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Molecular mechanisms underlying the assembly of the human proteasome and endogenous protein complexes

Award Number: R35GM147487
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/01/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2027

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Molecular mechanisms underlying the assembly of the human proteasome and endogenous protein complexes - Project Summary Advances in structural biology techniques including single particle cryogenic electron microscopy (cryo-EM) have enabled unprecedented molecular insights into the function of biological macromolecules. However, the study of many proteins and ribonucleoprotein complexes remains challenging due to current limitations in sample preparation approaches. Traditionally, proteins of interest are produced in over-expression systems within bacterial, insect, and mammalian cell lines. While this approach can allow the production and purification of proteins with high yields, it often requires substantial optimization that can limit the study of biomedically important membrane proteins and large protein complexes, especially where specific chaperones and cellular conditions are required that are difficult to replicate in vitro. To overcome these limitations, we are developing methodology to efficiently tag and purify endogenous proteins by leveraging advances in CRISPR/Cas gene editing. This approach enables us to investigate macromolecular complexes and their intricate assembly pathways under native and context-specific conditions that are relevant to human health and disease. We are interested in developing and applying the approach in three major areas of study: constitutive protein complexes, cell-type specific macromolecular assemblies, and cell state dependent membrane protein complexes. Using the proteasome as a model system, we will investigate the assembly pathway of proteasomal complexes by cryo-EM and mass spectrometry. This work will provide mechanistic insights into critical protein degradation machinery and help to establish important methodology for the study of endogenous protein assemblies. Next, we will expand our approach to the study of protein complexes in different cell types, including hematopoietic and epithelial cells. This goal will be achieved by developing efficient strategies to optimize CRISPR/Cas gene editing in specialized cell types, which will constitute an important step towards the study of proteins in their native states. Finally, we will examine the conditional assembly of protein complexes and membrane protein assemblies. For this direction, we will investigate proteins involved in nutrient sensing at the lysosome in conjunction with mTOR signaling. This work will provide molecular insights into the mechanisms regulating key metabolic pathways and shed light on how mTOR integrates different signals to promote cell growth and proliferation. Additionally, we will establish protocols for screening cellular and biochemical conditions to acquire context-specific protein assemblies. Altogether, these studies will provide mechanistic insights into remarkable molecular machines and develop important methodology that can be applied to the study of other biological systems. These methods will enable us to unravel the molecular mechanisms underlying the function of proteins in specific cellular environments and help advance structural biology towards understanding how biological macromolecules work in their native contexts.


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: 2025 ( Subtotal = $487,500 )
2025	2025	SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE	10901 N TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	4	7/29/2025	NON-COMPETING CONTINUATION	$487,500
														Subtotal = $487,500

Issue Date FY: 2024 ( Subtotal = $487,500 )
2024	2024	SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE	10901 N TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	3	7/23/2024	NON-COMPETING CONTINUATION	$487,500
														Subtotal = $487,500

Issue Date FY: 2023 ( Subtotal = $487,500 )
2023	2023	SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE	10901 N TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	2	7/20/2023	NON-COMPETING CONTINUATION	$487,500
														Subtotal = $487,500

Issue Date FY: 2022 ( Subtotal = $487,500 )
2022	2022	SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE	10901 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	1	7/22/2022	NEW	$487,500
														Subtotal = $487,500

Grand Total All Awards = $1,950,000

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Molecular mechanisms underlying the assembly of the human proteasome and endogenous protein complexes

Award Number: R35GM147487

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 08/01/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2027

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