2 research outputs found
Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay
Most
mammalian tissues contain a single proteasome species: constitutive
proteasomes. Tissues able to express, next to the constitutive proteasome
catalytic activities (β1c, β2c, β5c), the three
homologous activities, β1i, β2i and β5i, may contain
numerous distinct proteasome particles: immunoproteasomes (composed
of β1i, β2i and β5i) and mixed proteasomes containing
a mix of these activities. This work describes the development of
new subunit-selective activity-based probes and their use in an activity-based
protein profiling assay that allows the detection of various proteasome
particles. Tissue extracts are treated with subunit-specific probes
bearing distinct fluorophores and subunit-specific inhibitors. The
samples are resolved by native polyacrylamide gel electrophoresis,
after which fluorescence-resonance energy transfer (FRET) reports
on the nature of proteasomes present
Structure-Based Design of β5c Selective Inhibitors of Human Constitutive Proteasomes
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 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 <i>cis</i>- and <i>trans</i>-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