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Impact of gamma-glutamyl carboxylase processivity on vitamin K-dependent protein modification and function in human health and disease

Award Number: R01HL152678
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Dietary vitamin K is used by the gamma-glutamyl carboxylase to convert clusters of Glus to gamma- carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins in virtually all tissues of the body. The first VKD proteins identified were coagulation factors; however, the identification of nonhemostatic VKD proteins has revealed additional roles, e.g. the regulation of calcification. Carboxylation activates VKD proteins by generating a calcium-binding module required for their function, and a single gamma-glutamyl carboxylase modifies all VKD proteins. Naturally occurring mutations in the carboxylase cause two diseases: vitamin K clotting factor deficiency 1 that is associated with severe bleeding defects, and pseudoxanthoma elasticum-like (PXE-like) that is associated with mild bleeding but excessive soft tissue calcification. How these carboxylase mutations cause PXE-like was previously unknown. We studied two carboxylase mutations present in a PXE- like patient. Analysis of a VKD clotting factor (factor IX) and a VKD protein that inhibits calcification (Matrix Gla Protein) revealed partial carboxylation due to a defect in carboxylase processivity. Processivity refers to the carboxylase remaining bound to a VKD protein until the multiple Glu residues are carboxylated. We developed a novel assay to monitor processive carboxylation, and found that the wild type carboxylase shields the VKD protein, i.e. limiting access of other VKD proteins into the active site until the VKD protein is extensively carboxylated. In contrast, the PXE-like mutants allowed promiscuous access of VKD protein substrates into the active site, resulting in the production of partially carboxylated VKD proteins. Our studies also revealed that a single wild type carboxylase binds two VKD proteins at the same time. As tissues express multiple VKD proteins thought to have widely different affinities, how full carboxylation of all VKD proteins is achieved is an open question. Our long-term goal is to understand how partial VKD protein carboxylation impacts human physiology. Central questions are whether treatment with the anticoagulant warfarin, which limits VKD protein carboxylation, generates partially carboxylated proteins, and whether warfarin evokes PXE-like phenotypes. We will approach these questions using a combination of protein mapping and activity assays to determine how partial carboxylation by PXE-like carboxylases impacts VKD protein function (Aim 1), determine whether the carboxylation of a VKD protein is impacted by the presence of a different VKD protein (Aim 2), and examine the consequence of warfarin therapy and a PXE-like mutant on VKD protein carboxylation and function in vivo (Aim 3). Results from these studies will provide the first insights that link the extent of protein carboxylation to different phenotypic outcomes.


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: 2024 ( Subtotal = $516,155 )
2024	2024	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE OF CASE WESTERN RESERVE UNIVERSITY	9500 EUCLID AVE.	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	4	6/3/2024	NON-COMPETING CONTINUATION	$516,155
														Subtotal = $516,155

Issue Date FY: 2023 ( Subtotal = $526,689 )
2023	2023	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE OF CASE WESTERN RESERVE UNIVERSITY	9500 EUCLID AVE.	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	3	6/12/2023	NON-COMPETING CONTINUATION	$526,689
														Subtotal = $526,689

Issue Date FY: 2022 ( Subtotal = $526,689 )
2022	2022	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE/CWRU	9500 EUCLID AVE	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	2	5/25/2022	NON-COMPETING CONTINUATION	$526,689
														Subtotal = $526,689

Issue Date FY: 2021 ( Subtotal = $526,689 )
2021	2021	CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE/CWRU	9500 EUCLID AVE	CLEVELAND	OH	44195	CUYAHOGA	USA	Blood Diseases and Resources Research	000	1	7/21/2021	NEW	$526,689
														Subtotal = $526,689

Grand Total All Awards = $2,096,222

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Impact of gamma-glutamyl carboxylase processivity on vitamin K-dependent protein modification and function in human health and disease

Award Number: R01HL152678

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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