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The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.

Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

While exoskeleton technology is becoming more and more common for gait rehabilitation in children with neurological disorders, evaluation of gait performance still faces challenges and concerns. The reasoning behind evaluating the spinal locomotor output is that, while exoskeleton's guidance forces create the desired walking kinematics, they also affect sensorimotor interactions, which may lead to an abnormal spatiotemporal integration of activity in particular spinal segments and the risk of abnormalities in gait recovery. Therefore, traditional indicators based on kinematic or kinetic characteristics for optimizing exoskeleton controllers for gait rehabilitation may be supplemented by performance measures associated with the neural control mechanisms. The purpose of this study on a sample of children was to determine the basic features of lower limb muscle activity and to implement a method for assessing the neuromechanics of spinal locomotor output during exoskeleton-assisted gait. To this end, we assessed the effects of a robotic exoskeleton (ExoAtlet Bambini) on gait performance, by recording electromyographic activity of leg muscles and analyzing the corresponding spinal motor pool output. A slower walking setting (about 0.2 m/s) was chosen on the exoskeleton. The results showed that, even with slower walking, the level of muscle activation was roughly comparable during exoskeleton-assisted gait and normal walking. This suggests that, despite full assistance for leg movements, the child's locomotor controllers can interpret step-related afferent information promoting essential activity in leg muscles. This is most likely explained by the active nature of stepping in the exoskeleton (the child was not fully relaxed, experienced full foot loading and needed to maintain the upper trunk posture). In terms of the general muscle activity patterns, we identified notable variations for the proximal leg muscles, coactivation of the lumbar and sacral motor pools, and weak propulsion from the distal extensors at push-off. These changes led to the lack of characteristic lumbosacral oscillations of the center of motoneuron activity, normally associated with the pendulum mechanism of bipedal walking. This work shows promise as a useful technique for analyzing exoskeleton performance to help children develop their natural gait pattern and to guide system optimization in the future for inclusion into clinical care. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer is a highly aggressive malignancy with limited therapeutic options and a poor prognosis. Deubiquitinating enzymes (DUBs) have emerged as critical regulators of protein ubiquitination and proteasomal degradation, influencing various cellular processes relevant to cancer pathogenesis. In this study, the research progress between ovarian cancer and DUBs was mapped and visualized using bibliometrics, and the expression patterns and biological roles of DUBs in ovarian cancer were summarized. Methods: Studies related to DUBs in ovarian cancer were extracted from the Web of Science Core Collection (WoSCC) database. VOSviewer 1.6.20, CiteSpace 6.3.R1, and R4.3.3 were used for bibliometric analysis and visualization. Results: For analysis 243 articles were included in this study. The number of publications on DUBs in ovarian cancer has gradually increased each year. China, the United States, and the United Kingdom are at the center of this field of research. The Johns Hopkins University, Genentech, and Roche Holding are the main research institutions. David Komander, Zhihua Liu, and Richard Roden are the top authors in this field. The top five journals with the largest publication volumes in this field are Biochemical and Biophysical Research Communications, Journal of Biological Chemistry, PLOS One, Nature Communications, and Oncotarget. Keyword burst analysis identified five research areas: "deubiquitinating enzyme," "expression," "activation," "degradation," and "ubiquitin." In addition, we summarized the expression profiles and biological roles of DUBs in ovarian cancer, highlighting their roles in tumor initiation, growth, chemoresistance, and metastasis. Conclusion: An overview of the research progress is provided in this study on DUBs in ovarian cancer over the last three decades. It offers insight into the most cited papers and authors, core journals, and identified new trends. [ABSTRACT FROM AUTHOR]

Biped robots usually adopt feet with a rigid structure that simplifies walking on flat grounds and yet hinders ground adaptation in unstructured environments, thus jeopardizing stability. We recently explored in the SoftFoot the idea of adapting a robotic foot to ground irregularities along the sagittal plane. Building on the previous results, we propose in this paper a novel robotic foot able to adapt both in the sagittal and frontal planes, similarly to the human foot. It features five parallel modules with intrinsic longitudinal adaptability that can be combined in many possible designs through optional rigid or elastic connections. By following a methodological design approach, we narrow down the design space to five candidate foot designs and implement them on a modular system. Prototypes are tested experimentally via controlled application of force, through a robotic arm, onto a sensorized plate endowed with different obstacles. Their performance is compared, using also a rigid foot and the previous SoftFoot as a baseline. Analysis of footprint stability shows that the introduction of the transverse arch, by elastically connecting the five parallel modules, is advantageous for obstacle negotiation, especially when obstacles are located under the forefoot. In addition to biped robots' locomotion, this finding might also benefit lower-limb prostheses design. [ABSTRACT FROM AUTHOR]

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a "target" not necessarily connected to the "mechanism of action" thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level. [ABSTRACT FROM AUTHOR]

The efficacy of cancer immunotherapy depends on the fine interplay between tumoral immune checkpoints and host immune system. However, the up-to-date clinical performance of checkpoint blockers in cancer therapy revealed that higher-level regulation should be further investigated for better therapeutic outcomes. It is becoming increasingly evident that the expression of immune checkpoints is largely associated to the immunotherapeutic response and consequent prognosis. Deubiquitinating enzymes (DUBs) with their role of cleaving ubiquitin from proteins and other molecules, thus reversing ubiquitination-mediated protein degradation, modulate multiple cellular processes, including, but not limited to, transcriptional regulation, cell cycle progression, tissue development, and antiviral response. Accumulating evidence indicates that DUBs also have the critical influence on anticancer immunity, simply by stabilizing pivotal checkpoints or key regulators of T-cell functions. Therefore, this review summarizes the current knowledge about DUBs, highlights the secondary checkpoint-like role of DUBs in cancer immunity, in particular their direct effects on the stability control of pivotal checkpoints and key regulators of T-cell functions, and suggests the therapeutic potential of DUBs-based strategy in targeted immunotherapy for cancer. [ABSTRACT FROM AUTHOR]

Background: C type CpG oligodeoxynucleotides (CpG-C ODNs), possessing the features of both A type and B type CpG ODNs, exert a variety of immunostimulatory activities and have been demonstrated as an effective antitumor immunotherapy. Based on the structural characteristics, we designed 20 potential ODNs with the aim of synthesizing an optimal, novel CpG-C ODN specific to human and murine Toll-like receptor 9 (TLR9). We also sought to investigate the in vitro immunostimulatory and in vivo antitumor effects of the novel CpG-C ODN. Methods: Twenty potential CpG-C ODNs were screened for their ability to secrete interferon (IFN)-α, and interleukin (IL)-6 and tumor necrosis factor (TNF)-α production for the three most promising sequences were assayed in human peripheral blood mononuclear cells (PBMCs) by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array assay. The functions of human and mouse B cells, and cytokine production in mice induced by the most promising sequence, HP06T07, were determined by flow cytometry and ELISA. Growth and morphology of tumor tissues in in vivo murine models inoculated with CT26 cells were analyzed by a growth inhibition assay and immunohistochemistry, respectively. Results: Among the 20 designed ODNs, HP06T07 significantly induced IFN-α, IL-6, and TNF-α secretion, and promoted B-cell activation and proliferation in a dose-dependent manner in human PBMCs and mouse splenocytes in vitro. Intratumoral injection of HP06T07 notably suppressed tumor growth and prolonged survival in the CT26 subcutaneous mouse model in a dose-dependent manner. HP06T07 administered nine times at 2-day intervals (I2) eradicated tumor growth at both primary and distant sites of CT26 tumors. HP06T07 restrained tumor growth by increasing the infiltration of T cells, NK cells, and plasmacytoid dendritic cells (pDCs). Conclusions: HP06T07, a novel CpG-C ODN, shows potent immunostimulatory activity in vitro and suppresses tumor growth in the CT26 subcutaneous mouse model. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) antibodies may have detrimental effects in patients with metastatic colorectal cancer expressing oncogenic Rat sarcoma (RAS). Since a significant number of patients acquire RAS-mediated resistance during EGFR-directed treatment, understanding the molecular mechanism underlying these antibody-mediated tumor-promoting effects is of relevance to design more resistance-preventive treatment approaches. To test this, we set up a Ba/F3 cellular model system transformed to EGFR/RAS dependency to be able to study proliferation, RAS activity as well as MAPK signaling upon inhibition of wild-type RAS isoforms by therapeutic EGFR antibodies. Here, we show that the EGFR antibodies cetuximab and panitumumab induce paradoxical stimulation and enhance proliferation in cells expressing oncogenic RAS (KRAS G12V). These experiments clearly showed that the stimulatory effect is a direct result of the antibody-EGFR interaction leading to prolonged mitogen-activated protein-Kinase (MAPK) signaling. The effect was also induced by antibody-chemotherapy combinations but always depended on simultaneous low-level ligand-dependent EGFR pathway activation. Moreover, we observed significant growth retardation of RAS mutant cells after antibody withdrawal compatible with a drug-addiction phenotype. Our data suggests that EGFR antibodies paradoxically sustain MAPK signaling downstream of oncogenic RAS thereby driving proliferation of RAS mutant tumors or tumor subclones. The observed drug-addiction encourages fixed-duration or liquid-biopsy-guided drug holiday concepts to preventively clear RAS mutant subclones selected under EGFR-directed therapeutic pressure. [ABSTRACT FROM AUTHOR]

Mutations and altered expression of deubiquitinating enzymes (DUBs) have been found associated with many human diseases including cancers. In this study, Ubiquitin specific protease 1 (USP1) expression was found significantly increased in some colorectal cancers (CRC). The elevated USP1 level was associated with short overall survival of patients and with advanced stages of cancers. In cultured CRC cells, knockdown of USP1 induced growth arrest at G2/M of cell cycle and reduced the expression of anti-apoptotic proteins Bcl-2 and Mcl-1. Its knockdown also led to reduction of DNA-repair related substrates FANCD2 and ID1. Further investigations found that small molecular inhibitor of USP1 ML323 sensitized CRC cells to DNA-targeting chemotherapeutics, including doxorubicin, TOPI/II inhibitors, and PARP inhibitor, but not to 5-Fu. These results indicate that USP1 plays a critical in colorectal cancer cell survival and is a promising target for anti-colorectal cancer chemotherapy. Targeting USP1 may represent an effective strategy to regulate the DNA-repairing system. [ABSTRACT FROM AUTHOR]