Tag: Biochemistry

Identifying Areas of Enhanced Flexibility in the SARS-CoV-2 Spike Protein with Computational Methods

Presenter: Sonny Kusaka − Biochemistry

Faculty Mentor(s): Professor Marina Guenza

Session: (Virtual) Oral Panel—Health and Social Science

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has become one of the most well-known and influential viruses of the 21st century. This research utilizes three different computational methods with varying predicted levels of detail both to compare the methods against one another as well as to analyze atomistic molecular dynamics simulations of the SARS-CoV-2 spike protein to look for regions of enhanced flexibility. Previously established theoretical models of protein binding indicate a correlation between local flexibility and increased binding capabilities, the likes of which are of interest because they may be of importance for the protein in performing its biological function. As the computational methods increase in predicted accuracy, so too do the level of detail in the dynamics of the spike protein that they model. These results show enhanced flexibility of the spike protein in the functional regions that have been previously described and published in literature, other flexible regions not previously documented in literature that may be of interest, and promising results for the future of coarse-grain analysis of large multi-subunit proteins.

Strained Alkyne-Cycloparaphenylenes: a New Class of Clickable Fluorophores

Presenter(s): Anna Garrison — Biochemistry

Faculty Mentor(s): Julia Fehr

Session: (Virtual) Poster Presentation

Looking inside the human body is a critical tool researchers and physicians need in order to explore the human species, navigate and discover new diseases, or even begin to look at cures or treatment plans for illnesses. What most don’t know, is that the human body is dark and examining its intricate details is impossible without some sort of tagging method. Fluorophores are fluorescent chemical markers that can be used to illuminate and strategically fluoresce certain aspects of biological systems. [n]cycloparaphenylenes or “CPPs” are a unique class of macrocycles made of radially oriented pi systems. CPPs can be synthesized with various techniques to manipulate molecules with different electronic properties that yield varying size-dependent fluorescence. CPPs foster the potential to be used for biological imaging due to their tunable optical properties and reactivity. In this study, the use of macrocyclic angle-strained alkyne-containing CPPs is explored as their unique moiety can serve as the means of attachment to biological molecules through copper-free click chemistry. This study reports the first account of a strained-alkyne CPP clicked to a biological molecule. Additionally, it is demonstrated that strained-alkyne CPPs are not only nontoxic to living cells, but can also be used to fluoresce the cell surface of living human Jurkat cells utilizing a metabolic labelling approach. This is a less invasive cell labelling procedure that can be used to image live cells.

Single molecule polarization sweep spectroscopy

Presenter: Anabel Chang − Biochemistry

Faculty Mentor(s): Andrew Marcus, Jack Maurer

(In-Person) Poster Presentation

Local fluctuations of the sugar-phosphate backbones of DNA (a form of DNA ‘breathing’) play key roles in protein-DNA assembly and enzymatic function. By monitoring spectroscopic signals from single-molecules of DNA constructs labeled with optical probes rigidly inserted within the sugar- phosphate backbones at opposite positions within complementary single-strands, it is possible to study local conformational fluctuations within DNA at specific sites. Here we present an experimental single-molecule spectroscopic method, to monitor the local fluctuations of Cy3-labeled DNA constructs at varying positions near a single-stranded (ss)-double-stranded (ds)DNA junction. The method combines single-molecule total internal reflection fluorescence (TIRF) microscopy with polarized, phase-modulated optical excitation to detect linear optical signals. We use a linearly polarized continuous wave (cw) laser beam to excite the single-molecule sample, so that the emitted fluorescence contains information about the relative conformational changes of an exciton-coupled Cy3 dimer probe that labels the DNA sugar-phosphate backbones. Our results indicate that the local conformation of DNA at positions near ss-dsDNA junctions adopts four topologically-relevant macrostates. We apply a kinetic network model approach to interpret our observations of DNA breathing fluctuations at and near the ss-dsDNA junction.

Observation of Corneal Guttae by Plasma-FIB Microscope

Presenter: Mayurika Bhaskar − Biochemistry, Biology

Faculty Mentor(s): Hiro Uehara

(In-Person) Poster Presentation 

Fuchs Endothelial Corneal Dystrophy (FECD) is an inherited disease that leads to blindness. FECD is characterized with the thickening of the Descement’s membrane, corneal endothelial cell loss, and the formation of guttae (deposition of the extracellular matrix). Once corneal endothelial cells are lost, the cornea cannot maintain its transparency. Since these cells do not proliferate in vivo, the current treatment is through a cornea transplant, but this comes with risks such as infection and tissue rejection.

The purpose of my research is to observe the structure of guttae in FECD mice corneas to better understand its condition.

In this study, normal and FECD mouse corneas were compared. After euthanizing the mice, the eyeballs were harvested, and corneas were removed. Then they were stained with osmium tetroxide, fixed with epoxy resin, and microtomed. Finally, the sample was mounted on a 45-degree tilt and cut with a 45 nanoampere oxygen beam at a slice size of 50 nm by a Plasma-FIB microscope to obtain ~700 images. Some issues included the bending of the cornea once harvested and the time it took to image a sample.

We successfully 2D imaged the normal and FECD mouse corneas. The normal mouse corneal endothelium is smooth and thick, but FECD mouse cornea showed signs of bumps and thinness. I’m currently developing these images into 3D models to better analyze them. Overall, I hope that my work will provide information about guttae developed from FECD for future research.

OptiDicer reduces CUG RNA accumulation in corneal endothelial cells affected by Fuchs’ dystrophy

Presenter: Sanjana Basak − Biochemistry

Faculty Mentor(s): Julie Weise

(In-Person) Poster Presentation 

Fuchs’ endothelial corneal dystrophy (FECD) is a genetic disease which leads to eye pain, significant loss of vision and corneal lesions called guttae.

Late-onset FECD is characterized by the expanded repeat trinucleotide sequence (CTG)n (n>30- 40) in the TCF4 gene. The accumulation of CUG RNA in the nucleus forms cytotoxic RNA foci. Recently, we developed a recombinant variant of DICER, OptiDicer, which can degrade double-strand RNA through RNaseIII activity. In this study, we examined whether OptiDicer can decrease CUG RNA accumulation in corneal endothelial cells from patients with Fuchs dystrophy.

F35T cells, Human corneal endothelial cells from an FECD patient with (CTG)n n>1000, were used in this study. The cells were transfected with OptiDicer and a control, and then subjected to in situ hybridization in order to detect CUG RNA accumulation. The images were obtained with an EVOS fluorescence microscope, and the number of CUG RNA accumulation was counted. The average number of CUG RNA accumulation was 1.9+/-1.4 in OptiDicer-F35T and 2.9+/-1.7 in D2A-OptiDicer control F35T (p<0.001), respectively.

We found that OptiDicer significantly decreased CUG-RNA accumulation in late-onset FECD patient derived corneal endothelial cells, although the low transfection efficiency may underestimate OptiDicer effect. Our result suggests OptiDicer can be a potential treatment for long CUG RNA repeat derived FECD. Future studies will explore OptiDicer in other cell lines from FECD.

Synthesis of a Water-Soluble Macrocyclic Iron-Phosphine Complex

Presenter : Aditya Nathan

Mentor : Bryan Nell

Major : Biochemistry

Poster 7

Although society is progressing towards increased dependency on alternative sources of energy, natural gas remains as the one of the most relied upon sources of energy. A major contaminant of natural gas is dinitrogen. Our research focuses on the synthesis and characterization of a water-soluble macrocyclic iron-phosphine complex that is capable of reversibly binding dinitrogen. Our intended method for developing such a complex involves a multistep process beginning with a template synthesis, which involves the coordina- tion of open-chain phosphine ligands to a transition metal atom (specifically Ni(II), Pd(II), or Pt(II) for the sake of square planar geom- etry). Subsequently, the components would be linked/bridged together using base and an alpha/omega dihalide to form the macrocycle. The complex would then be demetallated using cyanide ion or a sulfide source and subsequently coordinated to Fe(II). In order to confer the complex with water-solubility, we plan on adding water-soluble functional groups to the side chains of the macrocycle. Thus far, we have been able to synthesize and characterize key intermediate complexes that serve as the precursor for the macrocycles. In addition, we have investigated methods for macrocyclizing the intermediate complexes.

Alkaline Synthesis of Amidines – A New Approach to Preparing Medicinally Relevant Small Molecules

Presenter : Muhammad Khalifa

Mentor : Michael Haley

Major : Biochemistry

Poster 16

Myotonic dystrophy (DM) is the most common adult form of muscular dystrophy. Recently, the small molecule pentamidine has been shown to relieve symptoms of DM in cell models; however, pentamidine is an inadequate drug for DM because of toxicity and bioavailability problems. Analogs of pentamidine, generically termed amidines, have proven to be significant candidates in the search for an effective cure for DM. It is therefore important to have access to the widest possible range of amidine structures for study against symptoms of DM. Existing methods of synthesizing amidines have largely depended upon reactions with acidic con- ditions; features of these reactions have limited the accessible range of amidines, especially substituted amidines. Here we outline a new method of preparing substituted amidines using alkaline conditions that features shorter reaction times, better yields, and better compatibility with many of our compounds of interest. Through synthesis and NMR characterization, we explore the range of usable starting materials, test the method’s selectivity in the presence of competing reactions, and demonstrate its application to the synthesis of several novel compounds. This method makes possible a host of new substituted amidine compounds that could prove useful in the search for a cure for DM, and provides a new, potentially more efficient, synthetic path to unsubstituted amidines for the same purpose.

Effects of Pentamidine Derivatives on Myotonic Dystrophy

Presenter : Jessica Choi

Mentor : Andy Berglund

Major : Biochemistry

Poster 8

Myotonic dystrophy (DM) is a genetic disorder caused by an expansion of the trinucleotide (CTG) repeats in myotonic dystrophy protein kinase (DMPK) gene. This disease is characterized by myotonia and is commonly presented as the inability to relax muscles after contraction. Currently, there is no known cure or treatment for this disease. However, a drug called, pentamidine, has been discovered to relieve the severity of the disease by decreasing the level of toxic RNA. More specifically, pentamidine has been demonstrated to rescue RNA splicing, which involves excising out introns and combining exons together in an mRNA sequence to ultimately provide a functioning RNA. Without proper splicing, mutations can give rise to serious diseases like DM. In order to decrease the severity of DM, a high concentration of this drug must be administered, which also inevitably results in a significant decrease in cell viability. Thus, designing a derivative of pentamidine with higher efficacy and lower toxicity is the primary goal of this project. Performing a simple substitution reaction (SN2) from a cyano group-containing core compounds and n-butyl lithium is an easy, yet powerful method to produce the derivatives. Some previously synthesized derivatives have shown promising results with less nega- tive effects on cell viability (less toxic) and increased levels of splicing rescue, although further study needs to continue to search for the most effective drug.the overall success of these organisms.

Predicted SH3 Binding Motif in Drosophila aPKC is Required for Proper Localization of aPKC During Asymmetric Cell Division of Neuroblasts

Presenter : Ryan Boileau

Mentor : Ken Prehoda

Major : Biochemistry, Human Physiology, Human Biology

Poster 22

Asymmetric cell division of Drosophila neural stem cells, neuroblasts, require the proper localization of factors that influence the orientation of cell divisions and future fates of mitotic progeny. Errors in the generation of this polarity could cause cells to overproliferate and become cancerous. In neuroblasts, atypical protein kinase C (aPKC) has been previously shown to be a key mediator in the genera- tion of apico-basal polarity by localizing to the apical cortex and restricting fate determinants Numb and Miranda to the basal cortex during cell division. This allows the dividing neuroblast to maintain pluripotency while also generating a daughter cell that differenti- ates into neurons. Although the mechanism of how aPKC restricts basal determinants has become transparent, we seek to evaluate how aPKC itself is apically localized. Using a combination of genetic and biochemical approaches, we have found that a predicted SH3 binding motif within aPKC is necessary for apical polarization. We hypothesize that an SH3 domain containing protein binds to aPKC at this site and plays a role in stabilizing apical localization. Future research will be focused on finding interacting partners of this SH3 binding motif using a candidate gene-based approach.

aPKC Induces Polarization of Numb by Inhibiting Cortical Targeting Sites

Presenter: Lyle McPherson

Mentor: Ken Prehoda

Poster: 23

Major: Biochemistry 

Cell polarity regulates important functions for metazoan cells, including epithelial, neuronal and stem cells. However, little is known about the molecular mechanisms that allow cells to establish cell polarity. aPKC, the kinase of the evolutionarily conserved Par complex, polarizes cellular proteins. In these polarized cells, protein polarization genetically downstream of aPKC maintains tissue integrity and establishes cell identity. For multiple aPKC substrates, phosphorylation induces protein polarization by displacing substrates from aPKCcontaining membrane domains. Despite the clear role of aPKC in establishing cell polarity the molecular mechanism by which aPKC’s kinase activity polarizes its substrates remains unclear. We characterized the polarization mechanism of Numb, an aPKC substrate, using cell biology and biochemistry. We identified lipidbinding sites within Numb that mediate its recruitment to the cellular cortex by binding to negatively charged phospholipids. Additionally, we found that specific amino acids within these sites are phosphorylated by aPKC to inhibit lipid binding. Our findings suggest one mechanism for aPKCmediated cell polarity where aPKC polarizes Numb by phosphorylating it to inhibit cortical association thereby resulting in its polarization. We are currently investigating other domains of Numb containing aPKC phosphorylation sites to further our understanding of the molecular mechanisms behind this process. The mechanism of Numb’s polarization by aPKC illustrates to us a way that a kinase can induce cell polarity by destabilizing a protein’s membrane association in specific regions of the cell.