Philosophy of Involvement of Students in Research
The main reason that I have for involving students in research is to share with them the wonderment and astonishing beauty of biochemistry and biology. Additionally, I am motivated by the intellectual challenge of being able to push the frontiers of scientific understanding. I also want students to obtain real intellectual and technical skills for use beyond the experience. For each student, this can be achieved in different ways and at different paces. To date, research in my lab has involved 10 undergraduate students (2-5/semester + 2-3 fulltime summer students), received internal and external funding, and was presented at a conference by three undergraduate students. I am currently preparing a manuscript, with five undergraduate co-authors, that will be submitted this spring.
Doctoral Research Experience: Metal-Binding Properties of Prion Proteins
My doctoral thesis focused on characterizing two properties of prion proteins: metal binding and reactivity. Prion proteins are the causal agent of Mad Cow disease, chronic wasting disease in deer, and Creutzfeldt–Jakob disease in humans. Classic symptoms of prion diseases are rapid development of dementia, difficultly walking, muscle stiffness, and hallucinations. Post-mortem examination of the brains of patients (and animals) suffering from prion disease have similar histology: aggregates of prion proteins, a holey appearance, and and a textural change to resemble that of a kitchen sponge, Figure 1, lower center. The unique feature of these diseases is that the propagation and transmission of the disease does not require any genetic information but rather comes from the misfolding and aggregation of prion proteins themselves. However, the role of metal binding of both the native and pathogenic state of prion proteins was not known. The goal of my research was to understand the properties of copper binding to prion proteins. We concluded from this work that the selective binding of copper by prion proteins prevented the reduction of copper’s oxidation state and may be important in protecting cells from oxidative stress.
At the end of my doctoral work, I realized that in order to truly understand the underlying causes of neurodegeneration, at both a cellular and molecular level, I needed to deepen my understanding of protein structure and protein degradation pathways. For this reason, I sought out an opportunity to work at the Carver College of Medicine nuclear magnetic resonance (NMR) facility at the University of Iowa. While I worked as scientific support staff in the facility, I gain the ability to structurally characterize proteins. This then followed to my postdoctoral research that focused on structure/function studies of the ubiquitin-binding receptors of a protein degradation pathway.
Post-Doctoral Research: Structure/Functional Characterization of Essential Protein-Protein Interactions of the Endocytic Pathway
The overall goal of my postdoctoral research was to discern the protein-protein interactions that are essential for the functioning of the endocytic pathway, which relies on ubiquitin as a ticket for entry. Ubiquitin (Ub) is a highly conserved, small protein that is covalently attached to target proteins. The attachment of Ub to cell surface proteins can mediate their internalization and signal their degradation. This is a powerful and versatile mechanism cells can use to respond to their environment and fine-tune the composition of the plasma membrane. Once Ub has been attached to a target protein, a host of other proteins (Ub-receptors) recognize the Ub moiety to affect a number of different outcomes. A major outcome for cell surface proteins is sorting into intralumenal vesicles at the endosome forming multivesicle bodies (MVB). The MVBs eventually fuse with the lytic compartment (the lysosome in mammals and the vacuole in yeast) delivering the vesicles and their contents for degradation. These sorting steps appear to be initiated by distinct Ub-sorting receptors at the endosome by the multimeric Endosomal Sorting Complex Required for Transport (ESCRT) complexes. Within the ESCRTs, there are components that bind Ub and have been proposed to act as Ub-sorting receptors, Figure 2.
My first efforts focused on testing the hypothesis that there were additional, unidentified ubiquitin binding domains (UBDs) within the ESCRT complexes. I identified and characterized an additional UBD within the ESCRT-I complex both in vitro and in vivo. This work was published in an excellent journal in the field3, was a subject of a mini-review in the same issue4, and was featured in Faculty of 10005. Subsequently, I have identified several additional UBDs within the ESCRTs. Current efforts are underway to examine the in vivo function of these UBDs, in isolation and in combination. Additionally, I am the corresponding author of a publication with an undergraduate mentee6 and co-authored an invited review article for the journal Traffic7. I also gained the necessary skills and knowledge to perform a variety of in vivo and in vitro assays in both a model eukaryote (baker’s yeast) and mammalian cell culture. I now have the skill set and knowledge to answer questions about the fundamental causes of neurodegeneration, interconnecting the molecular mechanisms of protein misfolding and the cellular responses to it.
Future Research: A Model to Understand the Molecular and Cellular Causes of Neurodegeneration.
There are three ongoing areas of investigation in my lab: (1) the cross talk between the ubiquitin-dependent (proteasome and lysosome) and ubiquitin-independent (autophagy) protein degradation pathways, (2) the connection between protein folding stability and metal ion homeostasis, and (3) mechanisms that regulate the degradative enzymes (proteases and lipases) within lysosome.
Several neurodegenerative diseases have similar molecular and cellular features. The first, shown in Figure 1, is the formation of protein aggregates, which can be exported from the cell by exosomes and affect neighboring cells. Additionally, the protein degradation pathways are unable to clear both the protein aggregates and perform their normal cellular functions. Further, damaged and dysfunctional mitochondria are present and the normal levels of of biologically important metal ions (iron, copper, zinc, and magnesium) are imbalanced, Figure 3. However, it is unclear if these different features are causative or a result of neurodegeneration. For example, many of the proteins found in protein aggregates are known to bind metal ions, similar to prion proteins and copper. Yet, is the aggregation caused by the metal ion imbalance or is the metal ion imbalance a consequence of the protein aggregation? Do the protein aggregates inactivate the protein degradation pathways or is the inability to properly clear protein result in the protein aggregates. Additionally, several of these characteristics are connected with ubiquitin; the protein aggregates and dysfunctional mitochondria are coated with Ub and several metal ion transporters are mistargeted for degradation by ubiquitin.
Aim 1: Dissecting the Targeting of Proteins between the Ubiquitin-Dependent and Ubiquitin-Independent Protein Degradation Pathways While there are still mysteries about the specifics of the MVB (membrane bound proteins) and proteasome (cytosolic proteins) degradative pathways, the data are convincing that Ub is required for targeting and entry. However, it is not clear the role that Ub might play in targeting in “Ub-independent” pathways, like autophagy. Autophagy, which translated literally to “self eating”, is the cells response to starvation and allows it to cannibalize materials for reuse. Experimentally, it has been difficult to study the role of Ub in autophagy with mammalian systems and, in fact, it has provided conflicting evidence. Some studies indicate that the ubiquitination of protein aggregates is the cell’s desperate attempt to remove toxic materials through all possible mechanisms while others indicate that the Ub is a genuine player in autophagy and only detectable when there is a block in autophagy. (Still others have proposed that the lack of unbound Ub is what leads to neuron death and not the protein aggregates themselves.) Baker’s yeast (Saccharomyces cerevisiae) have proven to be an excellent model system to provide insight into the basic mechanisms of autophagy and protein aggregation and these findings have directly translated in understanding of mammalian neurodegeneration. Studies have found that neurodegeneration is associated with changes in conserved eukaryotic pathways, rather than neuron-specific pathways. Additionally, the rapid growth and genetic manipulability of yeast allow for high-throughout assays to be preformed quickly. Preliminary studies, Figure 4, in my lab have identified a candidate yeast protein that is both a cargo of autophagy and is ubiquitinated in response to starvation. Current efforts are underway to identify the factors that influence the ubiquitination of this protein, components within the autophagy machinery that act as Ub-receptors, and affect of increased Ub levels on the toxicity of protein aggregrates. Aim 2: Dissect the Connection between Protein Folding and Stability and Metal Ion Homeostasis. Metal ions are essential for all forms of life and their unique properties allow them to provide indispensable functions for the proper folding and activity of half of all known proteins. However, free metal ions are associated with oxidative damage and cell toxicity. Hence, the concentration and transport of metal ions in cells are tightly controlled processes. The regulation, requirements for metal ion homeostasis, and transport of metal ions has been well detailed in baker’s yeast, which has provided amazing understanding into a variety of diseases resulting from metal ion imbalance (e.g. Wilson’s disease). Additionally, a number of prototypical neurodegenerative proteins for expression in yeast have been developed and characterized. I intend to combine these two tools with my abilities to structurally characterize proteins to look at the effects of metal ion binding on the dynamics and stability of these prototypical proteins in both in vivo and in vitro assays. Aim 3: Identify the Mechanisms that Regulate Degradative Enzymes of the Lytic Compartment. Despite the wealth of information that is known about the mechanisms that target proteins and vesicles to the vacuole/lysosome from a variety of cellular locations, only a small number of enzymes have been identified that are responsible for degrading targets once they arrive. Conceptually, there must be a swarm of lipases, proteases, and polysaccharidases that can catalyze the breakdown of each of their corresponding macromolecules with specificity and precision to leave the lytic compartment itself unharmed and intact. In mammalian cells, it has been proposed that integral membrane proteins, LAMPs, are heavily glycosylated on their lumenal side, forming a protective ‘shell’ preventing the lytic enzymes from coming into contact with the lysosomal membrane. However, the lysosome remains intact and functional without LAMP proteins and other eukaryotes do not have homologous proteins, indicate that this is not a conserved mechanism to regulate or sequester the lytic enzymes from destroying the lysosome. A final goal of my research is to identify the degradative enzymes of the lytic compartment using proteomics and then characterizing their function and regulation in vivo and in vitro.