Iron-sulfur cluster proteins are considered to be one of the most ubiquitous prosthetic groups since ancient times. Regulations of iron homeostasis through iron-sulfur proteins is of critical importance for cell development as free labile iron can cause DNA and lipid damage, lesions and ultimately cancer from production of reactive oxygen species (ROS). The unique NEET class of proteins harbors 3 Cys- 1 His ligand configuration having a consensus sequence of [C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H] that houses 2Fe-2S clusters. This thesis focuses on a monomeric human NEET protein MiNT found within the mitochondrial matrix as opposed to the dimeric mNT and NAF-1. Though MiNT has an upregulated expression in cancer cells and 2Fe-2S cluster properties are similar to mNT and NAF-1, not much is known about it. Characterization of MiNT’s structure function relationships are studied in subsequent chapters in order to understand the effects of its asymmetry by doing a point mutation on the histidine at position 75 and 113 individually and both. Ultra-violet visible spectroscopy (UV-Vis) and circular dichroism (CD) will be employed to further understand MiNT’s unique differences as a monomeric protein carrying both clusters. These assays show that there are effects on the stability by temperature and structure differences are analyzed through the differences of the CD spectra. Comparison of spectra between oxidized and reduced state in UV-Vis for all constructs show that a possible tryptophan residue is responsible for acting as an electron transfer hub between the two clusters in MiNT, not noticed in the other human NEET proteins.

Understanding the complex knotted topology in protein folding is an important structural and functional indication as increasing families of protein discovered to contain deeply knotted structure. Methyltransferases in SpoU-TrmD (SPOUT) family catalyze the post-translational methylation of RNA assisted by the cofactor, S-Adenosyl methionine (SAM), and have a deeply fold trefoil knot that associated with the dimer interface and ligand binding domain. Misfolding in neuronal ubiquitin C-terminal hydrolases (UCH), where SPOUT family protein associate with, can result in aggregation and cause neurodegenerative disease. “Minimalism” of knotted protein was used to investigate the unfolding/untying process mechanism and its functional relationship of the knot in a more straightforward manner. MTTTM and MTTSA are both SPOUT MTases that contain trefoil knot with similar structure but different stabilities. Thus, studying native and unfolded proteins can provide information about knot forming mechanism and functional approach.

MTTTM purification protocol was modified, and the purification result was analyzed by mass spectroscopy. Fluorescence spectroscopy was utilized to monitor the tertiary structure change of native and unfolded MTases by tracing tryptophane signal for MTTSA and tyrosine signal for MTTTM. Highly refined structural and dynamics information of native and unfolded MTTSA was obtained by NMR spectroscopy. Shifts in unfolded MTTSA emission spectrum maximum as well as tryptophane signal in 1H-15N HSQC spectrum were observed, indicating possible solvent exposure and change in microenvironment of tryptophane. The unusual peak and shoulder appeared in the unfolded MTTTM emission spectrum requires further investigation in tyrosine residues and its relationship with tertiary fold of the protein.

There exist three different NEET family proteins in human cells. Named for their shared characteristic amino acid sequence (Asp-Glu-Glu-Thr), they were first identified as pioglitazone drug targets, one of the thiazolidinedione class of insulin-sensitizing therapeutics for Type 2 diabetes. Although each share similar structural organization and coordinate two [2Fe-2S] clusters with a unique 3Cys:1His coordination chemistry, their cellular localization varies and their functional roles are dissimilar. MitoNEET (mNT, CISD1), was the first characterized and is an outer mitochondrial membrane-anchored dimer with the C-terminal iron-sulfur cluster domain in the cytosol. Nutrient Deprivation Autophagy Factor 1 (NAF-1, Miner1, CISD2) is anchored to the mitochondrial-associated membranes of the endoplasmic reticulum (ER) with the iron-sulfur cluster domain in the cytosol. The third human NEET family protein is Mitochondrial Inner NEET (MiNT, Miner2, CISD3), is distinguished among the three, as it is the only soluble NEET, exists as a monomer, and is found inside the mitochondrial matrix.

The NEET proteins are critical for cellular iron regulation and reactive oxygen species (ROS) protection, redox sensing, metabolic regulation, and are critical for maintenance of respiratory electron transfer machinery. A missense mutation in NAF-1 causes Wolfram syndrome 2 and other NEET pathologies include neurodegeneration, diabetes, premature aging, as well as play a role in the proliferation of several cancers. Collectively these indicated a crucial underlying cellular role for iron management, redox regulation, and metabolism.

Until recently the role that mNT and NAF-1 play in the cell was unresolved. Though plenty of in situ and in vivo findings were published, elucidation of the precise cellular role of these proteins proved fruitless to scientific investigation. The major clues to their putative role were that 3Cys:1His iron-sulfur coordination is utilized by other iron-sulfur cluster transfer proteins, and a NEET family protein found in Arabidopsis thaliana (At-NEET) transfers iron into the mitochondria. Furthermore, RNAi knockdown of At-NEET caused free iron overload in the cell and led to ROS accumulation. Both mNT and NAF-1 have recently been identified as key links between the iron-sulfur cluster (ISC) biogenesis pathways in the mitochondria and the cytosol by transferring [2Fe-2S] clusters to human anamorsin, an early key piece of machinery that is responsible for providing [2Fe-2S] to the cytosolic iron-sulfur assembly (CIA) pathway. It has also been shown that mNT can transfer clusters to NAF-1, establishing that these proteins connect the flow of iron-sulfur away from the mitochondria to cytosolic targets.

The method by which iron-sulfur clusters are exported out of the mitochondrion to the cytosol is currently now well characterized. To determine whether human mNT could translocate iron-sulfur clusters into the mitochondrion through the outer membrane voltage dependent anion channel (VDAC), we examined the ability of mNT to control membrane potential across a bilayer embedded with VDAC. We characterized the mNT-VDAC binding with microscale thermophoresis (MST) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) in addition to computational fragment-docking direct-coupling analysis (Fd-DCA) to propose a model for mNT-VDAC binding. Through these data, our model suggests that mNT interacts measurably with the inner VDAC channel in addition to controlling VDAC gating in a redox-dependent manner, while NAF-1 does neither.

To further identify and characterize cytosolic iron-sulfur protein transfer partners, we selected human glutaredoxin-3 (GRX3), a protein recently determined to carry [2Fe-2S] clusters and contribute to the CIA pathway. We identified that GRX3 selectively transfers clusters to mNT, but not NAF-1, further establishing diverged cellular roles for these two proteins. This is also the only protein reported to date that can transfer [2Fe-2S] clusters to mNT. These findings further solidify the role of the NEET proteins link the ISC and CIA, and demonstrate that there is directionality of iron-sulfur transfer at the interface of the ISC-CIA pathways. This directionality suggests a path for feedback and recycling of cytosolic iron-sulfur.

The Minimal Tied Trefoil from Thermotoga maritima (MTTTm) represents an ideal system for investigating knots in proteins. It is one of the smallest knotted proteins to contain a deep trefoil knot, and one of the smallest SPOUT methyltransferases. To date, however, little has been accomplished on this system probing into the knots purpose in the protein. The work presented here characterizes the role of the knot in MTTTm. There are two main facets for understanding the knot: its role in the native state, and its role in the denatured state. To probe these, the work following uses a myriad of biophysical techniques to further understand the knots role in both states. These experiments show that in the native state the knot helps to tether down what was expected to be a flexible loop, which if flexible would impact binding. As well, there exist a series of hydrophobic contacts beneath the knot that are critical for allostery, and would be disrupted if the knot were not there. In the denatured state, there appears to be a linkage between the knot and critical packing residues that would complicate refolding in vivo, but the knot helps to maintain those contacts to allow for easier refolding. The studies here give us more information on why the knot exists in MTTTm, and help us to further understand the role of knots in proteins as a whole.

ABSTRACT OF THE DISSERTATION

Biochemical and Structural Characterization of the NEET Protein Family and their Role in Iron Homeostasis and Disease

by

Colin Harrison Lipper

Doctor of Philosophy in Chemistry

University of California, San Diego, 2017

Professor Patricia A. Jennings, Chair

The NEET proteins are a unique family of iron-sulfur proteins that bind a [2Fe-2S] cluster with an unusual 3Cys-1His coordination. The family consists of three human members, mitoNEET (mNT), nutrient-deprivation autophagy factor-1 (NAF-1) and Mitochondrial inner NEET (MiNT). These proteins have important roles in the regulation of iron and reactive oxygen species homeostasis and are involved in the pathology of several human diseases including cancer, diabetes, Wolfram syndrome 2 and neurodegeneration. These proteins have the ability to act as redox-sensors and transfer their Fe/[2Fe-2S] clusters to apo-acceptor proteins. We discovered that both mNT and NAF-1 can donate their clusters to human Anamorsin, a protein that is essential for the cytosolic assembly of Fe-S clusters. This connects the mitochondrial iron-sulfur cluster assembly system (ISC) to the dependent cytosolic iron-sulfur cluster assembly system (CIA). Additionally we identify an oxidation-dependent interaction with the outer-mitochondrial membrane pore protein VDAC1. The VDAC1 channel permeability is gated by the interaction with mNT. This NEET protein can transfer its [2Fe-2S] cluster into the mitochondria, causing iron to accumulate. Addition of a VDAC inhibitor prevents this mitochondrial transfer in situ and inhibits the interaction between mNT and VDAC1 in vitro. This indicates that VDAC channels are a direct route for mitochondrial iron transfer from mNT. Moreover, we find using hydrogen deuterium exchange mass spectrometry that mNT binds across the VDAC1 channel on the cytosolic side. Until now, very little was known about the third NEET family member, MiNT. We determined the crystal structure of this protein, which is significantly different than the other two human family members. It is monomeric with two cluster-binding domains in a single polypeptide, while mNT and NAF-1 are each homodimers with one cluster-binding domain per protomer. We find that MiNT can transfer its [2Fe-2S] cluster to human mitochondrial matrix ferredoxins, which are important for mitochondrial assembly of both iron-sulfur clusters and heme. MiNT is upregulated in breast cancer cells; we find that suppression of MiNT expression in cancer cells results in accumulation of iron and reactive oxygen species, as well as decreased mitochondrial membrane potential. Taken together, our findings further our understanding of how the NEET proteins function at a molecular level in maintaining iron homeostasis.

Interleukin-36 receptor antagonist is a cytokine that recently has been implicated in psoriatic diseases. Genome wide association studies observed single point mutations in the IL36RN gene which were associated with psoriasis patients. Characterizing how IL-36Ra works at a molecular level is critical in determining how diseases states may arise. Two observations regarding IL-36Ra were studied in detail in this work. Firstly, IL-36Ra was observed to be highly sensitive to N-terminal cleavage, in that the removal of the initiator methionine can activate the cytokine to its full activity. Secondly, IL-36Ra contains a unique disulfide bond that is not found in any related family member.

Chapter 3 details the NMR (Nuclear Magnetic Resonance) assignments of IL-36Ra necessary to solve the solution structure and probe phenomena at a residue resolution.

Chapter 4 details our observations of the structural changes and dynamics changes in IL-36Ra when comparing full length IL-36Ra to the N-terminal methionine cleaved IL-36Ra. Using a combination of NMR and hydrogen/deuterium we observed subtle differences in the two isoforms. The structural studies implicated the barrel structure in allowing allosteric transmission from N-terminal face to the opposite side of the molecule. Additionally, our dynamics studies revealed changes in the motions of the loops in the cap of the protein that are responsible for receptor binding. The changes were likely mediated via the residues at the cap-barrel interface which allowed changes in the barrel architecture to affect the loops in the cap subdomain.

Chapter 5 details our observations of the structural changes as well as dynamics changes in IL-36Ra when comparing oxidized IL-36Ra to the reduced IL-36Ra. Our data shows that the reduced form is highly destabilized overall, and specifically in the strands local to the disulfide bond. However, H/D exchange revealed that the residue diametrically opposed to the disulfide increased in protection from exchange, whereas most other residues decreased after the removal of the disulfide bond. These observations allowed us to construct a model in which the barrel expands without the presence of a disulfide

Lastly, Chapter 6 details the changes in H/D protection when IL-36Ra interacts with its receptor IL-36R. Different exchange profiles were observed for the two N-terminal isoforms when interacting with IL-36R. Using the observations, we were able to build a model in which the fully active IL-36Ra likely tunes the receptor off rate through the N-terminal/2nd trefoil interface that does not exist in the less active isoform.

NEET proteins belong to a class of iron–sulfur proteins that harbor a unique labile [2Fe–2S] cluster and play a major role in preventing mitochondrial dysfunction. Loss of function in mitochondria, the key organelle responsible for cellular energy production, can result in many human pathologies including diabetes, neurodegeneration, and cancer. Despite the recent advances in molecular medicine, cancer remains a significant challenge as it is the second leading cause of death in developed countries. Therefore, additional therapies are needed to increase therapeutic options to cure diseases related to NEET proteins. Since the exciting discovery of the novel mitochondrial target, MiNT, for the thiazolidinedione (TZD) insulin sensitizing drug pioglitazone, the NEET family has emerged as an important class of human proteins that are implicated in multiple pathologies. There are only a few compounds that have thus far been discovered or designed as NEET ligands that control NEET [2Fe–2S] stability. The Jennings laboratory and collaborators have shown that NEET proteins act as central sensors/transducers of iron/ reactive oxygen species (ROS) metabolism were deregulation results in cell death. Specifically, recent studies have shown that small molecules can bind to NEETs destabilizing the [2Fe-2S] cluster, leading to release of iron, induction of ROS and cell death. This effect is selective for cancer cells that upregulate NEETs. These findings demonstrate, for the first time, the significance of NEETs as drug targets for a chemotherapeutic treatment of cancer. This thesis focuses on the role of NEET proteins, specifically MiNT(CISD3), in different diseases using drug binding to examine protein stability. To further detail the unique structural difference of MiNT, as it is a monomeric protein containing two Fe-S clusters, we use a combination of biochemical and biological assays to characterize the effects of ligand binding. Specifically, we use ultraviolet visible (UV-Vis) spectroscopy and circular dichroism (CD) to monitor cluster stability over time. Together, these assays use temperature and secondary structure analysis to determine stability effects because of ligand interactions. Given the broadened understanding of the MiNT function and the initial results from binding interactions highlighted here, it might be possible to begin the development of therapeutic agents that may be useful in mitigating several different diseases including neurodegeneration, breast cancer, diabetes, and inflammation.

SR proteins are a family of splicing factors that plays important roles in regulating alternative splicing. The function of SR proteins is heavily regulated by the extent of phosphorylation of its C-terminal RS domain. Cdc-2 like kinases (CLKs) are known to hyper-phosphorylate the RS domains and control the splicing functions of SR proteins. CLKs have a folded kinase domain and an N-terminus that is predicted to be disordered. The N-terminus plays vital roles in regulating the function of CLK1 in various aspects. The studies presented here elucidate how the N-terminus modulates three different aspects of CLK1 function: sub-cellular localization, self-association to form oligomers, and substrate phosphorylation. Although prior studies had shown that the N-terminus was important for modulating the nuclear import of CLK1, the mechanism for this change in subcellular localization had largely been uninvestigated. Here, we show that CLK1 lacks a short, classical Nuclear Localization Sequence (NLS), indicating that the nuclear import is not mediated by the classical importin / system. Instead, we show that CLK1 enters the nucleus by forming a complex with its physiological substrate SRSF1, an SR protein prototype, in the cytoplasm and transportin SR-2 (TRN-SR2) imports the kinase-substrate complex into the nucleus. Previous studies from our laboratory had shown that the N-terminus induces oligomerization of CLK1, which helps the kinase select its physiological substrates over non-physiological ones. The nature of the interactions underlying this oligomerization was investigated and our results show that CLK1 oligomerization is driven not only by self-association of the N-terminus (N-N interactions) but also by interactions between the N-terminus and the kinase domain (N-K interactions). While interactions between the N-termini are mediated solely by aromatic residues, interactions between the N-terminus and kinase domain are electrostatic in nature. Lastly, we also investigated the role of the N-terminus in regulating SRSF1 hyper-phosphorylation. Our results show a strong correlation between CLK1 quaternary structure and substrate phosphorylation activity. While substrate binding affinity is solely regulated by the length of the N-terminus, the velocity of hyper-phosphorylation is tightly regulated by the quaternary structure of CLK1 oligomers. Our studies demonstrate that the N-terminus of CLK1 is highly versatile. It is important not only for recognizing a broad range of RS domains for essential SR protein hyper-phosphorylation but also for CLK1 nuclear localization through substrate “piggybacking.”

Pulmonary arterial hypertension (PAH) is a devastating disease that is often misdiagnosed. PAH is characterized by the remodeling of the small resistance arteries in the lungs that leads to elevated right ventricular pressure (RVP) and cardiac failure. Endothelial cells can sense danger or damage to the arterial wall caused by mechanical injury or respiratory pathogens through a subset of cytokines with an alarmin function to signal proliferation and restore barrier integrity. In this study we hypothesize that under chronic pulmonary hypertension conditions Interleukin-33 (IL-33) signals pulmonary artery endothelial cells to initiate a hyperproliferative response and this cellular event contributes to the remodeling of the vascular wall. To test this hypothesis pulmonary hypertension was induced in C57Bl/6J (wildtype), IL-33 receptor gene deleted (ST2-/-) and the upstream adaptor protein, MYD88 gene deleted (MYD88-/-) mice by exposure to 10% O2 and weekly SU5416 injections (SUHX). Arterial wall thickness increased in wild-type mice, but not ST2-/- and MYD88-/- mice, under SUHX treatments. The percentage of proliferating endothelial cells increased in wild-type mice under SUHX conditions but, this endothelial proliferative response was absent in ST2-/- and MYD88-/- mice. The C-terminal, mature form of IL-33 increased in wildtype, ST2-/- and MYD88-/- mice under SUHX conditions. This response suggests an upregulation of IL-33 and that IL-33 signaling through the ST2 receptor is important for endothelial cell proliferation and vascular remodeling in the SUHX model of pulmonary hypertension.