29th Annual Conference
"Expanding the Frontiers in Biocatalytic Science"
October 22, 2022
The University of Iowa
Pharmacy Building, Iowa City, IA
Lilliana Radoshevich, Ph.D., Assistant Professor of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA
"Mapping the role of Unerstudied Ubiquitin-Like Proteins in Infection and Disease"
During infection the ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogenic proteins. While progress has been made in identifying sites of modification on target proteins, the molecular consequences of ISGylation on individual protein substrates are still unknown. Here by using a model of enhanced ISGylation, we identify that ISG15 modifies the ARP2/3 complex following both bacterial and viral infection. ISGylation significantly slows Listeria-actin comet tail speed leading to morphologically shorter and denser comet tails. For Vaccinia virus this results in reduced spread, whereas while Listeria is initially restricted, over time the bacteria divide while remaining tightly attached to actin filaments resulting in multi-headed comet tails that move as a group. These structures contribute to spread in both human and murine cells and in vivo in mice with unchecked ISGylation, which leads to increased mortality. ISG15 modification of the Arp2/3 complex also affects cortical actin density, cell motility, and adhesion. Furthermore, ISG15-deficient neonates have aberrant epidermal epithelia, which correlates with observed defects in wound healing in a subset of human patients who lack ISG15. Our discovery identifies a conserved molecular mechanism of ISG15 modification of the Arp2/3 complex which directly restricts pathogen spread.
Nigel F. Reuel, Ph.D., Associate Professor of Chemical and Biological Engineering, Jack R. and Carol A. Johnson Faculty Fellow, Director of Graduate Education, COE Entrepreneurial Fellow, Iowa State University of Science and Technology
"Challenges and Opportunities in Process Analytical Technologies (PAT) for Biomanufacturing"
This talk will cover recent advances by the Reuel group at Iowa State University to develop and commercialize novel sensors for PAT and control strategies. These operate on two transduction methods – fluorescent single walled carbon nanotubes (SWCNT) and radio frequency resonant sensors. The near infrared SWCNT probes have been used to measure antibody binding, glycosylation, and protein activity. The resonant sensors have been applied to contact-free measurement of anaerobic fermentation, adherent cell culture, and static cell culture. Progress on advanced, AI-assisted control methods for optimization and control of biomanufacturing will be highlighted. These advances will be presented in context of traditional measurement techniques to highlight current challenges and opportunities in this space. Learnings from commercialization efforts through Volvox Biologic Inc., Frugi Biotechnology Inc., Skroot Laboratory Inc., and Zymosense Inc. will be shared to highlight best practices of translating technologies from academia to commercial practice. In conclusion, a combined vision of advanced biomanufacturing research and industrial practice will be presented in the context of a recently submitted NSF EPSCoR proposal that seeks to build biomanufacturing capacity in Iowa.
Christina Taylor, PhD., Computational Protein Design Lead and Science Fellow, Bayer Crop Science, St. Louis, MO
"Computational Molecular Design in Plant Biotechnology at Bayer Crop Science"
In the near future, the world will face many challenges. A growing population, as well as climate change, creates challenges to our food supply. In my talk, I will discuss how we are addressing these challenges at Bayer Crop Science. Specifically, I will discuss how we are driving innovation and new products in the Computational Molecular Design Team in the Data Science and Analytics Group. The Computational Molecular Design Team designs proteins for the insect control and herbicide tolerance pipeline, designs synthetic elements for expression, and works with the Biotransformation Group. Highlights from projects in each of these areas will be discussed during the talk.
Thaddeus J. Wadas, Ph.D., Associate Professor of Radiology, Carver College of Medicine, University of Iowa, Iowa City, IA
"Targeting Fibroblast Activation Protein Alpha as a Robust Biomarker in Glioma"
Gliomas represent a diverse group of central nervous system cancers including astrocytomas, oligodendromas, ependymomas and the most aggressive, glioblastoma multiforme (GBM). Despite the use of surgery, radiotherapy and pharmacotherapy, prognosis for patients remains poor. This has led researchers to identify and validate new biomarkers that may be exploited for imaging and therapy so that patient outcomes will improve.
The dipeptidyl peptidase (DPP) family of proteins includes DPP4, DPP8, DPP9, and fibroblast activation protein alpha (FAP). FAP is unique among this enzyme family because of its endopeptidase activity, substrate selectivity and limited expression profile in normal benign tissues. Despite a limited expression profile in benign tissue, FAP is overexpressed on the surface of neuroepithelial cancer cells, and studies have revealed it enables glioma cell invasion by facilitating the degradation of the brain parenchyma. Additionally, FAP expression was observed on a variety of stromal cell populations within glioma tumors suggesting that targeting FAP for imaging and therapy may provide a comprehensive treatment strategy that simultaneously targets tumor cells and the pro-tumorigenic microenvironment of these neuroepithelial cancers.
This report describes the synthesis and evaluation of the PET radiopharmaceutical, [89Zr]Zr-Df-Bz-F19 mAb, which is the radiolabeled version of the anti-FAP monoclonal antibody F19 using in vitro assays and small animal PET imaging. Finally, this report will also describe novel anti-FAP proteins that are being developed to improve the targeting of glioma cells so that the delivery of diagnostic and therapeutic molecules to these tumors is enhanced.
Christopher A. Vakulskas, PhD. Senior Director, Enzyme Evolution, Integrated DNA Technologies, Coralville, Iowa
"An overview of IDT's enzyme engineering and genome editing nuclease content"
Some of the largest breakthroughs in modern biotechnology have come in the form of newly discovered or engineered proteins and enzymes. Critical examples include the CRISPR-Cas9 system that has revolutionized the field of genome engineering, the high-fidelity DNA polymerases and DNA modifying enzymes that enable next generation sequencing (NGS), and the antibodies or other proteins/peptides that enable breakthrough human therapeutics. While IDT is traditionally known to be a leader in DNA/RNA synthesis, it is less well known that it is also a leader in the protein engineering space. Here we describe some of the various protein engineering techniques that IDT has used to engineer best in class products in CRISPR, qPCR, and NGS product categories.
Divya Bhat, Ph.D. Candidate, Maria Spies Research Group, Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA
"Therapeutic Disruption of RAD52-DNA Complexation via Novel Drug-Like Inhibitors for the Treatment of BRCA2 Deficient Cancers"
RAD52 protein is a coveted target for anti-cancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2. Deficiencies in these genes’ function are observed in about 25% of breast and ovarian cancers and put patients at an increased risk of developing additional cancers, including prostate cancer, fallopian tube cancer, primary peritoneal cancer, and pancreatic cancer. Certain variants in BRCA genes can also cause Fanconi Anemia and associated childhood solid tumors as well as acute myeloid leukemia. Emerging structure activity relationships for RAD52 are complicated, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same space as EGC within the RAD52 oligomeric ring.
A FRET based assay showed that all six compounds can inhibit RAD52 function (IC50~23-1200μM), with two of the compounds (Z56 and Z99) selectively killing BRCA2 deficient cells, as was assessed via a cell proliferation assay. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA2-deficient cells only, Z99 inhibited both proteins and displayed some toxicity towards BRCA2-competent cells. Notably, Z56 specificity was also reflected in its ability to specifically interfere with cellular functions of RAD52 centered on protection of stalled DNA replication forks. Optimization of the Z99 scaffold resulted in a set of more powerful inhibitors (IC50~1.3-8 μM), which were more selective towards RAD52, showing similar efficacy when compared to PARP inhibitor Veliparib. These modified compounds were only toxic to BRCA2-deficient cells. RAD52 complexation by Z56, Z99 and its derivatives vis-à-vis RPA provides a roadmap for further improvement of our lead compound.
Nicholas A. Luedtke, Ph.D. Candidate, Christopher M. Cheatum Research Group, Department of Chemistry, College of Liberal Arts and Sciences, University of Iowa, Iowa City, IA
"Probing the invilvement of Distal Residues in Active Site Dynamics of Purine Nucleoside Phosphorylase"
During an enzyme-catalyzed chemical reaction there can be motions within the active site on the picosecond-femtosecond timescale. These dynamics modulate the environment of the substrate thus promoting bond making and breaking events during the chemical step of the reaction pathway. While the chemical reaction is isolated to the active site, to what extent are ps-fs dynamics of the active site coupled to distal residues? Understanding how the broader enzyme scaffold contributes to enzyme function will deconvolute structure function relationships.
Previous studies on human purine nucleoside phosphorylase (PNP) have implicated a series of distal residues as being responsible for promoting the chemical step by modulating active site oscillatory behavior. We believe these dynamics are experimentally observable in the active site and we should be able to detect differences in active site fs-ps dynamics as a function of distal mutations. Our active site dynamics measurements will rely on a genetically incorporated infrared probe, 4-cyanophenylalanine (CNF), placed in the vicinity of reaction center. Evaluation of the invasiveness of CNF at two locations (F159 and F200) have shown that PNP tolerates this probe with decreases in catalytic efficiency but minor changes in the active site packing discovered through x-ray crystallography. We are continuing to characterize CNF-labeled PNP as we approach our key spectroscopic measurements and are evaluating the applicability of this probe for other enzymes.
Justin Ling, Ph.D. Candidate, M. Todd Washington Research Group, Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA
"Fork-remodeling helicase Rad5 preferentially reverses replication forks with gaps in the leading strand"
DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases converts stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we developed a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates that can be extended by DNA polymerases during the subsequent steps of template switching.