2018-19 CBI Trainees

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Raymond Berkeley 

Chemistry and Biochemistry
Galia Debelouchina Lab

Raymond Berekeley

CBI Appointment Period: 2018-19
Grant Year: [4]

Chemical tools for the study of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) play key roles in numerous cellular processes. These proteins often condense into discrete intracellular droplets that are required for these proteins to carry out their function. Despite the central role of these protein droplets in cell biology and disease, the mechanisms that underlie protein phase separation are poorly understood. The dynamic nature of these protein droplets makes nuclear magnetic resonance (NMR) the ideal tool for their study, but the low sensitivity of NMR makes it difficult to study IDPs in their native environment. Our lab uses dynamic nuclear polarization to circumvent the sensitivity problem. My work focuses on the design of probes that are capable of delivering polarization agents to intracellular protein droplets in order to selectively enhance the NMR signal from these condensed proteins. These probes will enable the study of the structural basis for phase separation in cells without requiring any chemical modification to the proteins under study.

 

Albert Kakkis 

Chemistry and Biochemistry
Akif Tezcan Lab

Thomas Bartholow

CBI Appointment Period: 2017-19
Grant Year: [3,4]

A metal-mediated path to biological materials: Harnessing coordination chemistry for heteromeric protein assembly

The self-assembly of proteins into multi-component supramolecular architectures underpins Nature’s most complex transformations, from nitrogen fixation to photosynthesis. Synthetic multi-component assembly is a long-standing goal of protein engineers, as it introduces a repertoire of functions unattainable via single-component assemblies. Current methods rely on the computational design of non-covalent interactions across large interfaces (>1,000 Å2), which limits the generalizability and reversibility of the designed assemblies. We aim to develop a method for multi-component assembly that can 1) Rely on minimal computational design, 2) Be achieved using small interfaces, and 3) Be externally controlled. To meet these criteria, we will use metal-ligand and π-π stacking interactions, popular tools in the design of small molecule assemblies that are underutilized in protein assemblies. As a proof of concept, we will harness CuII-tetrahistidine coordination motifs and π-π stacking interactions between nitroarenes and tryptophan to design a heterodimeric assembly of four-helix bundle proteins. Successful design of multi-component assemblies using these interactions would expand the chemical toolbox for de novo protein design. 

 

Kelsey Krug

Chemistry and Biochemistry
Michael Burkart Lab

Kelsey Krug

CBI Appointment Period: 2017-19
Grant Year: [3,4]

Application of small molecule splice modulators

RNA splicing plays a central role in disease and cancer progression, but the complexity of this large-scale process has left the study of RNA splicing unexplored. Recently, the alteration of splicing by small molecule splicing inhibitors (SPLMs) has been identified as an avenue for chemical biological discovery. My project focuses on the combined use of splice modulators with other cell-cycle regulating clinical agents, Rigosertib and Alisertib, which inhibit polo-like kinase 1 (PLK-1) and aurora kinase (AK) respectively, to selectively control the expression of cell-cycle progression proteins. The goal of this effort is to combine a molecular and cellular understanding of cell cycle selectivity for splice modulation as a tool to enhance specific cell cycle events. This concept relies on the use of the splice modulator to downregulate the levels of PLK-1 and AK by altering their splicing, and then using the reduced levels of these proteins to enhance the efficacy of their associated inhibitors, Rigosertib and Alisertib. So far, I have looked for synergy between PLK-1 and AK inhibitors and the splice modulator FD-895 in G1-synchronized human colon carcinoma HCT116 cells. Using the MTT assay, I found that the combined use of FD-895 with Rigosertib or Alisertib led to reduced tumor cell proliferation compared to treatment with either compound on their own. This preliminary data indicates that splice modulation can affect cell cycle regulation. Next, I will use qPCR to evaluate PLK-1 and AK mRNA isolated from G1-synchronized HCT116 cells that have been treated with splice modulator FD-895, and then either Rigosertib or Alisertib.  By varying the treatment time with FD-895, I will determine an optimum length of treatment, the one that most reduces the levels of spliced PLK-1 and AK mRNA.

 

Brodie Ranzau 

Chemistry and Biochemistry
Alexis Komor Lab

Brodie Ranzau

CBI Appointment Period: 2018-19
Grant Year: [4]
San Diego Fellowship Recipient

Repurposing RNA-modifying enzymes for genome editing

Single-point mutations are the genotypic cause of the vast majority of genetic diseases. Genome editing is one method of treating genetic diseases by mutating the pathogenic allele into a healthy allele. Current genome editing techniques create double-stranded DNA breaks (DSBs) at target locations in the genome, which are recognized by DNA repair pathways that may incorporate a supplied DNA strand and lead to successful genome editing. This process is inefficient, especially when attempting to substitute a single base pair, and may create other harmful insertions or deletions at the DSB. My work aims to circumvent the use of DSBs by creating precision base editing tools that can chemically modify target nucleobases. Base editors capable of causing C-to-T and A-to-G mutations have already been created. By directing the evolution of RNA modifying enzymes, new base editors will be created that cover other modifications and mutations, vastly increasing the utility of these tools for editing the genome.

 

Ariana Remmel

Chemistry and Biochemistry
Kamil Godula Lab

Ariana Remmel

CBI Appointment Period: 2018-19
Grant Year: [4]
San Diego Fellowship Recipient

Design and Synthesis of Glycopolymers to Assess Sperm Binding to Mucus Glycans

Cervical mucus (CM) is an important barrier in pathogen elimination and sperm selection during human reproduction. This mucus is a viscous network primarily composed of glycoproteins called mucins. Mucins are comprised
of a protein backbone highly decorated with glycan chains (80% per weight of mucin molecule). The viscoelastic properties of CM and its production volume change dramatically throughout the menstrual cycle such that CM is thin and copious during ovulation, but scant and thick outside of ovulation. This is believed to limit sperm transit to the ovulatory window. However, these rheological changes also correlate with altered glycan expression, with more sialic acids present outside of ovulation and increased fucosylation during ovulation. While the mechanical properties of CM have been well studied with respect to sperm penetration, little is known about sperm interactions with the changing glycans of CM. It is well known, however, that sperm bind to glycan partners in the female reproductive tract to both form the oviduct reservoir and penetrate the egg. Given the prevalence of glycans in CM, it would appear that sperm surface interactions with mucus glycans could also affect sperm selection by the female host. This project aims to create chemical tools to better understand the interactions of human sperm with CM glycans.

 

Holly Sullivan 

Bioengineering
Karen Christman Lab

Holly Sullivan

CBI Appointment Period: 2018-19
Grant Year: [4]

Inhalation of polymeric nanoparticles for therapeutic delivery after myocardial infarction

Every year, nearly 800,000 people in American have a heart attack. Many of these patients will now be in heart failure— 5 million people are living with congestive heart failure, and on average they will die within 5 years of diagnosis. The mounting prevalence of heart disease related deaths creates the need for more efficient methods of treatment. Nanoparticles present a non-invasive, targeted method of delivering therapeutic small molecules and peptides to the infarcted region of heart post myocardial infarction (MI). Our work, in collaboration with the Gianneschi lab at Northwestern University, is aimed at developing degradable polymeric nanoparticles that localize in the infarct after being cleaved by matrix metalloproteinases that are upregulated after MI. The nanoparticles preferentially enter the infarct via the leaky vasculature and remain in the infarct following a morphological switch to micron-scale aggregates in response to enzymatic cleavage.  Inhalation of nanoparticles is highly translational and minimally invasive, making it a desirable form of administration moving forward.