2013 — 2017 |
Zhang, Fangliang |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Cadherin-Based Cell Adhesions by Talin Proteolysis and Arginylation @ University of Miami School of Medicine
DESCRIPTION (provided by applicant): The cadherin-based adherens junction (AJ) is a critical cell-cell adhesion apparatus, misregulation of which leads to development failures and cancers. The formation of AJs is known to be dependent on the assembly of a local actin network, but the regulation of this remains unclear. Our preliminary data suggest that talin, a protein known for its role in the regulation of the actin network associated with integrin-based cell-matrix adhesion, is also involved in the AJ through a novel talin proteolytic fragment containing the vinculin- and actin- binding domains and a dimerizing region (referred as the VAD fragment). This fragment also undergoes posttranslational arginylation in vivo. We hypothesize that the proteolysis alters the domain architecture of talin, allowing the resulting VAD fragment to localize to the cadherin- based AJ. There the fragment enhances local actin assembly with its functional domains and stabilizes the AJ. Furthermore, arginylation changes the affinities of the VAD fragment to its binding partners and enhances its physiological function in AJs. In this proposed study we will test this hypothesis in these three specific aims: (1) How is the talin VAD fragment recruited to the cadherin-mediated AJ, and how does it enhance actin assembly? (2) Is the formation of cadherin-based AJs dependent on the VAD fragment? (3) How does arginylation enhance the function of the VAD fragment? Through this study, we will elucidate a novel regulatory mechanism for cadherin-based cell-cell adhesions mediated by the novel processing of talin, which was traditionally considered only associated with integrin-based adhesions. Also this project will provide new insight into how the conserved but poorly understood posttranslational arginylation modification affects protein function in cells.
|
0.939 |
2020 — 2021 |
Zhang, Fangliang |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Oxidative Stress Response and Metabolic Reprogramming by Protein Posttranslational Arginylation @ University of Miami School of Medicine
Oxidative stress response and metabolic reprogramming by protein posttranslational arginylation Posttranslational arginylation is the addition of one extra arginine to a protein. This modification often leads to rapid protein degradation. In fungi and animals, arginylation is solely mediated by Arginyltransferase1 (ATE1). Multiple lines of evidence from our group and others have shown that ATE1 and its arginylation activity are essential for cellular response to a variety of oxidative stressors and the associated metabolic reprogramming including glycolysis. However, the molecular mechanisms by which arginylation regulate oxidative stress response (OSR) and metabolism remain unknown. This gap of knowledge makes it difficult to devise approaches to intervene in arginylation for the prevention or treatment of diseases such as inflammation, cardiovascular abnormalities, cancer, and aging-related maladies, which are often derived from dysregulated OSR and the associated metabolic alterations. Following from our recent studies where we showed that ATE1 and arginylation activity are increased in cells under acute oxidative stress, and downregulated upon chronic exposures to stressors, we aim to understand exactly how arginylation influences OSR. The lack of understanding for arginylation is largely due to major technical challenges in the field for identifying the majority of arginylation substrates, which degrade rapidly. To overcome this problem, we redesign a new approach combining indirect methods to identify the impact of arginylation on protein stability and direct methods to identify arginylation modification on proteins. The power of this new approach was demonstrated in our preliminary screening, in which we identified several new arginylation candidates including hypoxia-inducible factor 1? (HIF1?), a critical regulator of OSR, glycolysis, and mitochondrial respiration. Our data further suggested that the functional role of HIF1a is regulated by arginylation. Following from this breakthrough, the objective of this proposed study is to reveal how arginylation regulates oxidative stress response and associated metabolic reprogramming by affecting critical proteins including HIF1?. We will apply and expand our newly developed approach to identify additional arginylated proteins (Aim1), elucidate the effects of arginylation on key substrates including HIF1? in regulating stress response/metabolism (Aim 2), and using high-throughput methods to characterize the global impact of arginylation on different cellular pathways (Aim 3). In the end of this study, we are expected to reveal major molecular mechanisms of how posttranslational arginylation of HIF1? and other critical proteins regulates OSR and metabolism. We would also uncover global impacts of arginylation, which is mediated by a single enzyme ATE1, in many cellular pathways. A long-lasting impact is further anticipated by the discoveries of many new and unexpected targets of arginylation.
|
0.939 |
2021 |
Zhang, Fangliang |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Targeted Degradation of Proteins by Affinity Peptide Conjugated Ubiquitin (Apcu) @ University of Miami School of Medicine
Targeted degradation of proteins by affinity peptide conjugated ubiquitin (APCU) Protein degradation possesses many unique properties that cannot be covered by DNA/RNA manipulation tools. To understand the mechanism of biological processes, and to achieve desirable therapeutic effects, techniques designed to induce protein degradation are in critical needs. However, very few options are currently available for such purposes. Also, the existing tools have known limitations such as difficulty for adaptation or bell-shaped dose-response curve, or vulnerability for a negative regulation by the de- ubiquitination system. To expand the option for tools, and to overcome limitations of current methods, we propose to establish a new approach to induce protein degradation by conjugating a ubiquitin moiety with a molecule of high affinity to a target protein. For a proof or concept, peptides will first be used as the affinity molecule. This prototype is thus called affinity peptide conjugated ubiquitin (APCU). In our preliminary study, we designed a prototype APCU molecule with a peptide targeting MCL1, an oncoprotein that is over-expressed in many types of cancer. We found that this molecule can specifically reduce the level of MCL1 protein by promoting its degradation. In the proposed study, we will determine the action mechanism of the APCU approach and the optimal conditions for designing this types of molecules. We will also determine the dose response curve of the MCL1-targeting APCU and its sensitivity of de-ubiquitination system. Furthermore, to prepare for a wide usage and future in vivo applications, we will test the application of APCU on several other proteins and explore the feasibility of forming an APCU molecule with a conditional conjugation strategy. This proposed study, if secedes, will provide a highly valuable tool to specifically degrade a protein for mechanistic investigations and for potential therapeutic usages.
|
0.939 |