Life Sciences Commons^™

Purification Of 26s Proteasomes And Their Subcomplexes From Plants, Richard S. Marshall, David C. Gemperline, Richard D. Vierstra

Biology Faculty Publications & Presentations

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type …

Modulating Parkin E3 Ubiquitin Ligase Activity Using Phospho-Ubiquitin Variants, Susanna George

Electronic Thesis and Dissertation Repository

Parkin is a Parkinson’s disease-linked E3 ubiquitin (Ub) ligase that promotes mitophagy by ubiquitination of mitochondrial outer membrane proteins. Phosphorylation of Ub at Ser65 by the PTEN-induced putative kinase 1 activates parkin. The role of other Ub phosphorylation sites and the associated kinases remain unknown. We optimized genetic code expansion to produce pure site-specfically phosphorylated Ub (pUb) variants (pUb^S7, pUb^S12, pUb^S20, pUb^S65) and investigated their activity in a key neurodegenerative pathway. Purification of pUb^S7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was …

Autophagic Turnover Of Inactive 26s Proteasomes In Yeast Is Directed By The Ubiquitin Receptor Cue5 And The Hsp42 Chaperone, Richard S. Marshall, Fionn Mcloughlin, Richard D. Vierstra

Biology Faculty Publications & Presentations

Highlights

• The yeast 26S proteasome is degraded by Atg8-mediated autophagy
• Nitrogen starvation and inactivation stimulate proteaphagy via distinct pathways
• Proteasome inhibition is accompanied by extensive ubiquitylation of the complex
• Proteaphagy engages the Cue5 autophagy receptor and the Hsp42 chaperone

Summary

The autophagic clearance of 26S proteasomes (proteaphagy) is an important homeostatic mechanism within the ubiquitin system that modulates proteolytic capacity and eliminates damaged particles. Here, we define two proteaphagy routes in yeast that respond to either nitrogen starvation or particle inactivation. Whereas the core autophagic machineries required for Atg8 lipidation and vesiculation are essential for both routes, the upstream Atg1 …

The San1 Ubiquitin Ligase Functions Preferentially With Ubiquitin-Conjugating Enzyme Ubc1 During Protein Quality Control, Rebeca Lea Ibarra

UNLV Theses, Dissertations, Professional Papers, and Capstones

Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system (UPS). In the UPS, ubiquitin-conjugating enzymes and ubiquitin ligases append poly-ubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount since a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, …

Roles Of Ubiquitin And Stress In Diacetyl Chemosensation Of C. Elegans, Ellen Zocher, Nelson Ruth, Marissa Hogg

Scholars Week

Ubiquitin is a small protein that can be attached to other proteins in a cell, tagging them for destruction. The process of adding ubiquitin to a protein substrate (ubiquitination), and the subsequent trafficking and degradation of this substrate, is a principle regulator of the abundance and activity of many proteins across all forms of life. We are examining the role and dynamics of this regulatory system in the olfactory neurons of the model organism Caenorhabditis elegans, specifically the olfactory receptor protein ODR-10, which allows the worm to detect diacetyl, a volatile compound that is produced by the bacteria the worm …

Regulation Of Ampa-Type Glutamate Receptor Homolog Glr-1 By Erad Ubiquitin Ligases In C. Elegans, Sam Witus

Scholars Week

Endoplasmic reticulum-associated degradation (ERAD) maintains cellular health by removing misfolded proteins from the endoplasmic reticulum (ER). ERAD is ubiquitin-dependent, and ubiquitination of target proteins can be catalyzed by ER-resident E3 ubiquitin ligases. In C. elegans, genes for three putative ERAD E3 ubiquitin ligases have been identified: hrd-1, hrdl-1, and marc-6 (HRD-1, GP78/AMFR, and MARCH-6 in mammalian systems). In C. elegans, these three genes cooperate to maintain the overall health of animals during ER stress. We are testing the roles of hrd-1, hrdl-1, and marc-6 in the neurons of C. elegans. GLR-1 is a glutamate receptor that is expressed in a …

In Vitro Investigation Of The Effect Of Exogenous Ubiquitin On Processes Associated With Atherosclerosis, Chase W. Mussard

Undergraduate Honors Theses

Atherosclerosis, characterized by the build-up of cholesterol, immune cells and cellular debris within arterial walls, is accelerated following myocardial infarction by poorly understood mechanisms. Ubiquitin, a small, well-studied intracellular protein involved in protein turnover via the proteasome pathway, has recently been shown to exert extracellular effects on cardiac myocytes, in vitro, and in mice undergoing myocardial remodeling. This study investigates the potential role of extracellular ubiquitin in atherosclerosis by determining its effects on two critical atherosclerotic processes: the migration of vascular smooth muscles cells and the uptake of modified LDL by monocyte/macrophages in foam cell formation. In the presence …

Investigation Of Respiratory Syncytial Virus Structural Determinants And Exploitation Of The Host Ubiquitin System, Jillian Nicole Whelan

USF Tampa Graduate Theses and Dissertations

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal …

Ubiquitin-Specific Peptidase 10 (Usp10) Deubiquitinates And Stabilizes Muts Homolog 2 (Msh2) To Regulate Cellular Sensitivity To Dna Damage, Mu Zhang, Chen Hu, Dan Tong, Shengyan Xiang, Kendra Williams, Wenlong Bai, Guo-Min Li, Gerold Bepler, Xiaohong Zhang

Toxicology and Cancer Biology Faculty Publications

MSH2 is a key DNA mismatch repair protein, which plays an important role in genomic stability. In addition to its DNA repair function, MSH2 serves as a sensor for DNA base analogs-provoked DNA replication errors and binds to various DNA damage-induced adducts to trigger cell cycle arrest or apoptosis. Loss or depletion of MSH2 from cells renders resistance to certain DNA-damaging agents. Therefore, the level of MSH2 determines DNA damage response. Previous studies showed that the level of MSH2 protein is modulated by the ubiquitin-proteasome pathway, and histone deacetylase 6 (HDAC6) serves as an ubiquitin E3 ligase. However, the deubiquitinating …

Role Of Deubiquitinating Enzymes In Dna Repair, Younghoon Kee, Tony T. Huang

Molecular Biosciences Faculty Publications

Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling.

AΒ40 Reduces P-Glycoprotein At The Blood-Brain Barrier Through The Ubiquitin-Proteasome Pathway, Anika M. S. Hartz, Yu Zhong, Andrea Wolf, Harry Levine Iii, David S. Miller, Björn Bauer

Sanders-Brown Center on Aging Faculty Publications

Failure to clear amyloid-β (Aβ) from the brain is in part responsible for Aβ brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aβ across the blood–brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood–brain barrier in AD, which has been shown to be associated with Aβ brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We …

It Is All About (U)Biquitin: Role Of Altered Ubiquitin-Proteasome System And Uchl1 In Alzheimer Disease, Antonella Tramutola, Fabio Di Domenico, Eugenio Barone, Marzia Perluigi, D. Allan Butterfield

Chemistry Faculty Publications

Free radical-mediated damage to macromolecules and the resulting oxidative modification of different cellular components are a common feature of aging, and this process becomes much more pronounced in age-associated pathologies, including Alzheimer disease (AD). In particular, proteins are particularly sensitive to oxidative stress-induced damage and these irreversible modifications lead to the alteration of protein structure and function. In order to maintain cell homeostasis, these oxidized/damaged proteins have to be removed in order to prevent their toxic accumulation. It is generally accepted that the age-related accumulation of “aberrant” proteins results from both the increased occurrence of damage and the decreased efficiency …