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  • br Activity based probes br Characterization of coronavirus

    2020-02-05


    Activity-based probes
    Characterization of coronavirus-encoded DUBs with activity-based probes To study the activity of specific DUBs, investigators frequently take advantage of the ~10kDa (monoUb or Ubl ABP) or ~20kDa (DiUb ABP) increase in MW on probe labeling. SDS-PAGE analysis or blotting for an individual DUB, and comparing the intensities of the larger (labeled) band to the smaller (unlabeled) band, which allows the reactivity of the DUB toward the probe to be inferred. Here, we demonstrate how a typical experiment can be performed, by showing analysis of the MERS-CoV-encoded papain like cysteine protease (abbreviated PLpro), a viral DUB, upon incubation with our panel of ABPs. Additionally, having access to both “proximal” and “in-between” diUb probes we will demonstrate their use in the investigation of the binding interfaces that play a role in the specificity of the coronavirus-encoded DUBs MERS-CoV PLpro and SARS-CoV PLpro.
    Methods
    Conclusions and outlook The introduction of ABPs into living cells permit visualization and in-cell enzymology in a spatial, temporal, and substrate context, allowing study of the intrinsic regulation by cellular signaling events such as phosphorylation of DUBs to enhance their proteolytic activity as highlighted by the necessity of serine phosphorylation of OTUD5/DUBA (Huang et al., 2012). Most ABP profiling experiments are currently performed using either recombinant enzymes or cell lysates, although several methods allowing their biochemical study in a functional cellular environment are emerging, such as electroporation (Mulder et al., 2016) or the use of cell-penetrating peptides (Gui et al., 2018; Shahul Hameed, Sapmaz, Gjonaj, Merkx, & Ovaa, 2018).
    Introduction Research from the past several decades has implicated the posttranslational modifier ubiquitin (Ub) in the regulation of nearly all aspects of cellular signaling, including fundamental roles in proteasome-mediated protein degradation, 832 582 4016 progression, and immune responses. The breadth of cellular roles played by Ub stems in part from its ability to be further posttranslationally modified. Modification of Ub can take the form of additional ubiquitination at one or several of its eight possible amide linkage points, creating complex polyUb chains; by modification with a Ub-like modifier (UBL) such as NEDD8 or SUMO; or through the addition of small chemical groups such as phosphorylation or acetylation (Swatek & Komander, 2016). The combinatorial possibilities of these alterations are enormous and give rise to what is sometimes called the “Ub code” (Komander & Rape, 2012). While the significance for many aspects of the Ub code remain to be deciphered, it is clear from the immense body of work at hand that breakdown or dysregulation of Ub signaling can result in severe health-related consequences (Rape, 2018). Therefore, all aspects of the Ub system are under tight control by hundreds of enzymes that together constitute the “writers,” “readers,” and “erasers” of the Ub code. Writer enzymes, consisting of the E1 Ub-activating, E2 Ub-conjugating, and E3 Ub-ligating enzymes, regulate the synthesis of defined Ub signals on specific targets. Reader proteins recognize these signals and help elicit the desired cellular outcomes. Eraser enzymes, also known as deubiquitinases (DUBs), are key regulators of the Ub system. Humans encode approximately 100 DUB genes belonging to seven protease families (Haahr et al., 2018; Hermanns et al., 2018; Hewings et al., 2018; Kwasna et al., 2018; Mevissen & Komander, 2017). Additional proteases are specific toward UBL modifiers; herein we collectively refer to all Ub/UBL proteases as DUBs for simplicity. These specialized proteases hydrolyze the isopeptide or peptide linkage at the carboxy-terminus of the Ub/UBL modifier that links it to substrate primary amine groups, usually lysine side chains, thus reversing the action of the writer enzyme and recycling the Ub/UBL back into the free pool for future rounds of conjugation. DUBs can be exquisitely specific for discrete cellular targets, either by selecting particular forms of the modification (e.g., OTULIN (Keusekotten et al., 2013; Rivkin et al., 2013)), by recognizing the modified substrate (e.g., the SAGA complex (Morgan et al., 2016)), or via regulation of subcellular localization (e.g., USP30 (Bingol et al., 2014)) (reviewed in Mevissen & Komander, 2017). Owing to their roles as key regulators of the Ub signal, DUBs have recently become a popular target for pharmacological intervention (Gavory et al., 2018; Kategaya et al., 2017; Lamberto et al., 2017; Pozhidaeva et al., 2017; Turnbull et al., 2017) and show potential for “drugging the undruggable” (Huang & Dixit, 2016).