome. Mol Cell 61:327-328

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