Nε-acetylation of Residues K57 and K401 is a Potential Posttranslational Modulator of the Multiple Catalytic Activities of Protein Disulfide Isomerase

Despite its study since the 1960\u27s, very little is known regarding the regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI). A variety of conserved residues have been implicated as either important or vital for activity. This work ventures to identify a functional role for the highly conserved CGHC-flanking residues Lys57 and Lys401 of human PDI in vitro. Site-directed mutagenesis studies have revealed a role for the active site lysine residues in modulating the oxidoreductase activity of PDI in a pH-dependent manner. The effects of mutagenesis indicated that, along with the oxidoreductase activity, the kinetics of thiol-reductase and thiol-oxidase catalysis were also attenuated. Substitution of the aforementioned residues to glutamine, alanine or glutamic acid resulted in an enzyme variant 20 to 35% as efficient as wild type. This was found to translate to the decreased rate of electron shuttling between PDI and ERO1α to as little as 54% that of wild type. Consistent with this, molecular dynamic simulations suggest a role for Lys57 and Lys401 in differentially mediating the accessibility of the nucleophilic cysteine of each active site. The possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using Aspirin, coupled with mass spectrometry. A total of 18 acetyllysine (acK) residues were identified reproducibly, including acK57 and acK401. Altered enzyme kinetics as a result of acetylation by Aspirin corroborate with site-directed mutagenesis data. This provides a strong indication that acetylation of residues Lys57 and Lys401 is a potential modulator of the catalytic activities of PDI

Allosteric regulation of the deubiquitinase USP8 and applications of ubiquitin variant tool molecules.

Ubiquitination is an essential post-translational event for cellular homeostasis. The attachment of ubiquitin (Ub) to a protein is, in some capacity, involved in every molecular process in the cell. Dysregulation of such has compelling links to cancers, ageing-related diseases, intellectual disorders, and many more pathologies. Currently being tested are a slew of therapies targeting players of Ub signaling, and while some have been clinically approved for E3 ligases that mediate Ub conjugation, therapies available for deubiquitinases (DUBs), which mediate deconjugation, lag behind. This is partly due to the complexity of these enzymes. This work focuses on characterizing the activity of one such DUB, USP8, and aims to also test the application of its Ub variant (UbV) inhibitor, known as UbV8.2, in biosensing and interactomics. USP8 is of particular interest for its demonstrated roles in glioblastoma, Parkinson\u27s disease, and Cushing\u27s disease. In addition, it is unique for being the only USP intramolecularly autoinhibited by its auxiliary domain, but how this occurs is not well understood. Here, we characterized the mechanism of autoinhibition, revealing a WW-like inhibitory domain that, due to its low affinity for the catalytic domain, functions only in cis. Modelling indicates it competes with Ub by binding the S1 pocket in a novel way. We speculate that USP8 evolved this mechanism for low-level basal activity. UbVs are engineered proteins that bind targets with exceptional potency. These tool molecules can probe DUB biology and drive the discovery of novel inhibitors. UbV8.2 is the only USP8 inhibitor of its kind. We recognized that these binders could be leveraged for broad applications, thus we tested UbV8.2 in a proof-of-concept study and developed a novel electronic biosensor capable of detecting analyte (USP8) with a sub-nanomolar limit of detection. Despite the usefulness of UbV8.2 in vitro, we wondered about its fidelity in more complex settings in vivo. The pipeline of UbV development is high throughput, leading to inconsistencies when validating binders during selection. As such, we sought a method to rigorously validate UbVs in a cellular context. Based on in vivo proximity biotinylation, we named the method uBioID—the biotinylation of proteins in proximity to the UbV when expressed inside the cell. Should the UbV co-localize and bind its endogenous target, only proteins in the vicinity of the target, such as physical interactors, will be biotinylated and detected using mass spectrometry. A comparative study of UbV8.2 and another well-characterized UbV, UbV7.2, was performed. The findings showed, under our conditions, UbV8.2 is inadequate in the cellular milieu, as it is unable to engage its endogenous target, USP8. On the other hand, UbV7.2 showed robust target engagement, thus facilitating the identification of potential interactors of its cognate target, USP7. uBioID presents as a platform for both UbV validation and target interactomics. Achieved here is a deeper understanding of DUB regulatory mechanisms, coupled with new applications and validation methods for UbVs, thus lending critical information to aid expansion of the druggable space for this recalcitrant enzyme class

On the Study of Deubiquitinases: Using the Right Tools for the Job

Deubiquitinases (DUBs) have been the subject of intense scrutiny in recent years. Many of their diverse enzymatic mechanisms are well characterized in vitro; however, our understanding of these enzymes at the cellular level lags due to the lack of quality tool reagents. DUBs play a role in seemingly every biological process and are central to many human pathologies, thus rendering them very desirable and challenging therapeutic targets. This review aims to provide researchers entering the field of ubiquitination with knowledge of the pharmacological modulators and tool molecules available to study DUBs. A focus is placed on small molecule inhibitors, ubiquitin variants (UbVs), and activity-based probes (ABPs). Leveraging these tools to uncover DUB biology at the cellular level is of particular importance and may lead to significant breakthroughs. Despite significant drug discovery efforts, only approximately 15 chemical probe-quality small molecule inhibitors have been reported, hitting just 6 of about 100 DUB targets. UbV technology is a promising approach to rapidly expand the library of known DUB inhibitors and may be used as a combinatorial platform for structure-guided drug design

Using ubiquitin variants to understand the function of RFWD3 at the molecular level

Tagging of cellular proteins with marks, also known as post-translational modification, is a natural mechanism that controls the function of proteins and biological pathways. Among all protein modifications, ubiquitination is the second most abundant. It involves the attachment of ubiquitin, a small protein, unto a target protein. Once ubiquitinated, the structure of the target protein is drastically altered, resulting in a different fate. Ubiquitination is critical to DNA damage repair. Our research uses engineered ubiquitin mutants, known as ubiquitin variants (UbVs), to study the function of a ubiquitination enzyme, RFWD3, that is important for DNA damage repair. UbVs bind with the target protein they raised against tightly and specifically, thus perturb the activity of that enzyme and reveal its function. Heritable mutations that negatively affect the function of RFWD3 have been linked to a rare cancer known as Fanconi anemia (FA); making this a potential therapeutic target. Using UbVs specific for RFWD3 we aim to understand how it recognizes substrates and to elucidate the molecular details of its involvement in DNA damage repair. By screening a candidate library of UbVs engineered to bind the substrate-binding domain of RFWD3 we will identify the best binders for further analyses in cancer cells. Ultimately, the UbVs identified in this work have the potential to become a protein-based drug to treating DNA damage repair-related diseases

Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity

DataSheet2.XLSX

<p>Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys⁵⁷ and Lys⁴⁰¹ of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys⁵⁷ and Lys⁴⁰¹ was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys⁵⁷ and acLys⁴⁰¹. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.</p

DataSheet1.DOCX

<p>Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys⁵⁷ and Lys⁴⁰¹ of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys⁵⁷ and Lys⁴⁰¹ was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys⁵⁷ and acLys⁴⁰¹. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.</p

Building a Versatile Platform for the Detection of Protein–Protein Interactions Based on Organic Field-Effect Transistors

Detection and characterization of biomolecular interactions, such as protein–protein interactions (PPIs), are critical to a fundamental understanding of biochemical processes, thus being a driver of innovation for drug discovery, clinical diagnostics, and protein engineering. Among the many sensor types used to probe PPIs, organic field-effect transistors are particularly desirable due to their unique features, including tunability, sensitivity, low-power requirements, and multi-parameter readouts. This work describes the development of a biosensor based on organic field-effect transistors, covalently functionalized at the surface with an engineered ubiquitin variant for the specific and sensitive detection of ubiquitin-specific protease 8 (USP8). The resulting sensor was carefully characterized to reveal both electronic and solid-state properties. The sensing platform showed high sensitivity (sub-nanomolar analyte concentrations) and selectivity for USP8 and robust performance that suggests that it may be highly tunable. The sensing system introduced in this work provides a detection method for PPIs, which constitutes a promising platform for advanced biotechnology applications