107 research outputs found
Functional Analyses of ABHD17 Enzymes
Post-translational modifications (PTMs) play a crucial role in trafficking proteins for many location-dependent cellular functions. Protein S-acylation describes the addition of long chain fatty acids (predominantly the C16:0 palmitate) to cysteines via a thioester bond. This reversible modification thus allows for controlled regulation of protein membrane tethering during various cellular processes. Indeed, S-palmitoylated proteins include kinases, small GTPases and transmembrane receptors, which function in response to diverse signaling events. It is important to study the enzymes that catalyze this modification since it is involved in many essential pathways. Enzymes such as APT1 and APT2 have thoroughly been studied and shown to depalmitoylate a myriad of S-palmitoylated substrates and the development of selective inhibitors has accelerated the discovery of new substrates. ABHD17 has more recently been shown to depalmitoylate substrates in cell-based studies. However, not much is known about its substrate recognition mechanism and cellular function and my dissertation helps to bridge this gap in knowledge. The first chapter presents a detailed introduction to protein depalmitoylases, their different cellular roles and methods that have been developed to study them. The following chapter focuses on understanding the in vitro activity of ABHD17 and the development of potential inhibitors to study its function. The third chapter outlines a proteomics strategy that can be used to study S-palmitoylation of multiple proteins at a time and was applied to understand how ABHD17 regulates S-palmitoylation in cells. Finally, future experiments are proposed in Chapter 4 to further improve our understanding of how ABHD17 works and what its cellular function is.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153388/1/mcheusk_1.pd
Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions
Gas-phase ion/ion chemistry was coupled to ion mobility/mass spectrometry analysis to correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Aqueous solutions stabilizing the native state of ubiquitin yielded folded ubiquitin structures with CCS values consistent with previously published literature. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous conditions were covalently modified preferentially at the lysine 29 and arginine 54 positions, indicating that elements of three-dimensional structure were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol- and low-pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase
Attacking logo-based phishing website detectors with adversarial perturbations
Recent times have witnessed the rise of anti-phishing schemes powered by deep
learning (DL). In particular, logo-based phishing detectors rely on DL models
from Computer Vision to identify logos of well-known brands on webpages, to
detect malicious webpages that imitate a given brand. For instance, Siamese
networks have demonstrated notable performance for these tasks, enabling the
corresponding anti-phishing solutions to detect even "zero-day" phishing
webpages. In this work, we take the next step of studying the robustness of
logo-based phishing detectors against adversarial ML attacks. We propose a
novel attack exploiting generative adversarial perturbations to craft
"adversarial logos" that evade phishing detectors. We evaluate our attacks
through: (i) experiments on datasets containing real logos, to evaluate the
robustness of state-of-the-art phishing detectors; and (ii) user studies to
gauge whether our adversarial logos can deceive human eyes. The results show
that our proposed attack is capable of crafting perturbed logos subtle enough
to evade various DL models-achieving an evasion rate of up to 95%. Moreover,
users are not able to spot significant differences between generated
adversarial logos and original ones.Comment: To appear in ESORICS 202
Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions
Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes
The combination of ion/ion chemistry with commercially available ion mobility/mass spectrometry systems has allowed rich structural information to be obtained for gaseous protein ions. Recently, the simple modification of such an instrument with an electrospray reagent source has allowed three-dimensional gas-phase interrogation of protein structures through covalent and noncovalent interactions coupled with collision cross section measurements. However, the energetics of these processes have not yet been studied quantitatively. In this work, previously developed Monte Carlo simulations of ion temperatures inside traveling wave ion guides are used to characterize the energetics of the transition state of activated ubiquitin cation/sulfo-benzoyl-HOAt reagent anion long-lived complexes formed via ion/ion reactions. The ΔH‡ and ΔS‡ of major processes observed from collisional activation of long-lived gas-phase ion/ion complexes, namely collision induced unfolding (CIU), covalent bond formation, or neutral loss of the anionic reagent via intramolecular proton transfer, were determined. Covalent bond formation via ion/ion complexes was found to be significantly lower energy compared to unfolding and bond cleavage. The ΔG‡ values of activation of all three processes lie between 55 and 75 kJ/mol, easily accessible with moderate collisional activation. Bond formation is favored over reagent loss at lower activation energies, whereas reagent loss becomes competitive at higher collision energies. Though the ΔG‡ values between CIU of a precursor ion and covalent bond formation of its ion/ion product complex are comparable, our data suggest covalent bond formation does not require extensive isomerization
Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking
Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure
Multi-tissue integrative analysis of personal epigenomes
Evaluating the impact of genetic variants on transcriptional regulation is a central goal in biological science that has been constrained by reliance on a single reference genome. To address this, we constructed phased, diploid genomes for four cadaveric donors (using long-read sequencing) and systematically charted noncoding regulatory elements and transcriptional activity across more than 25 tissues from these donors. Integrative analysis revealed over a million variants with allele-specific activity, coordinated, locus-scale allelic imbalances, and structural variants impacting proximal chromatin structure. We relate the personal genome analysis to the ENCODE encyclopedia, annotating allele- and tissue-specific elements that are strongly enriched for variants impacting expression and disease phenotypes. These experimental and statistical approaches, and the corresponding EN-TEx resource, provide a framework for personalized functional genomics
- …