Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with ...eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.
Abstract only
Glutamate Dehydrogenase (GDH) catalyzes the reversible oxidative deamination of glutamate using NAD(P)
+
(H), and is involved in various metabolic pathways and energy cycles. GDH can be ...allosterically regulated by various nucleotides (ADP, GTP) as well as showing cofactor cooperativity. Previous work shows NADH binds to both the active site and a second regulatory (inhibitory) site while NADPH binds only to the active site. We hypothesize that changes in stability/flexibility correlate with inhibition and activation. To explore the allosteric mode for GDH, we have used Fluorescence‐based Thermal Shift Assays (FTSA), Circular Dichroism (CD), Thermal melts (Tm) and Limited Proteolysis using immobilized trypsin and tandem mass spectrometry (LP) to explore the stability (global and localized) of the protein in the presence or absence of various combinations of nucleotides and substrates (glutamate or 2‐oxo‐glutarate). CD and Tm studies indicate that while the overall structure of the protein is not changed by pH, the stability changes dramatically over the pH range 6–8.5 with a pKa of 7.4. ADP stabilizes the protein while GTP destabilizes the protein, with coligands having little effect. NADPH shows a small destabilizing effect, while NADH effects are complex depending upon concentration. Below the pKa, FbTSA experiments show that ADP and NADH binding to its regulatory site stabilize the protein significantly, while NAD(P)H binding to the active site, or GTP binding destabilize the protein, consistent with the conclusions reached from the CD thermal melt studies. With ADP, binding curves established by FTSA indicate biphasic binding in the presence or absence of cofactor bound at the active site suggesting negative cooperativity in ADP binding. The presence of glutamate has little effect. At pH 8, ADP and GTP have similar effects, while with NADH and NADPH destabilizes the protein. To determine localized regions of the protein responsible for these effects, we are using LP experiments and mapping regions of flexibility changes on the overall 3 dimensional structure of the protein.
Support or Funding Information
Work was funded by the USD Knapp Chair and the USD McNair program.
Toll-like receptor (TLR) ligand-binding domains comprise 18-25 tandem copies of a 24-residue motif known as the leucine-rich repeat (LRR). Unlike other LRR proteins, TLRs contain significant numbers ...of non-consensus LRR sequences, which makes their identification by computer domain search programs problematic. Here, we provide methods for identifying non-consensus LRRs. Using the location of these LRRs, hypothetical models are constructed based on the known molecular structures of homologous LRR proteins. However, when a hypothetical model for TLR3 is compared with the molecular structure solved by x-ray crystallography, the solenoid curvature, planarity, and conformations of the LRR insertions are incorrectly predicted. These differences illustrate how non-consensus LRR motifs influence TLR structure. Since the determination of molecular structures by crystallography requires substantial amounts of protein, we describe methods for producing milligram amounts of TLR3 extracellular domain (ECD) protein. The recombinant TLR3-ECD previously used to solve the molecular structure of TLR3-ECD has also been used to study the binding of TLR3-ECD to its ligand, double-stranded RNA (dsRNA). In the last section, we describe the preparation of defined TLR3 ligands and present methods for characterizing their interaction with TLR3-ECD.
Suppressor of
IKK
epsilon (
SIKE
) is associated with the type I interferon response of the innate immune system through
TANK
‐binding kinase 1 (
TBK
1). Originally characterized as an endogenous ...inhibitor of
TBK
1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that
SIKE
acts as a high‐affinity substrate of
TBK
1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and
SIKE CRISPR
/Cas9 knockout
HAP
1 cells showed an ~ 20% decrease in cell migration. Investigation of the
SIKE
interaction network through affinity purification/mass spectrometry showed that
SIKE
formed interactions with cytoskeletal proteins. In immunofluorescence assays, endogenous
SIKE
localized to cytosolic puncta in both epithelial and myeloid cells and to nuclear puncta in myeloid cells, while in epithelial cells additional staining occurred in stress fiber‐like structures and adjacent to the plasma membrane. Using cellular markers, co‐occurrence of
SIKE
fluorescence with actin, α‐actinin, and ezrin was detected. Reciprocal immunoprecipitation revealed a
SIKE
:tubulin interaction sensitive to the phosphorylation state of
SIKE
, but a
SIKE
:α‐actinin interaction was unchanged by
SIKE
phosphorylation.
In vitro
precipitation assays confirmed a direct
SIKE
interaction with tubulin and α‐actinin. These results indicate that
SIKE
may promote cell migration by directly associating with the cytoskeleton. In this role,
SIKE
may mediate cytoskeletal rearrangement necessary in innate immunity, but also link a key catalytic hub,
TBK
1, to the cytoskeleton.
Database
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the
PRIDE
1 partner repository with the dataset identifier
PXD
007262.
Periodontitis is the most common disease of microbial etiology in humans. Periopathogen survival is dependent upon evasion of complement-mediated destruction. Treponema denticola, an important ...contributor to periodontitis, evades killing by the alternative complement cascade by binding factor H (FH) to its surface. Bound FH is rapidly cleaved by the T. denticola protease, dentilisin. In this report, the structure of the T. denticola FH-binding protein, FhbB, was solved to 1.7 Å resolution. FhbB possesses a unique fold that imparts high thermostability. The kinetics of the FH/FhbB interaction were assessed using surface plasmon resonance. A KD value in the micromolar range (low affinity) was demonstrated, and rapid off kinetics were observed. Site-directed mutagenesis and sucrose octasulfate competition assays collectively indicate that the negatively charged face of FhbB binds within FH complement control protein module 7. This study provides significant new insight into the molecular basis of FH/FhbB interaction and advances our understanding of the role that T. denticola plays in the development and progression of periodontal disease.
Background: The Treponema denticola FhbB protein binds FH, a complement regulator.
Results: The structure of FhbB was solved, and its interaction with FH was further defined.
Conclusion: The structurally unique FhbB protein interacts with CCP7 of FH through electrostatic interactions.
Significance: The T. denticola/FH interaction may perturb complement regulation resulting in conditions that favor the development of periodontal disease.
Wound inducible transcript 3.0 (WIT3.0) is a novel cytoskeleton protein that regulates fibroblast migration and initiates rapid wound closure. WIT3 shares ~50% sequence identity with an innate immune ...protein of unknown function, Suppressor of IKKepsilon (SIKE), that also associates with cytoskeletal proteins. We hypothesize that SIKE and WIT3 will have similar biochemical and biophysical characteristics that contribute to a shared function. To assess these similarities, we undertook characterization of WIT3 structure utilizing circular dichroism, fluorescence‐based thermal shift assays, ligand binding studies using an ANS reporter, and crosslinking with BS3. Using PHYRE2, a WIT3 model was predicted that consisted of an alpha helical coil with 30% disordered regions. The secondary structure of WIT3 was assessed via circular dichroism. Wavelength spectra is consistent with an alpha helical protein and thermal melt data showed a linear unfolding pattern with Tm of 37°C, suggesting a minimally stable protein. As part of the PHYRE2 model, 3DLigandSite proposed a zinc binding site. To assess an interaction between WIT3 and divalent cations, WIT3‐ANS titration and fluorescence‐based thermal shift assays were completed +/‐ Mg, Mn, Ca, Ni and Zn. An interaction between WIT3 and Zn was confirmed where, in the presence of zinc, WIT3's affinity for ANS was 2 fold higher than in the absence of zinc. The initial comparison suggests that WIT3 and SIKE share similar helical structure and similar FTS melt curve topography indicating exposed hydrophobic residues prior to denaturation, but different structural stability and a potential divalent cation binding site on WIT3. Together, these data suggest that WIT3 and SIKE share several biochemical characteristics that may allow these two proteins to have complimentary function.
Support or Funding Information
Studies were funded in part by USD SURE.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Upon pathogen challenge multiple receptors both inside and on the surface of the cell, recognize pathogen associated molecular patterns (PAMPs) and initiate the production of proinflammatory, ...antiviral, and apoptotic responses. Pathways converge at key hubs that serve to amplify and regulate the signals, and are often responsible for determining the downstream response. TANK Binding Kinase 1 (TBK1) serves as a catalytic hub in the antiviral TLR3 mediated innate immune pathway. Suppressor of IKK epsilon (SIKE) is a recently identified high affinity alternative substrate of TBK1. It was initially found to inhibit TBK1 activation of type 1 interferon production. Upon subsequent study, it was found that SIKE was phosphorylated at six serine residues by TBK1. This phosphorylation of SIKE corresponds to the activation of the antiviral response, and releases SIKE from the SIKE:TBK1 interaction. The primary function of SIKE remains unknown. Examination of SIKE's interaction network has established direct interactions with cytoskeletal proteins including tubulin and α‐actinin. Migration assays have shown that chronic myelogenous leukemia (CML) cells in which SIKE has been knocked out migrate at a slower rate. Together, these studies suggest that SIKE plays a role in cytoskeletal rearrangements associated with innate immune responses such as migration and phagocytosis. The goal of this study is to define the interaction surface of SIKE as well as the binding affinities associated with these interactions. A quartz crystal microbalance with dissipation (QCM‐D) assay was developed to obtain binding affinities for the SIKE:cytoskeletal protein complexes. A gold sensor was utilized and functionalized with protein G and α‐His antibody to which 6xHis‐SIKE was immobilized. Increasing concentrations of binding partner were flowed over the chip to develop a binding curve. Prior to examining SIKE:cytoskeletal protein interactions, SIKE's oligomeric state was defined by crosslinking studies. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) shows that SIKE is a dimer. Chemical crosslinking followed by tandem mass spectrometry was employed to determine the dimeric interface of the SIKE dimer. BS3 was used in an excess of 20 and 100X. Data were analyzed with Mascot using xComb strategy. Residues 13, 66, 68, 110, and 169 were found to be accessible to BS3 modification, while in the dimer only residues 13, 110, 119, and 120 were found to be accessible. Residues 110 and 119 are found in multiple crosslinked peptides in both dimer and monomer samples. Residues 178 and 195 were found in crosslinked peptides unique to the dimer. These data have been applied to SIKE dimer models, generated by computational docking experiments to select for models consistent with the crosslinking restraints. These data define the dimer interface of SIKE as well as the surfaces available for SIKE cytoskeletal protein interactions. Together, these studies begin to reveal parameters that determine SIKE's interactions and the protein surfaces available to mediate these interactions.
Support or Funding Information
Studies were funded by USD SURE, Beckman Foundation, NIH‐NIAID R21.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Suppressor of IKKepsilon (SIKE) is phosphorylated in the signaling cascade that initiates the anti‐viral innate immune response mediated by Toll‐like receptor 3. Although shown to be associated with ...cytoskeletal proteins, the function of SIKE in association with the cytoskeleton or as part of the innate immune response is not yet known. To gain insight into SIKE's function, structural and biophysical characterization were undertaken. Fluorescence‐based thermal shift assays (FTSA) indicated that the SIKE structure displayed hydrophobic residues prior to thermal denaturation. With increasing temperature, additional hydrophobic character was displayed giving rise to unique FTS curves showing three transitions, initial binding of fluor, unfolding, and quenching of fluorescence, presumably by protein aggregation. From modeling software, a divalent cation binding site was predicted. Using FTSA, the addition of zinc and calcium appear to alter the thermal stability of SIKE. Using ANS as a reporter group or direct tryptophan fluorescence of SIKE, the binding affinity of SIKE to various divalent cations, zinc, magnesium, manganese and calcium, was assessed. Fluorescence quenching experiments with neutral and charged quenchers were used to probe the local environment of the lone Trp residue of SIKE. The quenching coefficient for the Trp in SIKE determined from acrylamide studies was significantly lower than that of free Trp in solution indicating that the residue is fairly protected. Together, these studies provide biophysical parameters with which to interrogate and refine our predicted SIKE structure that is being employed in interpreting how SIKE interacts with cytoskeletal proteins to mediate host defense mechanisms.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Course-based undergraduate research experiences (CUREs) are laboratory courses that integrate broadly relevant problems, discovery, use of the scientific process, collaboration, and iteration to ...provide more students with research experiences than is possible in individually mentored faculty laboratories. Members of the national Malate dehydrogenase CUREs Community (MCC) investigated the differences in student impacts between traditional laboratory courses (control), a short module CURE within traditional laboratory courses (mCURE), and CUREs lasting the entire course (cCURE). The sample included approximately 1,500 students taught by 22 faculty at 19 institutions. We investigated course structures for elements of a CURE and student outcomes including student knowledge, student learning, student attitudes, interest in future research, overall experience, future GPA, and retention in STEM. We also disaggregated the data to investigate whether underrepresented minority (URM) outcomes were different from White and Asian students. We found that the less time students spent in the CURE the less the course was reported to contain experiences indicative of a CURE. The cCURE imparted the largest impacts for experimental design, career interests, and plans to conduct future research, while the remaining outcomes were similar between the three conditions. The mCURE student outcomes were similar to control courses for most outcomes measured in this study. However, for experimental design, the mCURE was not significantly different than either the control or cCURE. Comparing URM and White/Asian student outcomes indicated no difference for condition, except for interest in future research. Notably, the URM students in the mCURE condition had significantly higher interest in conducting research in the future than White/Asian students.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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