Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

Melvin, Adam T.; Woss, Gregery S.; Park, Jessica H.; Waters, Marcey L.; Allbritton, Nancy L.

doi:10.1007/s12013-013-9621-9

Cited by 21 publications

(12 citation statements)

References 86 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…11 One limitation of peptide-based reporters is their rapid degradation by intracellular peptidases and proteases. Designing fluorescent peptide-based reporters that report the activity of the UPS by the detection of proteasome-mediated reporter degradation 2 can be challenging. Often, a significant amount of the reporter is degraded by cytosolic proteases before the activity of the UPS can be detected.…”

Section: Introductionmentioning

confidence: 99%

Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

Houston¹,

Melvin

Woss³

et al. 2017

ACS Omega

Self Cite

View full text Add to dashboard Cite

Regulation of the ubiquitin–proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, β-hairpin “protectides” exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable β-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH2], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS.

show abstract

Section: Introductionmentioning

confidence: 99%

Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

Houston¹,

Melvin

Woss³

et al. 2017

ACS Omega

Self Cite

View full text Add to dashboard Cite

show abstract

“…UPP is an important mechanism in the degradation of proteins (18,19), which has not been reported in previous studies of TRAIL-resistance mechanism in myeloma cells. Recent studies showed that the disorder of tumor cell regulatory function can be due to the inactivation of key regulators.…”

Section: Discussionmentioning

confidence: 72%

Target and resistance-related proteins of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand on myeloma cell lines

et al. 2016

View full text Add to dashboard Cite

Abstract. Recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) has become a potential therapeutic drug for multiple myeloma (MM). However, the exact targets and resistance mechanisms of rmhTRAIL on MM cells remain to be elucidated. The present study aimed to investigate the target and resistance-related proteins of rmhTRAIL on myeloma cell lines. A TRAIL-sensitive myeloma cell line, RPMI 8226, and a TRAIL-resistance one, U266, were chosen and the differentially expressed proteins between the two cell lines were analyzed prior and subsequent to rmhTRAIL administration by a liquid chromatography-tandem mass spectrometry method. The results showed that following TRAIL treatment, 6 apoptosis-related proteins, calpain small subunit 1 (CPNS1), peflin (PEF1), B-cell receptor-associated protein 31 (BAP31), apoptosis-associated speck-like protein containing CARD (ASC), BAG family molecular chaperone regulator 2 (BAG2) and chromobox protein homolog 3 (CBX3), were upregulated in RPMI 8226 cells while no change was identified in the U266 cells. Furthermore, small ubiquitin-related modifier 1 and several other ubiquitin proteasome pathway (UPP)-related proteins expressed higher levels in TRAIL-resistant cells U266 compared to the RPMI-8226 cells prior and subsequent to rmhTRAIL treatment. These results suggested that CPNS1, PEF1, BAP31, ASC, BAG2 and CBX3 were possibly target proteins of rmhTRAIL on RPMI 8226 cells, while UPP may have a vital role in mediating TRAIL-resistance in U266 cells.

show abstract

“…The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, and tightly regulated proteins. It also plays crucial roles in DNA repair, cell cycle regulation, cell migration, and the immune response (Melvin et al, 2013). Ubiquitin (Ub) is a small protein composed of 76 amino acids that is highly conserved in eukaryotes (Tomohiro and Mizuno, 1995).…”

Section: Expression Of Herc4 In Lung Cancer and Its Correlation With mentioning

confidence: 99%

Expression of HERC4 in Lung Cancer and its Correlation with Clinicopathological Parameters

Zeng

Chen²,

Zhou³

et al. 2015

Asian Pacific Journal of Cancer Prevention

View full text Add to dashboard Cite

Asian Pac J Cancer Prev, 16 (2), 513-517 IntroductionLung cancer is the leading cause of death of cancer world-wide, causing more than 1 million deaths per year (Jemal et al., 2010). It is a disease characterized by uncontrolled cell growth in tissues of the lung. The global cancer burden in annual cases is projected to double by 2050, and lung cancer is expected to remain the leading cause of all cancer deaths during that time (Brothers et al., 2013). Cigarette smoking remains the main risk factor for lung cancer, with 85%-90% percent of lung cancer cases caused by active or passive smoking. Conventional treatments for lung cancer are surgery, radiotherapy and chemotherapy. And the choice of method depends on the type, progress and relevant genes of lung cancer. Besides, targeted therapy, as a more accurate and specific treatment, has come into our field of vision for us to choose from. It won't cause any harm to adjacent normal lung cells when it uses specific drugs to identify and target cancer cells.Recently, genetic, transcriptomic and epigenomic biomarkers are emerging as tools for the early diagnosis of lung cancer both in the screening setting and diagnostic. Furthermore, epigenetics refers to heritable modifications

show abstract

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

Cited by 21 publications

References 86 publications

Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

Target and resistance-related proteins of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand on myeloma cell lines

Expression of HERC4 in Lung Cancer and its Correlation with Clinicopathological Parameters

Contact Info

Product

Resources

About