Activity-Based Chemical Proteomics Accelerates Inhibitor Development for Deubiquitylating Enzymes

Altun, Mikael; Kramer, Holger; Willems, Lianne I.; McDermott, Jeffrey L.; Leach, Craig A.; Goldenberg, Seth J.; Kumar, Krishna; Konietzny, Rebecca; Fischer, Román; Kogan, Edward; Mackeen, Muhammad Mukram Mohamed; McGouran, Joanna F.; Khoronenkova, Svetlana V.; Parsons, Jason L.; Dianov, Grigory L.; Nicholson, Benjamin; Kessler, Benedikt M.

doi:10.1016/j.chembiol.2011.08.018

Cited by 345 publications

(341 citation statements)

References 58 publications

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“…decrease in the correlation coefficient was seen after 6 h of proteasome inhibition in the presence of D12-PGJ2 (relative to 6 h of EtOH + D12-PGJ2 treatment), compared to the 5-fold decrease seen after 6 h of proteasome inhibition alone (relative to 6 h of EtOH treatment). We repeated these experiments with an additional pharmacological inhibitor of deubiquitinating enzymes, PR-619, 32,33 and obtained similar results (Fig. 3D-F).…”

Section: Right) (C)supporting

confidence: 60%

“…Cells were treated with 1 lg/ml MG132 (Sigma-Aldrich) to inhibit the proteasome, or with ethanol (EtOH) as a vehicle control. In experiments using a deubiquitinating enzyme inhibitor, cells were treated with either 20 lM D12-PGJ2 30,31 (Cayman Chemical) or 20 lM PR-619 32,33 (LifeSensors) together with either MG132 or EtOH. After appropriate drug treatments, cells were fixed in 3.7% formaldehyde (Polysciences) in PIPES buffer for 5 min.…”

Section: Cells and Pharmaceuticalsmentioning

confidence: 99%

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Using Antiubiquitin Antibodies to Probe the Ubiquitination State Within rhTRIM5α Cytoplasmic Bodies

Danielson

Hope

2013

AIDS Research and Human Retroviruses

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The first line of defense protecting rhesus macaques from HIV-1 is the restriction factor rhTRIM5a, which recognizes the capsid core of the virus early after entry and normally blocks infection prior to reverse transcription. Cytoplasmic bodies containing rhTRIM5a have been implicated in the ubiquitin-proteasome pathway, but the specific roles these structures play remain uncharacterized. Here, we examine the ubiquitination status of cytoplasmic body proteins. Using antibodies specific for different forms of ubiquitin, we show that ubiquitinated proteins are present in cytoplasmic bodies, and that this localization is altered after proteasome inhibition. A decrease in polyubiquitinated proteins localizing to cytoplasmic bodies was apparent after 1 h of proteasome inhibition, and greater differences were seen after extended proteasome inhibition. The decrease in polyubiquitin conjugates within cytoplasmic bodies was also observed when deubiquitinating enzymes were inhibited, suggesting that the removal of ubiquitin moieties from polyubiquitinated cytoplasmic body proteins after extended proteasome inhibition is not responsible for this phenomenon. Superresolution structured illumination microscopy revealed finer details of rhTRIM5a cytoplasmic bodies and the polyubiquitin conjugates that localize to these structures. Finally, linkage-specific polyubiquitin antibodies revealed that K48-linked ubiquitin chains localize to rhTRIM5a cytoplasmic bodies, implicating these structures in proteasomal degradation. Differential staining of cytoplasmic bodies seen with different polyubiquitin antibodies suggests that structural changes occur during proteasome inhibition that alter epitope availability. Taken together, it is likely that rhTRIM5a cytoplasmic bodies are involved in recruiting components of the ubiquitin-proteasome system to coordinate proteasomal destruction of a viral or cellular protein(s) during restriction of HIV-1.

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Section: Right) (C)supporting

confidence: 60%

Section: Cells and Pharmaceuticalsmentioning

confidence: 99%

Using Antiubiquitin Antibodies to Probe the Ubiquitination State Within rhTRIM5α Cytoplasmic Bodies

Danielson

Hope

2013

AIDS Research and Human Retroviruses

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“…One must bear in mind that a requirement for catalytic activity has not been shown in all cases, and some assays may reflect other aspects of a particular DUB's function. Nevertheless, several DUBs are emerging as attractive drug targets, for which first generation tool compounds have been developed (11,42). The stage is now set for the development of clinically useful therapies that build upon this body of knowledge.…”

Section: Discussionmentioning

confidence: 99%

Deubiquitylases From Genes to Organism

Clague

Barsukov

Coulson

et al. 2013

Physiological Reviews

375

384

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Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.

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“…DUBs represent a large group of enzymes, which oppose the actions of E3 ligases, they maintain ubiquitin homeostasis, edit polyubiquitin chains, and prevent proteasomal ubiquitin degradation together with the substrates (Amerik and Hochstrasser, 2004;Komander et al, 2009;Todi and Paulson, 2011). Using PR-619, a cell permeable, broad-range inhibitor of deubiquitylases and ubiquitinlike isopeptidases (Altun et al, 2011), we could show that DUB inhibition in oligodendroglial OLN-t40 cells caused the time-and concentration-dependent induction of HSPs. Protein aggregates were formed, positively stained by antibodies against ubiquitin, αB-crystallin, HSP70 and p62, which increased in size over time and appeared as large deposits near the cell nucleus (Seiberlich et al, 2012).…”

Section: Cellular Stress and Tau Aggregate Formation In Oligodendrocytesmentioning

confidence: 89%

Protein aggregate formation in oligodendrocytes: tau and the cytoskeleton at the intersection of neuroprotection and neurodegeneration

Richter‐Landsberg¹

2016

Biological Chemistry

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Abstract:Oligodendrocytes are dependent on an intact, dynamic microtubule (MT) network, which participates in the elaboration and stabilization of myelin forming extensions, and is essential for cellular sorting processes. The microtubule-associated protein tau is constituent of oligodendrocytes. During culture maturation it is developmentally regulated and important for MT stability, MT formation and intracellular trafficking. Downregulation of tau impairs process outgrowth and the transport of myelin basic protein (MBP) mRNA to the cell periphery. Cells fail to differentiate into MBP-expressing, sheet-forming oligodendrocytes. Tau-positive inclusions originating in oligodendrocytes and white matter pathology are prominent in frontotemporal dementias, such as Pick's disease, progressive supranuclear palsy and corticobasal degeneration. An impairment or overload of the proteolytic degradation systems, i.e. the ubiquitin proteasomal system and the lysosomal degradation pathway, has been connected to the formation of protein aggregates. Large protein aggregates are excluded from the proteasome and degraded by autophagy, which is a highly selective process and requires receptor proteins for ubiquitinated proteins, including histone deacetylase 6 (HDAC6). HDAC6 is present in oligodendrocytes, and α-tubulin and tau are substrates of HDAC6. In this review our current knowledge of the role of tau and protein aggregate formation in oligodendrocyte cell culture systems is summarized.

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Activity-Based Chemical Proteomics Accelerates Inhibitor Development for Deubiquitylating Enzymes

Cited by 345 publications

References 58 publications

Using Antiubiquitin Antibodies to Probe the Ubiquitination State Within rhTRIM5α Cytoplasmic Bodies

Using Antiubiquitin Antibodies to Probe the Ubiquitination State Within rhTRIM5α Cytoplasmic Bodies

Deubiquitylases From Genes to Organism

Protein aggregate formation in oligodendrocytes: tau and the cytoskeleton at the intersection of neuroprotection and neurodegeneration

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