Two‐Step Bioorthogonal Activity‐Based Protein Profiling of Individual Human Proteasome Catalytic Sites

Abstract

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two‐step activity‐based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene‐modified, mechanism‐based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for β1i, β2i, β5c, and β5i proved to be useful two‐step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse‐electron‐demand Diels–Alder (iEDDA) ligation. The compound developed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro. The compound developed for β2c proved not selective, but its azide‐containing analogue LU‐002c proved effective in labeling of β2c via azide–alkyne click ligation chemistry both in vitro and in situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit‐selective activity‐based proteasome probes, four of which report on proteasome activities in living cells.

Dysregulation of neuroproteasomes by ApoE isoforms drives endogenous Tau aggregation

“…We synthesized click-able analogs of Epoxomicin (Azide-Epoxomicin) that would allow us to add a broad range of linkers ( Fig 4A ). Modification of the non-reactive end of epoxomicin has been shown not to change the selectivity of Epoxomicin for the proteasome 51, 55, 56 .We then conjugated Biotin-PEG24-Alkyne to Epoxomicin, producing Biotin-PEG24-Epoxomicin, or iBEp ( Fig 4A ). We observe efficient conjugation by HPLC analysis ( Fig S4A, B, C, D ).…”

“…All molecules were HPLC-purified as single peaks and confirmed by LC/MS analysis ( Fig S4D, E ). Because epoxomicin covalently and irreversibly binds the catalytic β subunits of the proteasome, the biotin moiety on iBEp also serves as a tracer to identify which proteasomes are inhibited by monitoring biotin signal on an SDS-PAGE gel 51, 55, 56 . To test the permeability of our compounds, we incubated primary cortical neurons with iBEp over a time course and prepared both cytosolic and plasma membrane-enriched fractions.…”

“…However, in order to test this hypothesis, we required an NMPspecific inhibitor. We previously accomplished this by biotinylating the N-terminus of Epoxomicin, a covalent and highly selective inhibitor which exclusively targets the catalytic b subunits of the proteasome, with no off-target reactivity [49][50][51][52] . We previously found that this small modification rendered epoxomicin cell-impermeable for up to an hour 4 .…”

Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ

“…As fluorophores are often large and hydrophobic moieties, directly incorporating them into an ABP is likely to affect target binding and will impact the physiochemical properties of the ABP. 5,6 To circumvent this issue, one can incorporate small, bioorthogonal handles into ABPs. 5,7,8 Bioorthogonal tags are selected on their reactivity, chemoselectivity, bioorthogonality, and their size, which is ideally as small as possible.…”