Tag: Biochemistry

Determining How S100A9 Activates TLR4 Using Evolutionary and Biochemical Approach

Presenter: Jiayi Yin – Biochemistry

Faculty Mentor(s): Mike Harms, Sophia Phillips

Session: (In-Person) Poster Presentation

The immune system activates inflammation in response to both foreign pathogens and internal damage. Dysregulated inflammation can lead to many chronic diseases such as arthritis, inflammatory bowel disease, and some cancers. S100A9, a protein expressed in immune cells, has been found in high concentration in inflamed tissue of many of these chronic diseases. S100A9 strongly activates TLR4, a proinflammatory receptor, and thus activates pathological inflammation. Understanding how S100A9 interacts with TLR4 would be useful to create therapeutics to treat these diseases. My project is to use evolutionary and biochemical techniques to find out what sequence changes to S100A9 were important in its evolutionary history that led to greater proinflammatory activity. I will continue to characterize modern mammalian S100A9s that diverged more distantly from humans such as koala, platypus, and echidna, using recombinant protein expression and purification of S100A9 proteins from Escherichia coli followed by functional assays in human embryonic kidney cells. I will also couple these studies with further characterization of how TLR4 specificity and activity for endotoxin, the pathogenic ligand for which TLR4 evolved to recognize, changed in different species. These data will help us understand how the host protein S100A9 evolved inflammatory activity, and how TLR4 evolved to activate with a variety of ligands.

The Role of Ribosome-Associated Protein Quality Control in a Prion-Based Epigenetic State

Presenter: Phaedra Whitty – Biochemistry

Faculty Mentor(s): David Garcia

Session: (In-Person) Poster Presentation

A prion that has been discovered in yeast, [BIG+], allows cells to adopt a ‘live fast, die young’ strategy, accelerating growth rate at the cost of a shortened lifespan. Prions are heritable, alternatively- structured proteins that are implicated in many mammalian neurodegenerative diseases. However, they can also act as a beneficial epigenetic mechanism by altering gene expression in cells. The [BIG+] prion is a form of Pus4, an RNA-modifying enzyme conserved throughout nature, and has been shown to affect protein synthesis. The epigenetic state induced by it is characterized by accelerated cell proliferation, increased cell size, shortened lifespan, and increased translation activity. The mechanism of the [BIG+] prion remains unknown. This project investigates the ribosome-associated protein quality control pathway (RQC), a cellular system to monitor issues in translation, as a potential contributor to the [BIG+] phenotype. A genetic approach was taken to knock out each of four genes coding for proteins involved in RQC. These strains were used in luciferase reporter assays to examine the difference in translation phenotypes between mutant naïve and mutant [BIG+] strains. A notable change in the [BIG+] translation phenotype of mutants as compared to wild-type strains suggested the involvement of at least two RQC proteins, Hbs1 and Rli1, in the prion phenotype. These results are some of the first mechanistic insights into how this prion affects translation.

Exploring the Role of the Arp2 D-Loop in Activation of Arp2/3 Complex

Presenter: Maisie Topping – Biochemistry

Faculty Mentor(s): Brad Nolen, Heidy Narvaez Ortiz

Session: (In-Person) Poster Presentation

Branched networks in the actin cytoskeleton are critical for a variety of cellular processes including endocytosis. New branched actin filaments are nucleated by Arp2/3 complex, and the deregulation of this protein is related to diseases such as cancer. Arp2/3 complex is intrinsically inactive. During activation, the complex undergoes a conformational change that brings two of its subunits, the actin-related proteins Arp2 and Arp3, into a position that mimics two consecutive actin subunits within a filament, thereby creating a template for the new filament. When actin polymerizes into filaments, a portion of the protein called the D-loop helps to stabilize the filamentous structure, and the Arp2 and Arp3 subunits both contain a similar D-loop. A previously solved structure of Arp2/3 complex at a branch junction indicates that a contact between the D-loop of Arp2 and ArpC3 may be important for stabilizing the activated complex at the junction site. This project aimed to assess the importance of that contact in activation of Arp2/3 complex. We generated a strain of budding yeast with three mutations in the Arp2 D-loop, purified Arp2/3 complex from cells, and used pyrene actin polymerization assays to test the ability of the mutated complex to nucleate actin filaments compared to the wild type. The Arp2 triple mutant showed greatly decreased activity, indicating that the contacts between Arp2 and ArpC3 are important for the activation and function of Arp2/3 complex.

Developing an In Vivo Assay for Quantitative Analysis of Arp2/3 Complex Inhibitors

Presenter: Maisie Topping – Biochemistry

Faculty Mentor(s): Brad Nolen, Heidy Narvaez Ortiz

Session: (In-Person) Poster Presentation

Branched networks in the actin cytoskeleton are critical for a variety of cellular processes such as motility and endocytosis. New branched actin filaments are nucleated by Arp2/3 complex, and the deregulation of this protein is related to a variety of diseases including cancer. Several classes of small molecule inhibitors of Arp2/3 complex have been discovered, most of which function by blocking an activating conformational change of the complex. These molecules are useful tools because they allow researchers to turn off activity in different processes, and they have potential as drugs due to Arp2/3 complex’s increased activity in some diseases. These inhibitors have been characterized in vitro and have been used in experiments, but they have never been quantitatively analyzed in vivo. My project will develop an in vivo assay for quantitatively measuring the effects of Arp2/3 complex inhibitors on cytoskeleton dynamics. The assay will use Drosophila S2 cells expressing a low level of GFP-tagged actin and total internal reflection fluorescence (TIRF) microscopy to extract velocity data from the cell’s actin cytoskeleton before and after treatment with inhibitors. These experiments will lead to a better understanding of how Arp2/3 complex inhibitors affect living things because this assay is a better approximation of biological systems than the currently used in vitro methods. The different assays can be used in concert to provide a fuller characterization of these inhibitors.

An investigation of novel left-right patterning genes in zebrafish

Presenter: Maisey Schering – Biochemistry, Biology

Faculty Mentor(s): Katie Fisher, Daniel Grimes

Session: (In-Person) Poster Presentation

Breaking of left-right (L-R) symmetry is a fundamental part of animal development. To facilitate this, cell to cell communication via extracellular fluid flow plays a critical role. Failure of this communication results in developmental diseases such as congenital heart disease and abnormal L-R positioning of the organs, termed heterotaxia. Understanding the mechanisms by which fluid flow signals control asymmetry is essential for understanding how to treat these diseases. In embryonic development of zebrafish, the model organism of this project, asymmetric flow in Kupffer’s vesicle (KV) breaks L-R symmetry. The flow signal results in asymmetric repression of an mRNA, dand5, triggering asymmetrical development of the emerging organs. How cells sense and transduce fluid flow, leading to dand5 repression, is not understood. My mentor in the Grimes lab, Katie Fisher, performed a literature review that resulted in 90 novel candidate genes which might regulate L-R asymmetry. These genes are all expressed at the right time and place during development to control fluid flow signaling. We are using a CRISPR/Cas9 screen to identify which of these genes are essential for L-R patterning. Several genes of interest have been identified and homozygous lines with these mutations are currently being generated. I will describe our ongoing screening efforts and early results. By completion of this project, we will know how these novel genes act to ultimately control organ asymmetry.

Coursed-Grained Approach for the Protein Dynamics of the SARS-CoV-2 Spike Protein Variants

Presenter: Ruben Sanchez – Biochemistry, Biology

Faculty Mentor(s): Marina Guenza

Session: (In-Person) Poster Presentation

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) utilizes a spike protein to recognize the receptor protein Angiotensin-converting enzyme 2 (ACE2) of human cells to initiate COVID-19. It is known that the spike protein adopts an active (open) conformation from an inactive (closed) conformation to initiate its infectious cycle. But it is unknown whether the different variants have mutations that affect the protein dynamics of the spike protein. It was hypothesized that the amino acid mutations of more transmissible variants will have increased protein dynamics leading to a dramatized Monod-Wyman-Changeux model. Identifying and targeting these dynamics may lead to the development of pharmaceuticals that may inhibit the infectivity of the SARS-CoV-2 virus. Therefore, two variants of the spike protein were analyzed using molecular dynamic simulations and the Langevin Equation for Protein Dynamics (LE4PD) to quantitively analyze residue fluctuation within their respective spike proteins. LE4PD quantified the protein dynamics and demonstrated that the more infectious variants have higher fluctuations in their protein dynamics.

Mapping Sequence-Function Landscapes in the Dihydrofolate Reductase Family Coauthors: Calin Plesa, Samuel Hint

Presenter: Carmen Resnick – Biochemistry

Faculty Mentor(s): Calin Plesa

Session: (In-Person) Poster Presentation

Dihydrofolate reductase (DHFR) is an essential enzyme in the folic acid synthesis pathway and has been the subject of intense study in the past few decades. Despite the wide diversity of homologs, research attention has primarily focused on particular DHFR proteins and as their mutants. In this study we explore DHFR expression through a knockout E. coli strain ER2566 ΔfolAΔthyA. We focus on the ability of DHFR to both rescue metabolic function and tolerate treatment against the antibiotic trimethoprim, which will allow us to understand how antibiotic resistance emerges given many evolutionarily divergent starting points. Changes in the mutational landscape of DHFR allows for varying survival rates in the presence of antibiotic inhibitors. We conduct a broad mutational scan using a library of 5,000 DHFR homologs synthesized using DropSynth gene synthesis. Variant fitness is determined in a multiplex survival assay in the knockout strain which allows supplementation- dependent conditional selection. We aim to collect quantitative fitness data on which mutations impact DHFR activity, both in the presence and absence of inhibitors, to elucidate sequence-function relationships and understand how the fitness landscapes vary as a function of the evolutionary distance between homologs. This data can be applied towards the development of narrow-spectrum and targeted antibiotics and mitigation of resistance through understanding the pathways from which antibiotic resistance arises.

Alkaline phosphatase-activated hydrogen sulfide donors for bone regeneration

Presenter: Mia Ramos – Biochemistry

Faculty Mentor(s): Annie Gilbert, Mike Pluth

Session: (In-Person) Poster Presentation

Hydrogen sulfide (H2S) is a small gaseous signaling molecule that can provide a variety of important physiological effects. For example, H2S can promote angiogenesis, osteogenesis, and regulate inflammation. These regenerative effects of H2S make it an ideal therapeutic candidate for healing bone defects. The challenge with studying therapeutic effects of H2S in bone applications is that the direct delivery of H2S as a gas or inorganic sulfide salt lack spatial and temporal control. To address this challenge, small molecule H2S donors have been developed. Previously, the Pluth lab has developed caged thiocarbamates as a highly tunable class of COS-based H2S donors. Upon activation, caged thiocarbamates undergo a self-immolative cascade in the presence of specific environments or analytes to produce COS, which is rapidly converted to H2S by carbonic anhydrase. This strategy could be useful for localizing H2S delivery in bone healing sites. Alkaline phosphatase (ALP) is an enzyme that is present in large concentrations in bone fractures and could serve as an activator of H2S production from a phosphate protected caged thiocarbamate. Here, we developed an alkaline phosphatase-activated caged thiocarbamate COS/H2S donor to study H2S in bone healing applications. We anticipate the development of these ALP-activated H2S donors will serve as useful tools for investigating therapeutic effects of H2S in bone healing.

The Impact of Hyaluronic Acid Molecular Weight on Hydrogel Properties for Bone Regeneration

Presenter: Esther Aurelie Mozipo – Biochemistry

Faculty Mentor(s): Veronica Spaulding, Marian Hettiaratchi

Session: (In-Person) Poster Presentation

Large bone defects have difficulty healing without intervention, leading to nonunion fractures.1 Hydrogels are a promising solution to this problem due to their biocompatibility and potential as a drug delivery vehicle. Hyaluronic acid (HA) is a naturally-occurring polymer that can be functionalized to create a hydrogel. HA exists in our bodies in different molecular weights, which are involved in different biological processes.2 I investigated whether varying the molecular weight of the HA could have an effect on the properties of the HA hydrogel and cellular responses. HA hydrogels were synthesized via a hydrazone click reaction of aldehyde-modified HA(HA-Ox) and carbohydrazide-modified HA (CH-HA). The degree of modification (DOM) of the HA was determined using 1H NMR spectroscopy. The effect of HA on osteogenesis was determined by measuring alkaline phosphatase (ALP) activity of C1C12 skeletal myoblasts in HA solutions. The DOM of CH-HA at 40 kDa, 100 kDa, 700 kDa, and 1500 kDa was 25.4, 20.4, 7.8, and 0%, respectively while the DOM of HA-Ox at the same molecular weights was 13.7%, 12.6%, 7.0%, and 3.6%, respectively. C2C12 cells grown in unmodified 40 kDa, 100kDa, 700kDa, and 1500 kDa HA exhibited ALP activity comparable to C2C12 cells cultured in media only. However, in the presence of bone morphogenetic protein 2 (BMP-2), an osteoinductive protein, the 700 and 1500kDa HA inhibited BMP-2 induced ALP activity when compared to the 40 and 100kDa HA.

Decreasing Hunchback and Bicoid Levels in Pair1 Neurons Alters the Pair1 Circuit in Drosophila

Presenter: Amanda Linskens – Biochemistry

Faculty Mentor(s): Kristen Lee, Chris Doe

Session: (In-Person) Poster Presentation

Transcription factors (TFs) are essential for cell specification across multiple species, including humans. During Drosophila melanogaster development, neuroblasts produce neuronal progeny that acquires identity based on the temporal TF (tTF) present during birth. tTFs activate specific Homeodomain TFs (HDTFs), which are also important for determining neuronal identity. Thus, the cascade of tTFs in neuroblasts creates the diversity necessary for forming precise neural circuits. Although prior research shows that TFs generate variety, few studies have examined how these TFs influence circuit establishment. My study focuses on the Pair1 neurons, which initiate pausing in larvae through neural circuits. Prior research in our laboratory showed that the Pair1s derive from the tTF Hunchback (Hb), which activates the HDTF Bicoid (Bcd). Therefore, I hypothesized that manipulating Hb’s and Bcd’s expressions in Pair1 would alter the Pair1 circuit. To investigate this,

I expressed the green fluorescent protein (GFP) in the Pair1s and knocked down Hb and Bcd levels individually to assay circuit morphology and behavior. I found that decreasing Hb resulted in more Pair1 axonal connections, affecting behavior. Interestingly, I saw similar results when Bcd was knocked down in Pair1, but the phenotypes are weaker than those experienced with decreased Hb levels. These results suggest that tTF activation of HDTFs is vital for circuit establishment in the central nervous system.