Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Ritorto, Maria Stella; Ewan, Richard; Oliva, Alfonso; Knebel, Axel; Buhrlage, Sara J.; Wightman, Melanie; Kelly, Sharon M.; Wood, Nicola T.; Virdee, Satpal; Gray, Nathanael S.; Morrice, Nicholas A.; Alessi, Dario R.; Trost, Matthias

doi:10.1038/ncomms5763

Cited by 287 publications

(366 citation statements)

References 64 publications

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“…Similar results were found by Ritorto et al (2014) who compared the performance of the Acclaim® 120 C18 and the Acclaim® RSLC C18 PA2 to separate tryptic digested proteins from cell lysate. The researchers found that the Acclaim® RSLC C18 PA2 had higher efficiencies and exhibited higher polarity of selectivity.…”

Section: Hplc Columnsupporting

confidence: 69%

“…Based on prior tetracycline antibiotic research, we hypothesized that sample preparation techniques, namely an additional filtration step to remove remaining particulates that can interfere with HPLC performance (CDER, 1994), and HLPC characteristics, such as mobile phase solution (Jia, Xiao, Hu, Asami, & Kunikane, 2009), column type (Ritorto et al, 2014) and wavelength (Ng, & Linder, 2003), would influence antibiotic detection in water samples. We anticipated that analysis of larger sample volumes would improve antibiotic detection, as we would have a more material from which to develop a concentrate, while higher manure concentrations would decrease detection capabilities due to the presence of more impurities requiring removal to not inhibit HPLC performance.…”

Section: Secondary Sample Preparationmentioning

confidence: 99%

See 1 more Smart Citation

Development of a Methodology to Determine Antibiotic Concentrations in Water Samples Using High-Pressure Liquid Chromatography

Qualls¹,

Agouridis²,

Kulshrestha³

2017

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Section: Hplc Columnsupporting

confidence: 69%

Section: Secondary Sample Preparationmentioning

confidence: 99%

Development of a Methodology to Determine Antibiotic Concentrations in Water Samples Using High-Pressure Liquid Chromatography

Qualls¹,

Agouridis²,

Kulshrestha³

2017

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“…Usp8 shows some selectivity for recombinant K63-ubiquitin linkages in vitro (28,29). However, compared with other DUBs of the cysteine protease type, Usp8 as well the closely related enzyme Usp7 showed strong capacity to hydrolyze K63-linked ubiquitin on α-synuclein in vitro.…”

Section: Discussionmentioning

confidence: 99%

Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease

Alexopoulou

Lang

Perrett

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

108

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In Parkinson’s disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to α-synuclein trafficking or turnover and Parkinson’s pathogenesis. By comparative analysis in human postmortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with α-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein–induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein–induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease.

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“…We tested this in HepG2 cells, and despite obtaining effective silencing of USP7, both expression of IDOL and the response to DUB inhibition remained unaffected (data not shown). A potential explanation for this result is recent evidence indicating that HBX41-108 has a much broader substrate specificity and is able to inhibit a large panel of cysteine DUBs, similar to PR-619 (35,36). The fact that multiple DUBs are sensitive to these inhibitors has precluded us from identifying a specific DUB responsible for the induction of IDOL.…”

Section: Lxr␣␤mentioning

confidence: 86%

Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake

Nelson

Cook

Loregger

et al. 2016

Journal of Biological Chemistry

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Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxr␣␤ ؊/؊ MEFs.Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation.Elevated levels of plasma LDL represent a major risk factor for development of atherosclerosis and cardiovascular disease (1). Because of its ability to promote LDL uptake into cells, the LDL receptor (LDLR) 2 is a major determinant of plasma LDL levels (2). The pivotal role of the LDLR in LDL metabolism is exemplified by the fact that LDLR mutations account for most incidences of familial hypercholesterolemia, a disease characterized by reduced hepatic LDL clearance, elevated plasma cholesterol levels, and accelerated cardiovascular disease (1, 3).The LDLR is subject to tight transcriptional and post-transcriptional regulation, which is largely governed by the intracellular levels of cholesterol (4). At the level of transcription, these pathways are regulated by two nuclear transcription factor families: SREBP1 and SREBP2 (5-7), and the liver X receptor ␣ and ␤ (LXRs) (8, 9). When cellular sterol levels decline, SREBPs are activated to induce genes required for de novo cholesterol biosynthesis, as well as the LDLR to increase uptake of LDL cholesterol (4). In contrast, when sterol levels rise, LXRs become activated by their endogenous ligands. These ligands include oxidized cholesterol derivatives (oxysterols) and intermediates of the cholesterol synthes...

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Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Cited by 287 publications

References 64 publications

Development of a Methodology to Determine Antibiotic Concentrations in Water Samples Using High-Pressure Liquid Chromatography

Development of a Methodology to Determine Antibiotic Concentrations in Water Samples Using High-Pressure Liquid Chromatography

Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease

Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake

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