Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell.

The integration of diverse chemical tools like small-molecule inhibitors, activity-based probes (ABPs), and proteolysis targeting chimeras (PROTACs) advances clinical drug discovery and facilitates the exploration of various biological facets of targeted proteins. Here, we report the development of such a chemical toolbox for the human Parkinson disease protein 7 (PARK7/DJ-1) implicated in Parkinson's disease and cancers. By combining structure-guided design, miniaturized library synthesis, and high-throughput screening, we identified two potent compounds, JYQ-164 and JYQ-173, inhibiting PARK7 in vitro and in cells by covalently and selectively targeting its critical residue, Cys106. Leveraging JYQ-173, we further developed a cell-permeable Bodipy probe, JYQ-196, for covalent labeling of PARK7 in living cells and a first-in-class PARK7 degrader JYQ-194 that selectively induces its proteasomal degradation in human cells. Our study provides a valuable toolbox to enhance the understanding of PARK7 biology in cellular contexts and opens new opportunities for therapeutic interventions.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

EGFR endocytosis: more than meets the eye.

Behind the scenes of signaling cascades initiated by activated receptors, endocytosis determines the fate of internalized proteins through degradation in lysosomes or recycling. Over the years, significant progress has been made in understanding the mechanisms of endocytosis and deregulation in disease states. Here we review the role of the EGF-SNX3-EGFR axis in breast cancers with an extended discussion on deregulated EGFR endocytosis in cancer.

Total Chemical Synthesis of LC3A and LC3B Activity-Based Probes.

Autophagy is a conserved cellular process involved in the degradation of intercellular materials. During this process, double-membrane vesicles called autophagosomes engulf cytoplasmic components ready for degradation. A key component in the formation of autophagosomes are the autophagy-related (Atg) proteins, including microtubule-associated protein light chain 3A (LC3A) and 3B (LC3B). After the C-terminus of LC3 is conjugated to a phospholipid, it promotes the elongation of the phagosome and provides a docking station for the delivery of proteins ready for degradation. Since dysregulation of the autophagy pathway has been associated with a variety of human diseases, components of this process have been considered as potential therapeutic targets. However, the mechanistic details of LC3-specific ligases and deconjugation enzymes are far from unraveled and chemical tools for activity profiling could aid in affording more insights into this process. Herein, we describe a native chemical ligation approach for the synthesis of two LC3 activity-based probes (ABPs). Initial studies show that the probes covalently interact with the cysteine protease ATG4B, showcasing the potential of these probes to unravel mechanistic and structural details.

Chemical Toolkit for PARK7: Potent, Selective, and High-Throughput.

The multifunctional human Parkinson's disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson's disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors.

Inhibiting UCH-L5: Rational Design of a Cyclic Ubiquitin-Based Peptide Inhibitor.

The ubiquitin-proteasome system is an essential regulator of many cellular processes including controlling protein homeostasis. The degradation of proteins by the multi-subunit proteasome complex is tightly regulated through a series of checkpoints, amongst which are a set of deubiquitinating proteases (DUBs). The proteasome-associated DUBs, UCH-L5 (Ubiquitin carboxyl-terminal hydrolase isozyme L5) and USP14 (Ubiquitin-specific protease 14), and the integral-DUB in the proteasome, Rpn11, is known to regulate proteasomal degradation by deubiquitination of distinct substrates. Although selective inhibitors for USP14 and Rpn11 have been recently developed, there are no known inhibitors that selectively bind to UCH-L5. The X-ray structure of the Ubiquitin (Ub) bound to UCH-L5 shows a ß-sheet hairpin in Ub that contains a crucial hydrophobic patch involved in the interaction with UCH-L5. Herein, we designed and developed both a Ub sequence-based linear- and cyclic- ß-sheet hairpin peptide that was found to preferably inhibit UCH-L5. We show that these peptides have low micromolar IC50 values and the cyclic peptide competes with the activity-based UbVME (Ubiquitin-Vinyl-Methyl-Ester) probe for UCH-L5, binding in a concentration-dependent manner. We further establish the selectivity profile of the cyclic peptide for UCH-L5 compared to other members of the UCH-DUB family and other cysteine DUBs in cell lysate. Furthermore, the cyclic peptide infiltrated cells resulting in the accumulation of polyUb chains, and was found to be non-toxic at the concentrations used here. Taken together, our data suggest that the cyclic peptide permeates the cell membrane, inhibits UCH-L5 by possibly blocking its deubiquitinating function, and contributes to the accumulation of polyubiquitinated substrates. The implications of inhibiting UCH-L5 in the context of the 26S proteasome render it an attractive candidate for further development as a potential selective inhibitor for therapeutic purposes.

EGF-SNX3-EGFR axis drives tumor progression and metastasis in triple-negative breast cancers.

Epidermal growth factor receptor (EGFR) has critical roles in epithelial cell physiology. Over-expression and over-activation of EGFR have been implicated in diverse cancers, including triple-negative breast cancers (TNBCs), prompting anti-EGFR therapies. Therefore, developing potent therapies and addressing the inevitable drug resistance mechanisms necessitates deciphering of EGFR related networks. Here, we describe Sorting Nexin 3 (SNX3), a member of the recycling retromer complex, as a critical player in the epidermal growth factor (EGF) stimulated EGFR network in TNBCs. We show that SNX3 is an immediate and sustained target of EGF stimulation initially at the protein level and later at the transcriptional level, causing increased SNX3 abundance. Using a proximity labeling approach, we observed increased interaction of SNX3 and EGFR upon EGF stimulation. We also detected colocalization of SNX3 with early endosomes and endocytosed EGF. Moreover, we show that EGFR protein levels are sensitive to SNX3 loss. Transient RNAi models of SNX3 downregulation have a temporary reduction in EGFR levels. In contrast, long-term silencing forces cells to recover and overexpress EGFR mRNA and protein, resulting in increased proliferation, colony formation, migration, invasion in TNBC cells, and increased tumor growth and metastasis in syngeneic models. Consistent with these results, low SNX3 and high EGFR mRNA levels correlate with poor relapse-free survival in breast cancer patients. Overall, our results suggest that SNX3 is a critical player in the EGFR network in TNBCs with implications for other cancers dependent on EGFR activity.

Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-Terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos.

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 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.

USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7.

The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7-the logistical centerpiece of LE biology-as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.

How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

ALCAM is indirectly modulated by miR-125b in MCF7 cells.

MicroRNA (miRNA) deregulation is associated with various cancers. Among an expanding list of cancer-related miRNAs, deregulation of miR-125b has been well documented in many cancers including breast. Based on current knowledge, miR-125b is considered to be a tumor suppressor in breast cancers. While important messenger RNA (mRNA) targets have been defined for miR-125b, here, we aimed to further investigate direct/indirect consequences of miR-125b expression in breast cancer cells by using a transcriptome approach. Upon miR-125b expression, a total of 138 cancer-related genes were found to be differentially expressed in breast cancer cells. While only a few of these were predicted to be direct mRNA targets, majority of the gene expression changes were potentially downstream and indirect effects of miR-125b expression. Among these, activated leukocyte antigen molecule (ALCAM) mRNA and protein levels were found to be highly significantly increased upon miR-125b expression. Given the tumor suppressor role of miR-125b in our model system, upon silencing of ALCAM expression, cell proliferation rate re-increased in miR-125b-expressing cells. While ALCAM's possible context-dependent roles are not clear in breast cancer, a diverse expression pattern of ALCAM mRNA was detected in a panel of breast cancer patient samples. Differentially expressed/regulated cancer-related genes upon miR-125b expression along with the significant increase of ALCAM are of future interest to understand how deregulated expression of miR-125b may have a tumor suppressor role in breast and other cancers.

miR-125b targets ARID3B in breast cancer cells.

Mounting evidence suggests involvement of deregulated microRNA (miRNA) expression during the complex events of tumorigenesis. Among such deregulated miRNAs in cancer, miR-125b expression is reported to be consistently low in breast cancers. In this study, we screened a panel of breast cancer cell lines (BCCLs) for miR-125b expression and detected decreased expression in 14 of 19 BCCLs. Due to the heterogeneity of breast cancers, MCF7 cells were chosen as a model system for ERBB2 independent breast cancers to restore miR-125b expression (MCF7-125b) to investigate the phenotypical and related functional changes. Earlier, miR-125b was shown to regulate cell motility by targeting ERBB2 in ERBB2 overexpressing breast cancer cells. Here we showed decreased motility and migration in miR-125b expressing MCF7 cells, independent of ERBB2. MCF7-125b cells demonstrated profoundly decreased cytoplasmic protrusions detected by phalloidin staining of filamentous actin along with decreased motility and migration behaviors detected by in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Among possible numerous targets of miR-125b, we showed ARID3B (AT-rich interactive domain 3B) to be a novel target with roles in cell motility in breast cancer cells. When ARID3B was transiently silenced, the decreased cell migration was also observed. In light of these findings, miR-125b continues to emerge as an interesting regulator of cancer related phenotypes.

USP32 is an active, membrane-bound ubiquitin protease overexpressed in breast cancers.

USP32, on chromosomal band 17q23.1-17q23.2, is a highly conserved but uncharacterized gene that gave rise during evolution to a well-known hominoid-specific proto-oncogene, USP6. We investigated the expression profile of USP32 in human tissues and examined its functions to gain insight into this novel member of the well-conserved ubiquitination system. We detected ubiquitous USP32 expression across tissues and confirmed the predicted deubiquitination function owing to the presence of conserved peptidase signature aspargine, cysteine, histidine, and aspartic acid domains of ubiquitin-specific proteases. A Golgi localization of GFP-fused USP32 was detected by fluorescent protection assay and BODIPY-TR staining. In addition, stable silencing of USP32 caused a significant decrease in the proliferation and migration rate of cells. Based on these and the fact that USP32 maps to 17q23, which is commonly amplified in breast cancers, we analyzed USP32 expression in breast cancer cells. We detected high expression of USP32 in 50% (9 of 18) of breast cancer cell lines and 22% (9 of 41) of primary breast tumors compared to mammary epithelial cells. In summary, we report the preliminary characterization of this novel deubiquitinating enzyme on 17q23 and demonstrate its functional role in the ubiquitin system and its potential involvement in tumorigenesis.