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Developing a platform for incorporation of a fluorescent amino acid into proteins for live-cell imaging of protein trafficking events

Award Number: R15GM126576
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/Abstract It is estimated that thousands of diseases are caused by genetic mutation and subsequent aberrant protein localization within the cell, though pathological mechanisms are understood for only a fraction of all known mutations. Here we propose to engineer a widely-applicable platform which can visualize real-time protein trafficking events in living cells. First, we will rationally design a mutant library of Saccharomyces cerevisiae tryptophanyl-tRNA synthetases which will recognize a small coumarin fluorescent amino acid (ScCoumarin-RS). The S. cerevisiae enzyme is thought to be orthogonal to human tryptophan aminoacylation processes, yet still recognizable to the mammalian cell's translational machinery including elongation factors and the ribosome complex. We will also engineer a cognate S. cerevisiae amber suppressor coumarinyl tRNA, which will be charged with free coumarin by the ScCoumarin-RS. When cloned into a mammalian vector, these engineered biomolecules will insert the small coumarin fluorescent amino acid into a desired position within the polypeptide chain of the protein without perturbing its native structure or function. After confirming in cellulo functionality using a GFP reporter assay, we will apply the technology to a proof-of-concept mistrafficking cellular disease model of Léri-Weill dyschondrosteosis (LWD), a rare form of skeletal dysplasia caused by a genetic mutation in the SHOX gene. A single missense mutation abolishes the nuclear localization signal within the SHOXa transcription factor, and thus it accumulates in the cytoplasm instead of translocating to the nucleus. Our platform will visualize both the wild-type protein nuclear trafficking, as well as the mutant protein cytosolic localization. We will also quantify the kinetics of protein accumulation over an 18-hour time course. This novel platform technology has broad applicability to protein localization studies which can reveal the pathogenesis of mistrafficking pathways, validate suspected mistrafficking mutations, and enable a greater understanding of disease-related phenotypes at the subcellular level. The proposed project will allow two graduate students and two undergraduate students to perform research in the biomedical field. Many of these students are following a pre-med track coursework and would benefit from this unique opportunity to work at the leading edge of biomedical research, inspiring them to pursue biomedical careers.


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 = -$4,472 )
2023	2017	UNIVERSITY OF MASSACHUSETTS DARTMOUTH	285 OLD WESTPORT RD	NORTH DARTMOUTH	MA	02747	BRISTOL	USA	Biomedical Research and Research Training	000	1	10/1/2022	NEW	-$4,472
														Subtotal = -$4,472

Issue Date FY: 2022 ( Subtotal = $0 )
2022	2017	UNIVERSITY OF MASSACHUSETTS	285 OLD WESTPORT RD	NORTH DARTMOUTH	MA	02747	BRISTOL	USA	Biomedical Research and Research Training	000	1	3/9/2022	NEW	$0
														Subtotal = $0

Issue Date FY: 2021 ( Subtotal = $0 )
2021	2017	UNIVERSITY OF MASSACHUSETTS	285 OLD WESTPORT RD	NORTH DARTMOUTH	MA	02747	BRISTOL	USA	Biomedical Research and Research Training	000	1	7/23/2021	NEW	$0
														Subtotal = $0

Issue Date FY: 2017 ( Subtotal = $446,166 )
2017	2017	UNIVERSITY OF MASSACHUSETTS	285 OLD WESTPORT RD	NORTH DARTMOUTH	MA	02747	BRISTOL	USA	Biomedical Research and Research Training	001	1	9/15/2017	NEW	-$23,382
2017	2017	UNIVERSITY OF MASSACHUSETTS	285 OLD WESTPORT RD	NORTH DARTMOUTH	MA	02747	BRISTOL	USA	Biomedical Research and Research Training	000	1	9/13/2017	NEW	$469,548
														Subtotal = $446,166

Grand Total All Awards = $441,694

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Developing a platform for incorporation of a fluorescent amino acid into proteins for live-cell imaging of protein trafficking events

Award Number: R15GM126576

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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