Detection of ubiquitin–proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development

AbstractBackground: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin–proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics

Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection

A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MSE). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus non-infected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits

AbstractBackground: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, β1, β2 and β5, which are replaced in the γ-interferon-inducible immunoproteasome by a different set of catalytic subunits, β1i, β2i and β5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as ‘chymotryptic-like’ (β5), ‘tryptic-like’ (β2) and ‘peptidyl-glutamyl peptide hydrolyzing’ (β1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits.Results: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone (AdaAhx3L3VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels.Conclusions: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts

Targeting CDK2 overcomes melanoma resistance against BRAF and Hsp90 inhibitors

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment

Old-age muscle atrophy : Cellular mechanisms and behavioral consequenses

With advancing age, humans and rodents alike lose about one third of the skeletal muscle mass. A process referred to as old-age muscle atrophy or sarcopenia. Atrophy is a major contributor to disability and morbidity among elderly adults hence the aim of this thesis is to shed light on the molecular mechanisms underlying old-age associated muscle atrophy and behavioral changes related to age in a rat model. In Paper I, we characterized the growth patterns, survival and behavioral alterations linked to advancing age in the rat. The median survival age was, on average, between 29 30 months for both female and male Sprague Dawley (SD) rats. There was a gradual decline in locomotor activity and explorative behavior associated with age, while disturbances in both coordination and balance did not become evident until later times points. In old age, weight carrying capacity, limb movement and temperature threshold were also impaired. While body weight continues to increase over the better part of the life span of rats, the behavioral changes in old age associated with a decrease in both total body weight and, in particular, muscle mass. Dietary restriction (DR) was found to increase median life span expectancy and impede the development of sarcopenia, and to retard the pace of behavioral aging. In Paper II, we used two-dimensional gel electrophoresis and mass spectrometry techniques to determine changes in protein expression as well as cDNA profiling to assess transcriptional regulations in skeletal muscle of adult and aged male SD rats. Among the highly expressed proteins, thirty-five were differentially expressed in aged muscle. Proteins and mRNA transcripts involved in redox homeostasis and iron load were increased, representing novel components previously not associated with sarcopenia. Iron levels in tissue were elevated in senescence, paralleling an increase in transferrin. Proteins involved in redox homeostasis were found to display a complex pattern of changes involving increases in SOD1 and decreases in SOD2. Together these results suggest that an elevated iron load is a significant component of sarcopenia with a potential to be exploited clinically and that the mitochondria of aged striated muscle may be more vulnerable to radicals produced during cell respiration. Muscle atrophy, in many conditions, shares a common mechanism for up-regulation of the muscle-specific ubiquitin E3-ligases Atrogin-1 and MuRF1. E3-ligases are part of the ubiquitin proteasome system (UPS) utilized for protein degradation during muscle atrophy. In Paper III, we show that Atrogin-1 and MuRF1 are down-regulated in old age-associated muscle atrophy. Our results suggest that this is mediated by AKT-induced inactivation of FOXO4. DR impeded sarcopenia as well as both FOXO4 inactivation and up-regulation of Atrogin-1 and MuRF1 transcripts. Our findings allow us to conclude that sarcopenia is mechanistically different from acute atrophies induced by disuse, disease, and denervation. The 26S proteasome is responsible for most cytosolic proteolysis. Molecules that inhibit or specially tag proteasomes are helpful tools for analysis of the UPS. In Paper IV, we present a new class of proteasome inhibitors, considerably extended in comparison to the commonly used fluorescent substrates and peptide-based inhibitors. Modification of the most active compound, Ada-Ahx3L3VS, capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. N-terminal extension of peptide vinyl sulfones was found to have a profound influence on both their efficacy and selectivity as proteasome inhibitors. Results demonstrated that such extensions greatly enhanced inhibition and largely obliterated their selectivity towards individual catalytic activities. The role of the UPS in aging-related muscle atrophy is highly controversial. In Paper V, we showed an accumulation of assembled proteasome particles with a corresponding increase in both proteasomal activity and protein degradation in old age muscle atrophy. This was accompanied by a wide range of UPS enzyme-regulation, including an increase in the activity of deubiquitylating enzymes. The accumulation of proteasomes was found to correlate well with muscle wasting. Both the accumulation of proteasome particles as well as the progression of muscle atrophy, were impeded when the normal pattern of aging was challenged by DR. In contrast to many conditions with UPS-associated muscle catabolism, the accumulation of proteasomes during senile muscle atrophy is not caused by transcriptional induction, but rather by decreases in their degradation. The lysosomal pathway is a candidate for degrading proteasomes. In Paper V, we demonstrated that impaired lysosomal function, achieved through chloroquine treatment, induced accumulation of proteasomes in adult rats. This emphasizes the existence of a functional link between the lysosomal pathway and the UPS suggesting that a decline in lysosomal function may contribute to increased proteasomal proteolysis in old-age skeletal muscle atrophy

An N-terminal SIAH-interacting motif regulates the stability of the ubiquitin specific protease (USP)-19

The Ubiquitin Specific Protease-19 (USP19) regulates cell cycle progression and is involved in the cellular response to different types of stress, including the unfolded protein response (UPR), hypoxia and muscle atrophy. Using the unique N-terminal domain as bait in a yeast-two hybrid screen we have identified the ubiquitin ligases Seven In Absentia Homolog (SIAH)-1 and SIAH2 as binding partners of USP19. The interaction is mediated by a SIAH-consensus binding motif and promotes USP19 ubiquitylation and proteasome-dependent degradation. These findings identify USP19 as a common substrate of the SIAH ubiquitin ligases. © 2013 Elsevier Inc

The ER-resident ubiquitin-specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membrane-associated ubiquitin-conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin-specific protease (USP)19. We show that USP19 is a tail-anchored ubiquitin-specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T-cell receptor-α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non-catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane-anchored DUB involved in the turnover of ERAD substrates