Posttranslational modification of proteins in signal pathways is one of the most pivotal control points in vivo for the activation, inactivation, and degradation of factors governing nearly all physiological processes. The goal of this proposal is to define the molecular mechanism and in vivo functions of protein arginylation, a very poorly understood post-translational modification that affects an estimated 25% of the cellular proteome. Arginylation plays a key role in cell migration and developmental morphogenesis, and is implicated in major events of cell metabolism, physiology, and human disease. Our preliminary data strongly suggest that arginylation regulates its protein targets through rapid and reversible modulation of their activity and protein interactions and constitutes a truly essential and dynamic biological regulator, however virtually nothing is known about the substrate site specificity and the molecular mechanisms of this reaction. This proposal will address the mechanisms of the arginyl transfer enzyme (ATE1) and the regulation of its substrate specificity through an integrated approach that will provide the first mechanistic insights into this enigmatic posttranslational modification. We propose the following specific aims: (1) To determine site specificity and properties of N- terminal arginylation;(2) To test the mechanisms of arginylation at internal protein sites in intact proteins;and (3) To test the effect of cellular components on he rate and site specificity of arginylation by different ATE1 isoforms and modulate its activity in vivo. Together, the proposed studies will elucidate the molecular mechanism of a novel posttranslational modification with major biological significance. These studies will ultimately enable differential modulation of ATE1 activity and biological targeting, essential for exploring is therapeutic potential in critical arginylation-dependent human conditions, including heart disease, cancer, neurodegeneration, and aging. The results of our studies will open new possibilities of functional arginylation analysis and targeted manipulation of arginylation of key proteins during essential processes in normal physiology and disease.

Public Health Relevance

Posttranslational modifications play a key role in protein functions, and their abnormalities lead to severe disease states, including heart disease and cancer - the two leading causes of death in the United States. The current proposal will delineate the mechanistic basis for arginylation, a novel posttranslational modification implicated in heart disease and cancer, with the goal of enabling the development of new therapeutics that modulate different arginylation-dependent mechanisms in disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104003-02
Application #
8727072
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
2013-09-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$312,968
Indirect Cost
$95,760
Name
University of Pennsylvania
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Kashina, Anna (2014) Protein arginylation, a global biological regulator that targets actin cytoskeleton and the muscle. Anat Rec (Hoboken) 297:1630-6
Cornachione, Anabelle S; Leite, Felipe S; Wang, Junling et al. (2014) Arginylation of myosin heavy chain regulates skeletal muscle strength. Cell Rep 8:470-6
Wang, Junling; Han, Xuemei; Wong, Catherine C L et al. (2014) Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo. Chem Biol 21:331-7