A bright approach to the immunoproteasome: Development of LMP2/β1i-specific imaging probes

Abstract

While the constitutive, 26S proteasome plays an important role in regulating many important cellular processes, a variant form known as the immunoproteasome is thought to primarily function in adaptive immune responses. However, recent studies indicate an association of immunoproteasomes with many physiological disorders such as cancer, neurodegenerative, and inflammatory diseases. Despite this, the detailed functions of the immunoproteasome remain poorly understood. Immunoproteasome-specific probes are essential to gain insight into immunoproteasome function. Here, we describe for the first time the development of the cell-permeable, activity-based fluorescent probes, UK101-Fluor and UK101-B660, which selectively target the catalytically active LMP2/β1i subunit of the immunoproteasome. These probes will facilitate rapid detection of the cellular localization of catalytically active immunoproteasomes in living cells, providing a valuable tool to analyze immunoproteasome functions. Additionally, as LMP2/β1i may serve as a potential tumor biomarker, an LMP2/β1i-targeting fluorescent imaging probe may be applicable to a rapid readout assay to determine tumor LMP2/β1i levels.

Elucidating the Catalytic Subunit Composition of Distinct Proteasome Subtypes: A Crosslinking Approach Employing Bifunctional Activity‐Based Probes

“…[19, 25] To determine the feasibility of our crosslinking strategy, we first utilized two of these inhibitors readily available in our laboratory—the β1/β1i-selective inhibitor UKP1-3 [19] and the β5/β5i-selective inhibitor LKS01 [25c] —to generate the first peptide epoxyketone-based bifunctional proteasome probe. UKP1-3 and LKS01 are derivatives of the β1/β1i-selective inhibitor YU-102 [25a, 26] and the β5i-selective inhibitor IPSI, [25b] respectively, in which the P4 glycine side chain of YU-102 and the P2 tryptophan side chain of IPSI were replaced with that of lysine to facilitate their coupling via an amine-reactive linker. Modifications at these positions do not alter the subunit selectivities of these inhibitors.…”

“…Modifications at these positions do not alter the subunit selectivities of these inhibitors. [19, 25c, 25e] Coupling of UKP1-3 and LKS01 via a short hydrocarbon linker with an estimated length of ~11.4 Å produced UKP13-C 6 -LKS01 (probe 1 ) (Figure 2, Scheme 1). …”

“…The appearance of two crosslinked β1i bands suggested that β1i was likely crosslinked with two different subunits by probe 1 . In order to identify the other subunits crosslinked with β1i, we pretreated U266 lysates with the subunit-selective inhibitors NC-012 (β2/β2i-selective) [25f] or IPSI (β5i-selective) [25b] prior to treatment with probe 1 . Pretreatment with NC-012, but not with IPSI, was able to completely block β1i crosslinking, as indicated by the complete depletion of these two crosslinked β1i bands (Figure 3B), demonstrating that the subunits crosslinked with β1i by probe 1 are β2 and β2i.…”

“…Encouraged by the successful crosslinking of β1i with β2/β2i by probe 1 we synthesized UKP13-(PEG) 4 -NC012 (probe 3 ), in which, in effort to improve the β1/β1i-β2/β2i crosslinking efficiency, we replaced the LKS01 inhibitor and short C 6 hydrocarbon linker of probe 1 with the β2/β2i-selective inhibitor NC-012 [25f] and a more flexible and water-soluble PEG linker, respectively. The N-terminus of NC-012 was derivatized to facilitate linker attachment without compromising the inhibitor’s subunit selectivity [25f] .…”

Activity‐based probes for the multicatalytic proteasome

“…A slightly different scaffold was used in the b1i-selective fluorescein-labeled probe UK101-Fluor, which uses an acylated lysine in P2, and a large hydrophobic moiety attached to the epoxide to mimic the P1 0 position. UK101-Fluor and its BODIPY analog were shown to be suitable for imaging b1i activity in live cells by fluorescence microscopy [41].…”