Redox Biology

Dr. Donald Becker

DEPARTMENT OF BIOCHEMISTRY

Redox regulatory mechanisms in proline metabolism

Proline utilization A (PutA) is a membrane-associated flavoenzyme from gram-negative bacteria that oxidizes proline to glutamate using FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent L-glutamate-γ-semialdehyde (GSAL) dehydrogenase (GSALDH) domains. The PRODH and GSALDH domains are connected via a 40 A cavity in which the intermediate of the reaction, GSAL, is channeled during catalysis. In Escherichia coli, PutA also has DNA binding activity and is an autogenous transcriptional repressor of the proline utilization (put) regulon. A major focus in the Becker Lab is understanding communication between domains in PutA proteins and how PutA proteins regulate DNA binding and catalysis. REU students will develop and test important hypotheses of PutA redox regulation and gain experience with electrochemistry, molecular biology, protein purification, enzymology, and spectroscopic methods.

Dr. Lindsey Crawford

DEPARTMENT OF BIOCHEMISTRY

Viral manipulation of cell fate and redox metabolism

Chronic viral infection plays key roles in the manipulation of essential cellular functions including the fundamental processes of proliferation, differentiation, metabolism, and redox function. Understanding how specific viruses control these processes provides novel insights into how fundamental human cell biology works. The Crawford Lab focuses on a common human herpesvirus and how the proteins from an understudied viral gene region (RL11) regulate cell signaling, mitochondrial function, and cell-cell interactions with the corresponding implications for viral latency, reactivation, pathogenesis, and cellular functions. REU students will develop and test individual hypotheses on the function of a specific RL11 protein or protein domain in controlling cell fate, with emphasis on redox biology, metabolism, and mitochondrial structure. They will be immersed in an interdisciplinary group and receive training in biochemistry, virology, immunology, stem cell biology, and scientific design, analysis, methodology, and presentation.

Dr. Katarzyna Glowacka

DEPARTMENT OF BIOCHEMISTRY

Redox regulation of photosynthesis processes

Plants experience rapid fluctuations in the light, resulting from both cloud and leaf movements. In the absence of buffering mechanisms, rapid increases in light intensity can produce singlet oxygen and other ROS that can damage cells. Plants have evolved non-photochemical quenching (NPQ) to safely dissipate excess energy absorbed as heat. Uniquely, NPQ is regulated by redox and regulates the redox of other proteins including the first stable acceptor of the electron transport chain, quinone A (QA). 

A main interest of the Glowacka Lab is to understand how NPQ is regulated under control and stress conditions. The Glowacka Group has recently determined that uncharacterized thioredoxins (TRXs) are involved in the regulation of NPQ. TRXs are oxidoreductases that regulate the stability and activity of proteins via reversible modification of thiols. REU students in the Glowacka Group will be guided to develop individual hypotheses on regulation of the photochemical and non-photochemical quenching by TRXs. To test the hypotheses, the REU students will use multidisciplinary approaches, including fluorescence imaging to quantify the kinetics of NPQ, photosystem II, and QA redox states and spectrometry to assess photosystem I redox state and ATPsynthases activity.

Dr. Yihe Huang

DEPARTMENT OF BIOCHEMISTRY

Structural and evolutionary mechanisms of transmembrane signal transduction

Frizzled receptors, a group of transmembrane proteins, are key mediators in animal development and regeneration and are linked to redox sensing. However, the molecular principles underlying redox sensing by Frizzled receptors are largely unknown. Studies have suggested that Frizzled receptors and their transmembrane complexes sense redox signals through interactions with the thioredoxin-like protein nucleoredoxin (NRX). 

REU students in the Huang Lab will receive training in biochemical, biophysical, and structural approaches to develop hypotheses on the interactions between NRX and Frizzled receptors and their complexes. To test these hypotheses, students will design and conduct experiments including size exclusion chromatography, pull-down assays, mutagenesis, and cryo-EM studies. These research experiences will provide REU students with rich opportunities to develop their scientific skills in biophysics and structural biology.

Dr. Oleh Khalimonchuk

DEPARTMENT OF BIOCHEMISTRY

Redox regulation of mitochondrial processes

The Khalimonchuk Lab seeks to understand processes that preserve normal mitochondrial functions during homeostatic challenges, such as changes in nutrient availability, oxidative damage, and protein misfolding. The current research focus is to delineate the roles of several conserved redox-regulated proteases that are critical for mitochondrial protein quality control and energy metabolism. REU students will investigate mitochondrial proteases such as metallopeptidase Oma1 and serine proteases CLPXP and LACTB to elucidate the mechanisms of redox-tuned activation of mitochondrial protein quality control. 

Using yeast and mammalian cell culture systems, state-of-the-art genetics and biochemical approaches, and various physiological and imaging techniques, REU students will develop and test hypotheses by: 1) assessing the impact of site-specific mutations of redox-sensitive residues on the function, stability, and activity of mitochondrial proteases; 2) analyzing physical and genetic interactions of said enzymes by proteomic approaches and reverse genetic screens; and 3) determining physiological penalties of the proteases’ inactivation and relevant signaling cues associated with the deficit.

Dr. Jaekwon Lee

DEPARTMENT OF BIOCHEMISTRY

Mechanistic insights into homeostasis and functional roles of inorganic elements

Inorganic elements are vital nutrients that support many biological processes, including protein structure and function, maintenance of membrane potential, and cell signaling. However, excess accumulation or uncontrolled chemical reactions involving these elements can be toxic (e.g., generation of ROS). Dr. Lee's research program is dedicated to identifying and characterizing the molecular factors involved in the uptake, utilization, and detoxification of inorganic elements (e.g., Cu, Fe, K) and their functional roles and toxicities in yeast and mammalian cells. Under this program, REU students will investigate the mechanisms underlying lipid metabolism disorder in mouse liver cells and budding yeast due to copper deficiency induced by knocking out the Ctr1 copper importer gene.

Dr. Toshihiro Obata

DEPARTMENT OF BIOCHEMISTRY

Effects of intracellular redox states on multienzyme complexes of central carbon metabolism

A complex of enzymes catalyzing sequential reactions of a metabolic pathway, called a ‘metabolon’, channel metabolic intermediates of the enzymes and enhance pathway reaction efficiency. Enzymes of central carbon metabolism, including glycolysis, oxidative pentose phosphate pathway, and the tricarboxylic acid cycle, form dynamic metabolons whose association is related to cellular metabolic status. Since central carbon metabolism is closely related to the production and consumption of redox molecules such as NADH, NADPH, and FADH2, component enzymes are often redox regulated. The Obata laboratory hypothesis is that the metabolons in the central carbon metabolism are regulated according to the subcellular redox states. The REU students will generate Saccharomyces cerevisiae reporter cell lines to monitor redox state of specific subcellular compartments and multienzyme complex formation using an established gene-editing procedures.

Dr. Xinghui Sun

DEPARTMENT OF BIOCHEMISTRY

Protein post-translational modification in redox processes, cell death, and vascular disease

Dr. Sun’s group is interested in elucidating the uncharacterized roles of protein neddylation in regulating cell death, redox homeostasis, and vascular integrity. Protein neddylation is one type of post-translational modification that regulates protein function and stability. The Sun Lab hypothesizes that the inhibition of neddylation causes reductive stress, activates inflammasome, and impairs endothelial integrity resulting from the persistent activation of the nuclear factor erythroid 2–related factor 2, a target of Cullin3 RING E3 ligase. The Sun Lab employs various approaches including molecular and cellular techniques, mouse models of human diseases, immunohistochemistry, multi-omics (e.g., genomics, transcriptomics, proteomics), and bioinformatics to discover new knowledge in biochemistry, vascular biology, and human diseases, which provide an excellent training opportunity for students interested in basic cellular and molecular biology research. 

Specifically, REU students will have opportunities to examine the role of neddylation in: 1) redox homeostasis by doing cell culture, molecular cloning, immunoprecipitation, and mass spectrometry; 2) redox-regulated cell death of human endothelial cells by doing biochemical assay, cell culture, flow cytometry, and confocal imaging; and 3) redox-regulated atherosclerosis in mice by doing real-time qPCR, immunostaining, and histological analysis.

Dr. Mark Wilson

DEPARTMENT OF BIOCHEMISTRY

Conformational dynamics and enzyme catalysis

Enzyme catalysis requires a delicate balance of structural stability and conformational dynamics. The non-equilibrium conformational dynamics of enzymes has traditionally been inaccessible to detailed structural characterization by X-ray crystallography or nuclear magnetic resonance because both techniques have been limited to near-equilibrium states. The advent of serial X-ray crystallography at X-ray free electron laser and synchrotron sources has opened a new window to non-equilibrium structural biology, allowing enzymes to be characterized in detail as they catalyze reactions in crystals in real time. The Wilson Group develops and applies new methods to study enzyme catalysis in various systems using time-resolved X-ray crystallography and cryo-EM. The central goals of this work are to explore new enzyme mechanisms and study functional enzyme conformational changes during catalysis by direct structural observation. 

Current projects focus on cysteine-dependent enzymes of the DJ-1 superfamily, where the Lab addresses mechanistic questions concerning the identity of the true substrate for human DJ-1, the identity of mandatory intermediates in isocyanide hydratase catalysis, and the role of electrostatic and quantum mechanical effects in enzyme catalysis in these and other proteins. Students working in the Wilson Lab will develop hypotheses about the function of DJ-1 superfamily (and other) enzymes and be immersed in protein biochemistry, conventional and time-resolved X-ray crystallography, and cryo-EM methods at the bench and computer.

Dr. Tatsuya Yamada

DEPARTMENT OF BIOCHEMISTRY

Neural basis of redox control in hepatocytes

As the primary organ for processing nutrients and xenobiotics, the liver undergoes various redox reactions. Mitochondria occupy the central position in redox metabolism due to their primary role in processing innumerable metabolites. The liver is densely innervated and receives topdown inputs from the central nervous system that modulate hepatic mitochondrial functions and structures. The Yamada Lab is dedicated to unraveling the communication mechanisms between the liver and nervous system that control the hepatic mitochondrial redox response. 

REU students will postulate their concepts and investigate how neural inputs are translated into intracellular signals that trigger subsequent metabolic reactions. They will decide which signaling cascade to focus on and design experiments to test whether the signaling cascade is activated or repressed by neural inputs by analyzing the phosphorylation status of proteins involved in the cascade. Thus, REU students will have a unique opportunity to conduct hypothesis-driven research while gaining technical skills in molecular biology, biochemistry, and physiological analysis.

Dr. Limei Zhang

DEPARTMENT OF BIOCHEMISTRY

Iron-sulfur proteins in redox stress response

Transition metals play essential roles in a broad range of biological processes and are widely used in bacteria as redox stress sensors. A major research goal of the Zhang Group is to advance mechanistic understandings of the structural basis for iron-sulfur cluster-containing transcription factors involved in bacterial stress response and antibiotic resistance, including the WhiBLike (Wbl) transcription factors from Mycobacterium tuberculosis. REU students will use various structural and computational tools to develop hypotheses on the key residues that fine-tune the redox reactivities of the iron-sulfur cluster and DNA binding in the Wbl proteins. They will then design and conduct experiments to test the hypotheses. Projects will provide a unique, hypothesis-driven research experience for REU students in molecular biology, redox protein biochemistry, and structural biology.