Activity-Based Profiling Reveals Reactivity of the Murine Thymoproteasome-Specific Subunit β5t

Florea, Bogdan I.; Verdoes, Martijn; Li, Nan; Linden, Wouter A. van der; Geurink, Paul P.; Elst, Hans van den; Hofmann, Tanja; Ru, Arnoud de; Veelen, Peter A. van; Tanaka, Keiji; Sasaki, Katsuhiro; Murata, Shigeo; Dulk, Hans den; Brouwer, Jaap; Ossendorp, Ferry; Kisselev, Alexei F.; Overkleeft, Herman S.

doi:10.1016/j.chembiol.2010.05.027

Cited by 76 publications

(83 citation statements)

References 26 publications

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“…Expression of thymoproteasomes by cortical TECs is essential for positive selection of most, although not all, CD8 thymocytes (27). Because of their low chymotrypsin-like activity, thymoproteasomes are predicted to generate peptides that bind MHC I molecules with low affinity and, therefore, yield unstable MHCpeptide complexes (59,63). It would seem plausible that being more stable, thymoproteasome-independent MHCI-peptide complexes would elicit stronger TCR signals.…”

Section: Discussionmentioning

confidence: 99%

Development and Function of Innate Polyclonal TCRαβ+ CD8+ Thymocytes

Rafei

Hardy

Williams

et al. 2011

The Journal of Immunology

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Innate CD8 T cells are found in mutant mouse models, but whether they are produced in a normal thymus remains controversial. Using the RAG2p-GFP mouse model, we found that ∼10% of TCRαβ+ CD4−CD8+ thymocytes were innate polyclonal T cells (GFP+CD44hi). Relative to conventional T cells, innate CD8 thymocytes displayed increased cell surface amounts of B7-H1, CD2, CD5, CD38, IL-2Rβ, and IL-4Rα and downmodulation of TCRβ. Moreover, they overexpressed several transcripts, including T-bet, Id3, Klf2, and, most of all, Eomes. Innate CD8 thymocytes were positively selected, mainly by nonhematopoietic MHCIa+ cells. They rapidly produced high levels of IFN-γ upon stimulation and readily proliferated in response to IL-2 and IL-4. Furthermore, low numbers of innate CD8 thymocytes were sufficient to help conventional CD8 T cells expand and secrete cytokine following Ag recognition. This helper effect depended on CD44-mediated interactions between innate and conventional CD8 T cells. We concluded that innate TCRαβ+ CD8 T cells represent a sizeable proportion of normal thymocytes whose development and function differ in many ways from those of conventional CD8 T cells.

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Section: Discussionmentioning

confidence: 99%

Development and Function of Innate Polyclonal TCRαβ+ CD8+ Thymocytes

Rafei

Hardy

Williams

et al. 2011

The Journal of Immunology

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“…Imaging of the ABPs signal in adherent C2C12 cells confirmed that the MVB127 signal in cells was not reduced in Epox‐treated cells (Figure 4C). The MVB127 non‐specific signal was mostly localized to membrane structures because of its lipophilic property 18. The LW124 non‐specific signal was found in the nucleus (Figure 4C).…”

Section: Resultsmentioning

confidence: 98%

“…We recently reported that in the C2C12 cell culture, β5 protein levels are lower than β2;14 this indicates that β5 activity in C2C12 is higher than β2 activity. Also, in mouse thymus, the β5 signal is higher compared with the other subunits 18. Thus, specific proteasomal activity in cells and tissues should include subunit‐specific analysis.…”

Section: Resultsmentioning

confidence: 99%

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Proteasomal activity‐based probes mark protein homeostasis in muscles

Raz¹,

Raz²,

Soriano

et al. 2017

J cachexia sarcopenia muscle

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BackgroundProtein homeostasis, primarily regulated by the ubiquitin–proteasome system is crucial for proper function of cells. In tissues of post‐mitotic cells, the impaired ubiquitin–proteasome system is found in a wide range of neuromuscular disorders. Activity‐based probes (ABPs) measure proteasomal proteolytic subunits and can be used to report protein homeostasis. Despite the crucial role of the proteasome in neuromuscular pathologies, ABPs were not employed in muscle cells and tissues, and measurement of proteasomal activity was carried out in vitro using low‐throughput procedures.MethodsWe screened six ABPs for specific application in muscle cell culture using high throughput call‐based imaging procedures. We then determined an in situ proteasomal activity in myofibers of muscle cryosections.ResultsWe demonstrate that LWA300, a pan‐reactive proteasomal probe, is most suitable to report proteasomal activity in muscle cells using cell‐based bio‐imaging. We found that proteasomal activity is two‐fold and three‐fold enhanced in fused muscle cell culture compared with non‐fused cells. Moreover, we found that proteasomal activity can discriminate between muscles. Across muscles, a relative higher proteasomal activity was found in hybrid myofibers whereas fast‐twitch myofibers displayed lower activity.ConclusionsOur study demonstrates that proteasomal activity differ between muscles and between myofiber types. We suggest that ABPs can be used to report disease progression and treatment efficacy.

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“…The analysis was performed as previously reported. [42] Peptides were desalted on stage tips [43] and analyzed with a trap-elute system on C18 reversed phase nano LC with a 45 min 10-60% ACN/0.1% formic acid gradient, hyphenated to a Thermo LTQ-orbitrap mass spectrometer using a top 3 data dependent protocol at 60.000 resolution, m/z range 300-2000, 1000 msec fill time in the Orbitrap, for MS/MS fragmentation 35 units of CID energy, 120 msec max fill time, AGC 50 e3 and a threshold of 750 counts. Ions of z = 2+ and higher were selected to be fragmented twice within 10 sec prior to exclusion for 150 sec.…”

Section: Proteomicsmentioning

confidence: 99%

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

et al. 2017

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Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a Bodipy fluorophore at C6 that allows visualizing active enzyme in cells and tissues. The detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intracerebroventricular infusion of the ABP. In conclusion, the GALCspecific ABP should find broad applications in diagnosis, drug development and evaluation of therapy of Krabbe disease.

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Activity-Based Profiling Reveals Reactivity of the Murine Thymoproteasome-Specific Subunit β5t

Cited by 76 publications

References 26 publications

Development and Function of Innate Polyclonal TCRαβ+ CD8+ Thymocytes

Development and Function of Innate Polyclonal TCRαβ+ CD8+ Thymocytes

Proteasomal activity‐based probes mark protein homeostasis in muscles

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

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