Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases

Bavikar, Sudhir N.; Spasser, Liat; Haj‐Yahya, Mahmood; Karthikeyan, Subramanian Vedhanarayanan; Moyal, Tal; Kumar, K. S. Ajish; Brik, Ashraf

doi:10.1002/anie.201106430

Cited by 104 publications

(55 citation statements)

References 69 publications

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“…The ubiquitination method used in this paper is efficient, but leads to attachment of one or more heterogeneous polyubiquitin chains to the substrate, which makes it difficult to know the precise concentration of ubiquitinated substrate and can therefore lead to uncertainty in the determined K M and V max values. Other methods allow the synthesis of substrate proteins with precisely defined ubiquitin modifications [24,26,29]. Nevertheless, together, these data demonstrate that the assay described in this paper can easily be used to characterize proteasomal degradation by standard kinetic approaches.…”

Section: Resultsmentioning

confidence: 80%

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

Bhattacharyya

Renn

et al. 2016

Analytical Biochemistry

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The 26S proteasome is the molecular machine at the center of the ubiquitin–proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.

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Section: Resultsmentioning

confidence: 80%

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

Bhattacharyya

Renn

et al. 2016

Analytical Biochemistry

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“…Native ubiquitination of histone H2 by expressed protein ligation revealed a direct correlation to an increased methylation of H3 [17 ]. A combination of genetic code expansion, orthogonal amino acid protection and final aminolysis of ubiquitin thioester furnished a native di-ubiquitin-conjugate [18]; alternatively, peptide synthesis and sequential native chemical ligations provided access to ubiquitinated peptides with varying lengths of specific ubiquitin chains [19]. These studies showed that deubiquitinases have a higher activity towards shorter chains as well as Lys29-linked and Lys48-linked poly-ubiquitins.…”

Section: Chemical Installation Of Posttranslational Modifications (Ptms)mentioning

confidence: 99%

More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins

Schumacher

Hackenberger

2014

Current Opinion in Chemical Biology

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“…[134] Auf diese Weise gelang es uns, einen Satz ubiquitinierter Peptide unterschiedlicher Kettenlängen (Mono-, Di-, Tri-und Tetra-) und Verknüpfungsarten (K63, K38) in einem effizienten, konvergenten Prozess herzustellen (Abbildung 6). [134] Auf diese Weise gelang es uns, einen Satz ubiquitinierter Peptide unterschiedlicher Kettenlängen (Mono-, Di-, Tri-und Tetra-) und Verknüpfungsarten (K63, K38) in einem effizienten, konvergenten Prozess herzustellen (Abbildung 6).…”

Section: Angewandte Aufsätzeunclassified

Die Chemie und Biologie des Ubiquitin‐Signals

Spasser

Brik

2012

Angewandte Chemie

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Die Ubiquitinierung ist eine der häufigsten posttranslationalen Modifikationen in Eukaryoten und spielt bei einer Vielzahl zellulärer Prozesse eine Rolle, die zumeist als Signal für den proteasomalen Abbau bekannt sind. Vor kurzem stellte sich heraus, dass das Ubiquitin‐Signal wesentlich komplexer ist und durch die Ubiquitin‐Komponente und das Substrat bestimmt wird. Die bemerkenswerte Mannigfaltigkeit des Ubiquitin‐Signalprozesses hat enorme Bemühungen zur Erforschung der Rolle der Ubiqitinierung in biologischen Prozessen ausgelöst. Trotz der seit mehr als drei Jahrzehnten andauernden Untersuchungen sind aber noch mehrere wichtige Fragestellungen offen. Eine große Hürde ist das Unvermögen, homogene Ubiquitin‐Biokonjugate durch Isolierung aus Zellen oder durch enzymatische Mechanismen zugänglich zu machen. Jüngste Erfolge bei chemischen und semisynthetischen Strategien bieten zunehmend Lösungen für diese Herausforderungen, indem sie die Produktion großer Mengen homogener Ubiquitinkonjugate ermöglichen. In diesem Aufsatz diskutierten wir die fundamentalen biologischen Aspekte des Ubiquitin‐Signals und stellen neuartige nichtenzymatische Ansätze vor, die diese Hindernisse umgehen.

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Chemical Synthesis of Ubiquitinated Peptides with Varying Lengths and Types of Ubiquitin Chains to Explore the Activity of Deubiquitinases

Cited by 104 publications

References 69 publications

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins

Die Chemie und Biologie des Ubiquitin‐Signals

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