Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of... Show moreMany reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity. Show less
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),... Show moreBioorthogonal 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. This paper describes 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 designed for β1i, β2i, β5c and β5i proved to be useful two-step ABPs that effectively label their designed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (IEDDA) ligation. The compound designed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro . The compound designed 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 reporting on proteasome activities in living cells. Show less
Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the β1c, β1i, β5c, and... Show more Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the β1c, β1i, β5c, and β5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting β2c or β2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the β2c- and β2i-selective compounds LU-002c (4; IC50 β2c: 8 nM, IC50 β2i/β2c: 40-fold) and LU-002i (5; IC50 β2i: 220 nM, IC50 β2c/β2i: 45-fold), respectively. Co-crystal structures with β2 humanized yeast proteasomes visualize protein–ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of β2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors. Show less
This work reports the development of highly potent and selective inhibitors of the β5c catalytic activity of human constitutive proteasomes. The work describes the design principles, large... Show moreThis work reports the development of highly potent and selective inhibitors of the β5c catalytic activity of human constitutive proteasomes. The work describes the design principles, large hydrophobic P3 residue and small hydrophobic P1 residue, that led to the synthesis of a panel of peptide epoxyketones; their evaluation and the selection of the most promising compounds for further analyses. Structure-activity relationships detail how in a logical order the β1c/i, β2c/i, and β5i activities became resistant to inhibition as compounds were diversified stepwise. The most effective compounds were obtained as a mixture of cis- and trans-biscyclohexyl isomers, and enantioselective synthesis resolved this issue. Studies on yeast proteasome structures complexed with some of the compounds provide a rationale for the potency and specificity. Substitution of the N-terminus in the most potent compound for a more soluble equivalent led to a cell-permeable molecule that selectively and efficiently blocks β5c in cells expressing both constitutive proteasomes and immunoproteasomes. Show less
Bruin, G. de; Xin, B.T.; Kraus, M.; Stelt, M. van der; Marel, G.A. van der; Kisselev, A.F.; ... ; Overkleeft, H.S. 2016
