1,720,973 research outputs found
Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis
No full textHuman frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule
In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01
Full text linkRieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.</p
Protein Motifs Employed by Photosynthetic Organisms to Protect from Reactive Oxygen Species
No full textPhotosynthesis is essential for all living organisms on Earth because it involves reactions that transform the light energy from the Sun into chemical energy that we can use. Furthermore, and importantly, it is a process that produces oxygen, which provides aerobic organisms the ability to respire. However, along with these benefits, comes danger: during the process of photosynthesis, reactive oxygen species (ROS) are also generated. These toxic molecules are the inevitable consequences of aerobic life and have the potential to damage DNA, protein, and lipids in cells. Although photosynthetic organisms have a wide array of enzymes to directly resolve ROS, they also have indirect methods to maintain redox homeostasis. For example, thioredoxin proteins contain two Cys residues, which reduce disulfide bonds of enzymes in the Calvin cycle and pentose phosphate pathway. This thesis examines two Cys-rich proteins that are found in photosynthetic organisms and elucidates the molecular basis of how these Cys residues contribute to regulation.
In the heterocystous cyanobacterium Nostoc sp. PCC7120, RexT is an ArsR-SmtB transcriptional regulator of thioredoxin. Although several proteins in the family are observed to bind metal ions, RexT instead senses H2O2 using redox active Cys residues. However, prior to our work on RexT, there was a lack of understanding regarding the molecular details of how the DNA binding activity of RexT is modulated by changes in Cys residue oxidation state. To address this critical gap in our molecular understanding of RexT, we solved structures of RexT in its reduced and H2O2-treated states using X-ray crystallography. Different from that of reduced RexT, the H2O2-treated RexT forms a vicinal disulfide bond. Formation of this vicinal disulfide bond was further investigated using site-directed mutagenesis, electrophoretic mobility shift, and H2O2 consumption assays. Through our structural and biochemical analysis, we identified important residues that interact with H2O2 to produce vicinal disulfide bond and characterized an ArsR-SmtB transcriptional regulator which employs redox active Cys residues to regulate its DNA binding activity.
Another photosynthetic protein that we investigated in the work described within this thesis is chlorophyllase. Although chlorophyll (Chl) pigments are important for photosynthetic organisms to absorb light from the Sun, excess Chl molecules can be phototoxic and generate ROS. Key to the removal of Chl pigments in response to danger, or induced stress is chlorophyllase. This enzyme is involved in hydrolyzing the phytol tail of Chl a to produce chlorophyllide a. Traditionally, chlorophyllase, which was identified more than one century ago was implicated in degradation of Chl, but now, we know that it is responsible for degrading Chl in times of abiotic stress. Prior to our work, however, there was no structural understanding of how chlorophyllase is regulated to only degrade Chl under these conditions. In this thesis, we present the first X-ray crystallographic structure of chlorophyllase from Triticum aestivum. This structure shows a dimeric α/β hydrolase fold, a catalytic triad residues in the active site, and five disulfide bonds. Remarkably, using a combination of thermal shift assays, site-directed mutagenesis, and colorimetric assay, we showed that these bonds are important the hydrolysis activity and stability of chlorophyllase. This data is consistent with a model in which the redox state of the conserved chlorophyllase Cys residues is a switch that regulates activity.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/177807/1/mjlink_1.pd
Borane-Appended Ligand Design Strategies for Small Molecule Capture and Reactivity
No full textIn nature, metalloenzymes act as the premier catalysts for small molecule activation and transformation. The high activity and selectivity of enzymatic processes are, in part, owed to the complex system of acidic residues that make up the secondary and tertiary structures surrounding metal active sites. Synthetic systems that mimic these interactions, by employing either Brønsted or Lewis acids, can be used to gain further insight into small molecule transformations. The results described herein build upon existing literature of intramolecular ligand systems that feature moderately acidic Lewis acids that are pre-organized to interact with target metal-substrate complexes.
In this thesis, metal-ligand complexes incorporating secondary sphere borane Lewis acids are presented. Two ligands have been prepared: 1) a bidentate pyridine-pyrazole ligand framework that bears an appended trialkylborane at varied tether lengths from the metal center (explored in chapter 2 and chapter 5) and 2) a tridentate, tetrahedral enforcing 1,4,7-triazacyclononane ligand framework that bears an appended trialkylborane with a 3-carbon tether length (explored in chapter 3, chapter 4, and chapter 5). Metalation with first-row transition metals Zn(II), Fe(II), Cu(I), and Co(II) resulted in a series of intramolecular transition metal-borane platforms that were employed for the capture and transformation of biologically and industrially-relevant small molecules.
The Zn(II)-N2H4 complexes investigated in chapter 2 provide important insight into the effect of Lewis acid binding to reactive substrates that contain acidic N-H peaks: additive Lewis acid effects from the metal center and appended borane lower the acidic group’s pKa, making deprotonation accessible. Reactions to transform hydrazine at Zn(II) and Fe(II) highlight the importance of flexibility in the system when a 3-carbon appended Lewis acid tether is employed. The importance of flexibility in this system is extended to the weakly basic substrate thiophenolate in chapter 5, where the 3-carbon tether is once again highlighted as the ideal length for an intramolecularly appended Lewis acid to form a cooperative interaction with the metal-bound substrate.
The chemical insight gained through these substrate binding studies was extended to the Cu(I) system explored in chapter 3. Importantly, the appended borane within the secondary coordination sphere enables capture of an otherwise unstable copper hydride. Reactivity investigations with this novel complex uncovered divergent reaction pathways that can be accessed by tuning the Cu oxidation state and, ultimately, a unique mechanism for phenylacetylene reduction was discovered. Chapter 4 extends the reactivity of intramolecular borane-bound metal hydrides to new Co(II) complexes that were found to be active for the hydrogenation of terminal alkenes under catalytic hydrogenation conditions.
An overall feature of the work described in this thesis is the formation of mononuclear cooperatively bound metal-substrate-boron complexes; chapter 5 provides fundamental guidelines for such substrate binding across a range of substrates. A key result uncovered here is that the 3-carbon tether for the appended borane displays ideal flexibility and mobility to 1) capture both inert and reactive substrates that bind with μ-1,1 and μ-1,2 binding modes and 2) switch between these modes. Collectively, the studies presented in this thesis demonstrate the important principles that appended borane Lewis acids can help stabilize reactive small molecules at transition metal centers that otherwise are unstable, and these intramolecular systems can regulate small molecule reactivity and facilitate novel reaction pathways.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/193486/1/enorwine_1.pd
Probing the Mechanism of Viral Inhibition by the Radical S-adenosyl-L-methionine (SAM) Dependent Enzyme- Viperin
Full text linkViperin (Virus Inhibitory Protein; Endoplasmic Reticulum associated, INterferon inducible) is an endoplasmic reticulum (ER)-associated antiviral responsive protein that is highly up-regulated in eukaryotic cells upon viral infection. Viperin is a radical S-adenosyl-L-methionine (SAM) enzyme, that catalyses the synthesis of antiviral nucleotide 3’-deoxy-3’, 4’-didehydro-CTP (ddhCTP) exploiting radical SAM chemistry. However, the modulation of its catalytic activity by other intracellular proteins is not well understood and needs further investigation. In this dissertation, I use enzymology-based approaches to investigate how viperin’s enzymatic activity is regulated through its interaction with various cellular and viral proteins that are involved in cellular metabolic and signalling pathways and viral replication. I showed that viperin can reduce the intracellular expression level of the cholesterol biosynthetic enzyme, farnesyl pyrophosphate synthase (FPPS). This, in turn perturbing the intracellular cholesterol synthesis, thereby retarding budding of enveloped viruses from cholesterol-rich lipid rafts of host cell membranes. I also undertook a proteomics study that revealed that viperin interacts with several other endogenous cholesterol biosynthetic enzymes. I also demonstrated that viperin promotes the degradation of viral non-structural protein A (NS5A) from hepatitis C virus through proteasome-mediated degradation in the presence of sterol-regulatory protein VAP-33. In turn, co-expression of viperin with VAP-33 and NS5A reduced the specific activity of viperin by ~ 3-fold. Lastly, this study showed that viperin is activated by innate immune signalling proteins kinase IRAK1 and ubiquitin ligase TRAF6, as it facilitates the ubiquitination of IRAK1 by TRAF6. The results provide valuable insights into the mechanism of action of viperin in regulating these target proteins and its significance as a SAM-dependent enzyme.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155178/1/soumigho_1.pd
Engineering Biocatalysts for Oxidative Cross-Coupling Reaction
No full textThe precision and efficiency offered by enzyme catalysis has propelled the incorporation of biocatalytic strategies into synthetic campaigns in academic and industrial spheres. Nevertheless, biocatalytic methods remain prohibitively underdeveloped compared to the vast repertoire of their small molecule-based counterparts, including for the formation of carbon–carbon (C–C) bonds. Oxidative coupling reactions offer a direct and complexity-generating approach to C–C bond formation; however, controlling the selectivity of these reactions remains an outstanding challenge in organic synthesis. In contrast, Nature has evolved a wealth of cytochrome P450 enzymes that catalyze selective oxidative C–C coupling reactions in the biosynthesis of biaryl natural products. Inspired by this catalyst-controlled selectivity, the work presented in this dissertation describes a platform for biocatalytic biaryl bond formation using P450 catalysts. Using a yeast-based platform for whole-cell biocatalysis, we demonstrate the ability of a fungal P450 to catalyze unnatural cross-coupling reactions on a panel of phenolic substrates. Moreover, over five rounds of directed evolution, we engineer the fungal P450 to possess the desired reactivity and site-selectivity by improving a low-yielding, unselective reaction 92-fold for the target cross-coupled product. We further expanded the biocatalytic platform by profiling the cross-coupling activity of a library of natural P450s using a bioinformatics-guided approach, leading to the identification of a bacterial P450 capable of cross-coupling substituted naphthols. Despite demonstrating promiscuous activity, the P450 offered little to no stereocontrol in cross-coupling reactions. To overcome this limitation, the P450 was engineered for improved atroposelectivity, ultimately providing a biocatalyst capable of cross-couplings with up to 95:5 er in the formation of privileged chiral biaryl ligands. Altogether, this streamlined method for constructing sterically hindered biaryl bonds provides a programmable platform for assembling biaryl molecules with catalyst-controlled reactivity and selectivity.PhDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/174503/1/zetzsche_1.pd
Structure and Mechanism of PhdC, a Prenylated Flavin Oxidative Maturase
No full textUbiD-like (de)carboxylase enzymes employ prenylated-FMN (prFMN) as a cofactor to catalyze (de)carboxylation reactions on otherwise unreactive aromatic rings and conjugated double bonds. UbiD-like enzymes are typically encoded along with a UbiX-like prFMN synthase, forming a two-enzyme system. UbiD-like enzymes are attractive for biocatalysis applications but are often difficult to obtain as active holoenzymes. Phenazine-1-carboxylic acid decarboxylase (PhdA) is one such case: even when co-expressed with its cognate prenylated-FMN synthase (PhdB), PhdA is largely obtained as inactive apoenzyme.
We show that a third previously unannotated protein, PhdC, encoded in the same operon, functions as a maturase to catalyze the oxidation of prFMN to the catalytically active form. Heterologous expression in E. coli of PhdA, PhdB and PhdC allowed highly active holo-PhdA to be purified. In vitro reconstitution of apo-PhdA with prFMN oxidized by PhdC gives fully active holo-PhdA. PhdC also facilitated the installation of prFMN in furan-1,4 dicarboxylate decarboxylase, HmfF, suggesting that this enzyme may have general utility in the production of active holo-UbiD-like enzymes. Using purified proteins, we show that PhdC uses molecular oxygen or cytochrome C to oxidize the prFMN semiquinone radical, normally a dead-end form of prFMN, to the active cofactor.
To elucidate the mechanism of this oxidative maturation, crystallographic and mutagenic studies were performed, revealing key active site residues in contact with prFMN and their roles in facilitating the maturation of prFMN. Of these residues, E134 may act as a general base for the deprotonation of prFMN. The variant E134Q is unable to mature prFMN using oxygen but retains maturation activity with cytochrome C. Alanine variants of E45 and S42 were active but showed impaired rates of turnover. Kinetic experiments with isotopically-labeled prFMN revealed a kinetic isotope effect of 1.6 ± 0.2 for wildtype PhdC but a much greater KIE was observed in the E45A and S42A mutants, highlighting the importance of these residues in facilitating the deprotonation of prFMN.
PhdC represents the first in a new class of prFMN maturases, but many more homologous proteins have been observed to co-occur with genes encoding for UbiX and UbiD-like proteins. Over 700 homologues were identified, that were mainly present in prokaryotes, but with some archaeal members as well. Informed by the structural and mechanistic characterization of PhdC, two conserved motifs were discovered in PhdC homologues. Both motifs correspond to the triad of residues E134, E45, and S42, which are important for catalysis in PhdC. In some homologs these motifs show intriguing differences in the residues corresponding to E134 of PhdC which may result in variations in activity between these homologous proteins. With this in mind, several homologous proteins were selected for cloning with the aim of better understanding the maturation of prFMN, discovering more efficient maturases, and possibly uncovering new prFMN biochemistry entirely.
Overall, this work has laid the groundwork for the study of a new facet to prFMN biochemistry: prFMN maturation. Maturase proteins have the potential to improve the activity of UbiD-like enzymes by increasing the expression of active, holo-enzyme forms.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/199527/1/ddirocco_1.pd
Designing Chemical- and Light-Activated Protein Switches for Regulating Peptide Functions
No full textPeptides serve important biological functions including neuromodulation, hormonal regulation, cell signaling, protein localization, and enzyme inhibition. The ability to modulate peptide functions with precision is invaluable in biological research. Genetic tools have offered precise control over biological systems with cell-type specificity, and chemogenetic and optogenetic techniques have expanded this control, providing high temporal and spatial resolution through small molecules and light as signaling inputs. While these methods have been extensively applied to regulate protein functions, their application in controlling peptide functions is less explored.
This thesis describes the engineering of chemogenetic and optogenetic protein domains for regulating peptide functions. These domains offer versatile control over various peptides, modulating biological processes through orthogonal signal inputs from small molecules and light. A directed evolution platform for optimizing these domains is also introduced.
For chemogenetic control over peptides, we developed the chemically activated protein domains (CAPs) for controlling the accessibility of both the N- and C-terminal portions of functional peptides. CAPs were developed through directed evolution of an FK506 binding protein (FKBP). By fusing a peptide to one or both CAPs, the peptide’s function is blocked until a small molecule displaces them from the FKBP ligand binding site. CAPs are generally applicable to a range of short peptides, including a protease cleavage site (TEVcs), a dimerization-inducing heptapeptide (SsrA), a nuclear localization signal peptide (NLS), and an opioid peptide (enkephalin), with a chemical dependence up to 156-fold. We show that the CAPs system can be utilized in cell cultures and multiple organs in living animals.
The second light, oxygen, voltage sensing domain from Avena sativa phototropin 1 (AsLOV2) has been widely applied to modulate the activity of various peptides by light. However, due to geometry restrictions, AsLOV2 is not applicable for peptides whose functions requires fusion-free N-terminus. We re-engineered AsLOV2 using circular permutation strategy to generate cpLOV. This modification allows modulation of the C-terminal accessibility of functional peptides while leaving the N-terminus unfused. Using the same strategy as CAPs and showcased by TEVcs, functional peptides can be fused to both AsLOV2 and cpLOV tandemly to reduce the basal activity and tune the dynamic range.
To further optimize these chemical- and light-switchable protein domains, we established an efficient yeast surface based directed evolution platform. This platform simultaneously exhibits activation and leakage signals on the same yeast cell, enabling further optimization of CAPs' caging efficiency. The improved CAPs were then applied to regulate three neuropeptides: enkephalin, pituitary adenylate cyclase-activating polypeptide (PACAP), and α-melanocyte-stimulating hormone (α-MSH), showcasing their broad applicability in modulating peptide functions.
Potential future work includes the optimization of the developed switchable protein domains, expanding the scope of using CAPs to modulate other neuropeptides, and development of orthogonal switchable protein domains.
This thesis contributes significantly to the field of peptide function modulation, offering novel chemogenetic and optogenetic tools and methodologies that have profound implications for biological research.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/193325/1/shenjiaq_1.pd
Designing Metalloenzymes by Controlling the First and Second Coordination Spheres Using Non-Traditional Design Strategies
No full textThis thesis extends or introduces innovative approaches to de novo design to interrogate metal binding, structure, and catalytic activity using a copper nitrite reductase (CuNiR) model. These designs will be used to investigate, at a molecular level, how the first and second coordination spheres influence the copper’s coordination number and, in turn, modify nitrite reduction in a well-folded 3-stranded α-helical coiled coil (3SCC). The non-coded amino acids 3’Pyridyl alanine [3’Py] and 4’Pyridyl alanine [4’Py] side chains were first designed into the 3SCC forming sequence [Ac-G WKALEEK (LKALEEK)2 PyKALEEK G-NH2]3 (i.e., TRIW) to enforce the-nitrogen of histidine within the core of the assembly (i.e., authentic pyridine type nitrogens). The pyridyl sites formed 3-coordinate cuprous sites and 4- or 5-coordinate cupric geometries which yielded an unnatural type 2 copper center (T2Cu). The cuprous state favored a more 2-coordinate linear geometry by designing space above the active site through the mutation of the leucine layer to an alanine layer (L19A), highlighting the impact of distant residues on protein structure around a metal. CuNiR kinetics were investigated under conditions favoring the binding of nitrite to the cuprous state (i.e., Abraham’s mechanism) which is the alternate, unexplored pathway of native CuNiRs. Compared to the natural histidine site, the pyridine ligands enhanced CuNiR activity by a factor of 400. Interestingly, while the kcat values were maximized (~3 s-1), the catalytic efficiency was doubled (kcat/Km= 5 M-1 s-1 vs. 11 M-1 s-1) which depended on the ligand’s orientation within the active site. The catalytic efficiency is further enhanced by shifting to a 2-coordinate Cu(I) enzyme which resulted in a decrease in the Km from 600 mM to 50 mM and kcat/Km of 39 M-1 s-1. Detailed structural experiments and inhibition kinetics resulted in competitive inhibition between Cl and NO2 providing strong evidence of nitrite binding to the reduced enzyme during the catalytic cycle. This demonstrated the formation of a Cu(I)NO2 adduct and highlighted the Cu(I) enzyme as the important oxidation state controlling catalytic efficiency. It is argued that NO2 displaces one pyridine ligand in the 3-coordinate cuprous complex (verses forming a 4-coordinate Cu(I)(Py)3-NO2), analogous to what is observed with the Cu(I)Cl adducts as a result of the steric constraints of the interior of the core. Collectively, the rationale for a 3-coordinate Cu(I)-NO2 enzyme-substrate complex is explored within the context of the sterics between the primary and secondary coordination spheres.
This work will also describe an alternative, novel strategy to repack side chains within the 3SCC using 3-residue non-canonical inserts (i.e., stammer) to overwind the coiled coil to explore nitrite reduction. Stammers will be explored in terms of their ability to change the metal-coordination number or rotate solvent-exposed residues (i.e., glutamate) into the core in proximity of the copper center. While this approach generates 3-coordinate cuprous enzymes for both the 3’Py and 4’Py ligands, 2-coordinate resting states are achieved by implementing two different stammers for either ligand (i.e., AEA and AE3’Py), all of which are distinct from the cuprous states of the Leu/Ala designs in terms of their electronic structure. Each stammer explored resulted in slight variations in the kinetics which impacted only the Km values in the range of 100-400 mM. This study will emphasize yet another approach to controlling the coordination number of copper active sites to tune the catalytic efficiency.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/196043/1/wcpitts_1.pd
Activation of Protein Ubiquitination by the Antiviral Enzyme, Viperin
No full textViperin (Virus Inhibitory Protein; Endoplasmic Reticulum-associated, Interferon iNducible) is an interferon-stimulated gene that is upregulated as a part of the innate immune response to viral infection. It has been shown to restrict the replication of a broad range of human viruses including influenza, hepatitis C, human immunodeficiency, Dengue, West Nile, Zika, and tick-borne encephalitis viruses. However, the mechanism with which viperin restricts infection varies dependent upon the type of virus. Viperin is a member of the radical S-adenosylmethionine (SAM) enzyme superfamily, and recently was shown to catalyze the dehydration of cytidine triphosphate (CTP) to form the antiviral nucleotide 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP) through a SAM-dependent radical mechanism. ddhCTP acts as a chain terminating inhibitor of viral genome replication of some, but not all, viral RNA-dependent RNA polymerases. These recent findings are exciting but do not fully account for viperin’s antiviral activity against most other viruses.
Viperin is also known to play a key role in the Toll-like receptor 7 and 9(TLR-7/9) immune signaling pathways. It recruits signaling proteins to lipid bodies, and thereby facilitates the downstream activation of numerous genes. However, evidence for activation of downstream genes comes from studies conducted with proteins transiently expressed in mammalian cells, and the interpretation of such data is complicated by the potential involvement of other cellular proteins. Therefore, this study focuses on reconstituting viperin’s interactions with two enzymes involved in TRL-7/9 signaling in vitro using purified proteins: TRAF6 (tumor necrosis factor receptor associated factor 6) and IRAK1 (interleukin receptor associated kinase 1). TRAF6 is an E3 ubiquitin ligase that catalyzes K63-linked polyubiquitination of a broad range of substrate proteins which are involved in several signaling pathways including TLR7/9, NF-kB, and MAPK signaling cascades. In addition, TRAF6 itself is auto-ubiquitinated to recruit downstream kinases into signaling complexes.
Here, I describe the recombinant expression and purification of various domains of TRAF6, the ‘death’ domain of IRAK1, and an N-terminal truncation of viperin (viperin-ΔN50) from E. coli. This has allowed the interaction between viperin and TRAF6 to be directly demonstrated. It also allowed the auto-ubiquitination activity of TRAF6 to be reconstituted in vitro using purified enzymes. Using this system, viperin was shown to activate TRAF6 ubiquitin ligase activity, which provides a biochemical mechanism to explain viperin’s role in potentiating innate immune signaling.
The interaction of viperin with IRAK1 has also been studied. IRAK1 is a serine/threonine kinase that is involved in TLR7/9 pathways. Viperin is predicted to facilitate the ubiquitination of IRAK1 by TRAF6 to activate the production of type I interferons. Using truncated IRAK1 constructs, transiently expressed in HEK293T cells, the interactions of IRAK1 with viperin was localized to the ‘death’ domain of IRAK1. Unfortunately, attempts to express and purify this IRAK1 domain in its soluble form in E. coli to facilitate in vitro studies proved unsuccessful.
Lastly, I developed a method to purify the full-length, membrane associated form of viperin using lipid nanodiscs to maintain a membrane-like environment. These experiments represent the first time that full-length viperin has been purified in its active form. This work thus provides a new platform to facilitate structural studies on full-length viperin and study its interaction with other membrane-associated proteins that may contribute to its antiviral activity.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/171349/1/ayepatel_1.pd
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