Cell-Permeable Activity-Based Ubiquitin Probes Enable Intracellular Profiling of Human Deubiquitinases.

Advancement in our knowledge of deubiquitinases (DUBs) and their biological functions requires biochemical tools permitting interrogation of DUB activities under physiologically relevant conditions. Activity-based DUB probes (DUB ABPs) have been widely used in investigating the function and activity of DUBs. However, most ubiquitin (Ub)-based DUB ABPs are not cell-permeable, limiting their utility to purified proteins and cell lysates. Lysis of cells usually leads to dilution of the cytoplasm and disruption of the normal cellular organization, which may alter the activity of many DUBs and DUB complexes. Here, we report a new class of cell-permeable DUB ABPs that enable intracellular DUB profiling. We used a semisynthetic approach to generate modular ubiquitin-based DUB probes containing a reactive warhead for covalent trapping of DUBs with a catalytic cysteine. We employed cell-penetrating peptides (CPPs), particualrly cyclic polyarginine (cR10), to deliver the DUB ABPs into cells, as confirmed using live-cell fluorescence microscopy and DUB ABPs containing a fluorophore at the C-terminus of Ub. In comparison to TAT, enhanced intacellular delivery was observed through conjugation of a cyclic polyarginine (cR10) to the N-terminus of ubiquitin via a disulfide linkage. Using the new cell-permeable DUB ABPs, we carried out DUB profiling in intact HeLa cells, and identified active DUBs using immunocapture and label-free quantitative mass spectrometry. Additionally, we demonstrated that the cell-permeable DUB ABPs can be used in assessing the inhibition of DUBs by small-molecule inhibitors in intact cells. Our results indicate that cell-permeable DUB ABPs hold great promise in providing a better understanding of the cellular functions of DUBs and advancing drug discovery efforts targeting human DUBs.

Incorporation of ubiquitin into the rat brain mitochondria is accompanied by increased proteolytic digestibility of MAO.

Incubation of rat brain mitochondria with ubiquitin followed by mitochondria sedimentation was accompanied by reduction of ubiquitin content in the supernatant only when ATP was included into the incubation mixture. Subsequent incubation of resedimented mitochondria revealed higher sensitivity to trypsin of MAO-A in ubiquitin-incorporated mitochondria. In control mitochondria (initially incubated with ATP) 0.5 mg/ml trypsin caused a decrease of MAO-A activity by 32.2 +/- 4.2%, whereas in ubiquitin-incorporated mitochondria (initially incubated with ATP + ubiquitin) reduction of MAO-A activity was significantly higher (51.4 +/- 2.5%, p < 0.02). Activity of MAO-B was resistant to trypsinolysis and incorporation of ubiquitin did not influence sensitivity of MAO-B to trypsin. Although there is no direct evidence yet that mitochondrial MAO is a target for ubiquitination the increased sensitivity to trypsinolysis of MAO-A suggests that incorporation of ubiquitin into mitochondria may increase susceptibility of MAO to certain proteases involved into degradation of these enzymes.

Expression of ubiquitin protein in each organ at death from hypothermia.

To examine the expression of heat shock protein (hsp) in each organ on autopsy in 11 deaths from hypothermia, we performed immunological staining of specimens from each organ using an antibody to hsp, ubiquitin (Ub). Staining of the liver, kidneys, lungs and pancreas revealed a high level of Ub expression in the cytoplasm and nuclei. However, staining of the same specimens of all controls revealed no Ub expression in the cytoplasm or nuclei. This finding suggests that cells are affected by stress even at a low temperature, and may be important in clarifying the cellular kinetics in death from hypothermia.