Presentations

Presenting a lightning talk at the Seattle Cell Science Symposium 2018 — Allen Institute

Presenting a lightning talk at the Seattle Cell Science Symposium 2018 — Allen Institute

EMRE Dependence in the Metazoan Mitochondrial Uniporter

University of Washington Pharmacology Department, Spring Rotation Talk

May 2018

Mitochondria house approximately one third of cellular metabolic reactions, which are tightly regulated to meet cellular demands. Calcium signaling has emerged as an important regulator of mitochondrial metabolism. Calcium stimulates the activities of calcium-responsive tricarboxylic acid (TCA) cycle enzymes and mitochondrial metabolite carriers, and increases energy production. Calcium entry into the mitochondria is mediated by mitochondrial calcium uniporter complex (uniplex), a calcium channel that resides in the inner mitochondrial membrane, and is composed of at least five proteins: MCU forms the pore of the channel, and four other proteins, MCUb, EMRE, MICU1 and MICU2, have regulatory roles. In addition to regulating metabolism, uniplex has been suggested to play an important role in a variety of cellular processes, and has been implicated in diseases such as cancer and neurodegenerative diseases. We are interested in interrogating the mechanism of regulation of MCU to better understand its role in physiology and disease. Interestingly, although MCU is the pore forming subunit of the uniplex, its ability to form a functional calcium channel by itself shows evolutionary divergence. In metazoans, MCU requires EMRE to conduct calcium. In contrast, in non-metazoan organisms that do not have an EMRE homolog, MCU alone is sufficient to form a functional channel. We exploited this species-specific EMRE dependence of MCU to understand the role of EMRE in the metazoan uniplex. First, we selected slime mold Dictyostelium discoideum MCU (DdMCU) and human MCU (HsMCU) to represent a non-metazoan and metazoan species, respectively. DdMCU has been shown to have robust uniporter activity and is sufficient to form a functional channel. We then swapped HsMCU domains with that of DdMCU domains to identify DdMCU sequences that would allow HsMCU to function independent of EMRE. These efforts led to the identification of a small, 10 amino acid region of DdMCU that would make HsMCU function without EMRE. Using crosslinking, we showed that MCU and EMRE interact directly in this region. In recent MCU structural studies, this region appears to be flexible, suggesting that EMRE binding to this region may stabilize MCU, allowing calcium conductance.

 

Snipping the Link: Using Truncation and Digestion to Promote D53 Crystallization

University of Washington Pharmacology Department, Winter Rotation Talk

March 2018

The most recently discovered class of plant hormones, strigolactones, help plants respond to their environment by regulating plant architecture and plants’ symbiotic relationships with soil microbes such as mycorrhizae. While better understanding of the strigolactone pathway will offer cues for the optimization of crop yields, understanding the molecular structures and cascades involved in this pathway also promises to shed light on novel drug design strategies. Protein D53 has emerged as a key regulator of the strigolactone pathway: on its own, D53 represses the pathway, but when complexed with proteins D3 and D14, D53 itself is degraded, allowing strigolactone signaling to proceed. Recent research suggests that D53’s D2 domain is crucial for this complexing, making this protein domain an appealing research target. Unfortunately, solving D53 D2 domain’s structure remains challenging, as the domain contains a large disordered linker that connects its two orderly, rigid regions. I presented two different approaches to crystallizing D53 D2: 1) an engineering approach that utilizes a series of constructs to decrease D53 D2’s linker, and 2) an empirical strategy that uses trypsin protease to carefully digest the linker.  

 

A Phosphoproteomic Method for High Throughput Kinase Domain Characterization

University of Washington Pharmacology Department, Fall Rotation Talk

December 2017

Approximately 518 kinases have been identified in the human genome. The importance of kinases has been extensively demonstrated in various human disease including cancer and diabetes. However, approximately 130 kinases remain functionally uncharacterized. This lack of knowledge leads to empirical blind spots and hampers diagnostic efforts. If the phosphorylation targets or motifs of every kinase were characterized, researchers could use these phospho-signatures to determine which kinases – and which pathways – contribute to a particular pathology. This capability would dramatically increase the power and accuracy of precision medicine. In this project, I developed a small-scale, time-efficient kinase domain expression and purification protocol. I determined kinase activity upon both model substrates and HeLa lysate using Promega’s ADP-Glo Kinase Assay. Following kinase activity confirmation, phosphopeptides in kinase-phosphorylated HeLa lysate were enriched using Immobilized Metal Affinity Chromatography (IMAC) and identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometry data analysis has demonstrated this workflow’s ability to identify previously characterized kinase phosphorylation sites. Following optimization, this workflow could likely be used to characterize dozens of kinase domains and full-length kinases batchwise.

Posters

Walking Dan and Paula, two graduate students associated with my department, through my poster at ASCB | EMBO 2018

Walking Dan and Paula, two graduate students associated with my department, through my poster at ASCB | EMBO 2018

EMRE Dependence in the Metazoan Mitochondrial Uniporter

Fred Hutch Metabolism Retreat

May 2019

Link to Poster

See description above, under “Presentations”

EMRE Dependence in the Metazoan Mitochondrial Uniporter

University of Washington Pharmacology Department, annual retreat

September 2018

See description above, under “Presentations”

EMRE Dependence in the Metazoan Mitochondrial Uniporter

ASCB | EMBO 2018 Meeting

December 2018

See description above, under “Presentations”

EMRE Dependence in the Metazoan Mitochondrial Uniporter

Seattle Cell Science Symposium 2018 — Allen Institute

December 2018

See description above, under “Presentations”

I also presented a 1 minute “lightning talk” at this symposium.