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Chemically Probing and Regulating Misfolding and Aggregation of Intrinsically Disordered Proteins in Membraneless Organelles

Award Number: R35GM133484
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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PROJECT SUMMARY Membraneless organelles have important functions in cellular physiology and pathology. Recent studies show that these organelles are formed through liquid-liquid phase separation of intrinsically disordered proteins (IDPs) and RNA molecules. IDPs phase separate into liquid droplets in test tubes and form P bodies or stress granules in stressed cells. Both mutant and wild type forms of several IDPs are found aggregated in neurons and associated with neurodegenerative disorders. However, very little is known about how IDPs misfold and aggregate in these organelles and how this process can be regulated. Lack of this knowledge is attributed to the current method that is used to monitor membraneless organelles in live cells: this process is visualized through imaging fluorescent protein-tagged IDPs to analyze changes of their location and diffusion rate before and after organelle formation. Nonetheless, this method does not reveal whether IDPs misfold or aggregate within the organelle, because the morphology remains unchanged before and after IDPs aggregation. To overcome this challenge, the PI has developed a novel imaging method, hereinafter named AggTag (aggregation tag), to enable fluorogenic detection (turn-on fluorescence) of misfolded soluble oligomers both in test tubes and live cells. In this MIRA proposal, the PI plans to further develop the AggTag method with new probes that can distinguish soluble oligomers from insoluble aggregates using orthogonal fluorescent signals (Project 1). This unprecedented resolution will allow the PI to ask how IDPs misfold and aggregate in phase separated droplets. The PI have begun this direction with a focus on a group of intrinsically disordered RNA binding proteins (RBPs), which harbor RNA binding domains (RBD) and disordered prion-like domains (PLD). While PLD has been the primary focus in literatures, preliminary data have led to a novel hypothesis that whether RBD misfolds contributes to whether RBP misfolds during and after formation of droplets. This hypothesis will be tested both in vitro and in live cells, using a combination of the AggTag method and biochemical assays (Project 2). Finally, the PI will develop chemical strategies to control phase separation and membraneless organelles. Although LLPS can be prevented and dissolved by small molecules, disruption of the liquid droplets could obstruct their physiological functions. Till now, no small molecules have been discovered to promote formation of liquid droplets and prevent RBP misfolding. Preliminary data indicate that sugar phosphates are a novel class of molecules that promote droplet formation, stabilize liquid droplets, and prevent RBP misfolding in droplets. Based on these results, the PI will use efforts from multiple disciplines to understand the mechanisms underlying the observed effects of sugar phosphates and further develop them into a class of chemical regulators with proper selectivity and efficacy (Project 3). In summary, the proposed research will provide an enabling technology to visualize misfolding and aggregation of proteins in membraneless organelles and generate novel chemical compounds to regulate this disease-related process.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = -$291,384 )
2023	2021	THE PENNSYLVANIA STATE UNIVERSITY	201 OLD MAIN	UNIVERSITY PARK	PA	16802	CENTRE	USA	Biomedical Research and Research Training	000	3	6/13/2023	NON-COMPETING CONTINUATION	-$291,384
														Subtotal = -$291,384

Issue Date FY: 2022 ( Subtotal = $0 )
2022	2021	PENNSYLVANIA STATE UNIVERSITY, THE	201 OLD MAIN	UNIVERSITY PARK	PA	16802	CENTRE	USA	Biomedical Research and Research Training	000	3	9/23/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2021 ( Subtotal = $391,596 )
2021	2021	PENNSYLVANIA STATE UNIVERSITY, THE	201 OLD MAIN	UNIVERSITY PARK	PA	16802	CENTRE	USA	Biomedical Research and Research Training	000	3	8/4/2021	NON-COMPETING CONTINUATION	$391,596
														Subtotal = $391,596

Issue Date FY: 2020 ( Subtotal = $391,907 )
2020	2020	PENNSYLVANIA STATE UNIVERSITY, THE	201 OLD MAIN	UNIVERSITY PARK	PA	16802	CENTRE	USA	Biomedical Research and Research Training	000	2	7/1/2020	NON-COMPETING CONTINUATION	$391,907
														Subtotal = $391,907

Issue Date FY: 2019 ( Subtotal = $392,205 )
2019	2019	PENNSYLVANIA STATE UNIVERSITY, THE	201 OLD MAIN	UNIVERSITY PARK	PA	16802	CENTRE	USA	Biomedical Research and Research Training	000	1	8/9/2019	NEW	$392,205
														Subtotal = $392,205

Grand Total All Awards = $884,324

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Chemically Probing and Regulating Misfolding and Aggregation of Intrinsically Disordered Proteins in Membraneless Organelles

Award Number: R35GM133484

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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