IUSM-Purdue TREAT-AD Center Target Enabling Report Proline-rich Tyrosine Kinase 2 beta (PTK2β): Protein Constructs, Cryo-EM, Biophysical Assay, and Biochemical Assay

A Target Enabling report detailing the Protein Construct, Cryo-EM, Biophysical Assay and Biochemical Assay work around PTK2

IUSM-Purdue TREAT-AD Center Target Enabling Report Proline-rich Tyrosine Kinase 2 beta (PTK2β): Protein Constructs, Cryo-EM, Biophysical Assay, and Biochemical Assay

A Target Enabling report detailing the Protein Construct, Cryo-EM, Biophysical Assay and Biochemical Assay work around PTK2

IUSM-Purdue TREAT-AD Center Target Enabling Report LYN, Src family tyrosine-protein kinase: Protein Constructs, Cryo-EM, and Biochemical Assay

A Target Enabling Report detailing Protein Constructs, Cryo-EM and Biochemical assay for LY

IUSM-Purdue TREAT-AD Center Target Enabling Report LYN, Src family tyrosine-protein kinase: Protein Constructs, Cryo-EM, and Biochemical Assay

A Target Enabling Report detailing Protein Constructs, Cryo-EM and Biochemical assay for LY

Elucidating the Role of HAUSP Ubiquitin Like Domains in the Catalytic Function of USP7

Ubiquitin specific proteases (USPs) are a class of enzymes involved in myriad cellular processes. One USP of great interest due to its oncogenic properties is USP7. In normal conditions USP7 is closely regulated due to its responsibility for destabilizing the tumor suppressor, p53, through the deubiquitination of MDM2. In multiple myeloma cases, it appears the regulation of USP7 subsides, as it is largely overexpressed, leading to the inappropriate degradation of p53. Inhibition of USP7 could, therefore, prove a viable target for cancer therapy. A greater understanding of USP7’s function and structure can lead to more insight into how this enzyme could be inhibited. USP7 is composed of the TRAF, catalytic and 5 HUBL domains. Previous work has shown that the catalytic activity of USP7 is greatly reduced in the absence of the HUBL 4 and 5 (H-45) domains. However, it is unclear if the other HUBL domains have specific roles in USP7 activity. To evaluate the individual HUBL domain roles in USP7s activity, constructs containing the full length HUBL domain, as well as just H-45 truncations were obtained. Each construct was expressed in E. coli BL21 (DE3) cells and purified by chromatography. These constructs were left with their respective histidine tags in order to evaluate the kinetics of their interactions in trans with the catalytic domain using the Fortebio Octet Red 384 system. Kinetic assays using the ubiquitin rhodamine substrate showed that the histidine tagged proteins are still able to activate the catalytic domain of USP7. Optimization of the Fortebio Octet Red 384 system suggested that the catalytic domain bound nonspecifically to the Anti-Penta-His (HIS1K) obscuring the off binding rates of the HUBL protein. Further truncations of the HUBL domains including H1, H2, H3, H1-2 were successfully subcloned using recombinant cloning techniques and will be analyzed using the Octet system

Engineering DUB-deficient Viral Proteases from FIPV and PEDV Coronaviruses

Coronaviruses form a class of viral pathogens lethal to humans and livestock. This issue is compounded by a lack of commercially available treatments or vaccines. In 2014, porcine epidemic diarrhea virus (PEDV) emerged in the United States and accounted for an estimated 7 million porcine deaths. Deaths of humans, companion animals, and livestock caused by coronaviruses highlight the need for therapeutic strategies to combat this devastating disease. One strategy involves engineering papain-like protease 2 (PLP2), an enzyme conserved among coronavirus species that is critical for virus replication and pathogenesis. PLP2’s de-ubiquitinating (DUB) activity aids in the suppression of the host’s innate antiviral immune response. By targeting and disrupting ubiquitin binding in PLP2 and thus its DUB activity, the virus would no longer be able to antagonize the innate immune response. To this end, we introduced informed single-point mutations in PEDV and in feline infectious peritonitis virus (FIPV) PLP2s using structure-guided mutagenesis. We then characterized the kinetic activity of the resulting mutants in vitro using fluorescent peptide and ubiquitin substrates. Through these studies, we were able to evaluate the relationship between PLP2-ubiquitin binding and DUB activity. Preliminary data analysis suggests that residues outside the active site of PLP2 and within the ubiquitin-binding interface are necessary for DUB activity; these residues can be selectively disrupted to abolish DUB activity relative to the wild-type. These results describe a series of DUB-deficient PLP2 mutants that can be leveraged as tools for use in future coronavirus research. Such tools will allow creation of an attenuated virus strain that could aid in vaccine and drug design

Study of Coronavirus Protease Using CFP-YFP Fluorescent Assay

Middle Eastern Respiratory Syndrome (MERS) is an emerging viral disease originating in the Arabian Peninsula with a current mortality rate of nearly fifty percent throughout Europe and Asia according to the World Health Organization. Characterization of this disease is being done to understand the basis of viral replication. One target for viral inhibition are replication proteases. Replication proteases are enzymes that cleave proteins specific to cell growth and reproduction that form the viral replicase complex making them an ideal target for viral replication inhibition. First, replication proteases were characterized using a fluorescence resonance energy transfer (FRET) construct by measuring the amount of fluorescence emitted during enzymatic activity. This construct produces a measurable change in fluorescent activity to analyze the rate at which replication proteases cleave proteins essential for viral growth. Once this assay was completed, data was extracted and enzymatic kinetic calculations were performed to continue further analysis of enzymatic activity. The results produced from these experiments will allow a comparison of replication proteases specific to MERS with other viral replication proteases. Further analysis will be done to measure varying cleavage rates of different coronaviruses. This study produces conclusive results for the characterization of MERS replication proteases that are essential in further development of inhibitor molecules

Structural and mechanistic analysis of trans-3-chloroacrylic acid dehalogenase activity

The X-ray structure of a noncovalently modified trans-3-chloroacrylic acid dehalogenase with a substrate-homolog acetate bound in the active site has been determined to 1.7 Å resolution. Elucidation of catalytically important water is reported and multiple conformations of the catalytic residue αGlu52 are observed

Scattering layers and vertical distribution of oceanic animals off Oregon

This paper reviews some of the distributional features of vertically migrating micronekton off Oregon; describes a new, conducting-cable, midwater-trawl system using an eight-net, opening-closing cod-end unit; and gives some preliminary results on trawl catches relative to sound-scattering layers. A variable complex of organisms, including euphausiids, a sergestid shrimp, and mesopelagic fishes, was often common in 12- and 38.5-kHz scattering layers. The depth range of many species was broad, and sometimes the largest catches were made at depths above or below scattering layers. Variability was large among nets that fished either horizontally or vertically during single tows