In JoVE (1)
Other Publications (7)
- Antioxidants & Redox Signaling
- Acta Crystallographica. Section D, Biological Crystallography
- Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry
- The Journal of Biological Chemistry
- Proceedings of the National Academy of Sciences of the United States of America
- Neurology(R) Neuroimmunology & Neuroinflammation
- Nature Communications
Articles by Pedro Brugarolas in JoVE
Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases Pedro Brugarolas1, Mohammed Bhuiyan2, Anna Kucharski2, Richard Freifelder2 1Department of Neurology, The University of Chicago, 2Department of Radiology, The University of Chicago We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.
Other articles by Pedro Brugarolas on PubMed
Redox Signaling in Human Pathogens Antioxidants & Redox Signaling. Mar, 2011 | Pubmed ID: 20578795 In recent studies of human bacterial pathogens, oxidation sensing and regulation have been shown to impact very diverse pathways that extend beyond inducing antioxidant genes in the bacteria. In fact, some redox-sensitive regulatory proteins act as major regulators of bacteria's adaptability to oxidative stress, an ability that originates from immune host response as well as antibiotic stress. Such proteins play particularly important roles in pathogenic bacteria S. aureus, P. aeruginosa, and M. tuberculosis in part because reactive oxygen species and reactive nitrogen species present significant challenges for pathogens during infection. Herein, we review recent progress toward the identification and understanding of oxidation sensing and regulation in human pathogens. The newly identified redox switches in pathogens are a focus of this review. We will cover several reactive oxygen species-sensing global regulators in both gram-positive and gram-negative pathogenic bacteria in detail. The following discussion of the mechanisms that these proteins employ to sense redox signals through covalent modification of redox active amino acid residues or associated metalloprotein centers will provide further understanding of bacteria pathogenesis, antibiotic resistance, and host-pathogen interaction.
Structural and Biochemical Characterization of N5-carboxyaminoimidazole Ribonucleotide Synthetase and N5-carboxyaminoimidazole Ribonucleotide Mutase from Staphylococcus Aureus Acta Crystallographica. Section D, Biological Crystallography. Aug, 2011 | Pubmed ID: 21795812 With the rapid rise of methicillin-resistant Staphylococcus aureus infections, new strategies against S. aureus are urgently needed. De novo purine biosynthesis is a promising yet unexploited target, insofar as abundant evidence has shown that bacteria with compromised purine biosynthesis are attenuated. Fundamental differences exist within the process by which humans and bacteria convert 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In bacteria, this transformation occurs through a two-step conversion catalyzed by PurK and PurE; in humans, it is mediated by a one-step conversion catalyzed by class II PurE. Thus, these bacterial enzymes are potential targets for selective antibiotic development. Here, the first comprehensive structural and biochemical characterization of PurK and PurE from S. aureus is presented. Structural analysis of S. aureus PurK reveals a nonconserved phenylalanine near the AIR-binding site that occupies the putative position of the imidazole ring of AIR. Mutation of this phenylalanine to isoleucine or tryptophan reduced the enzyme efficiency by around tenfold. The K(m) for bicarbonate was determined for the first time for a PurK enzyme and was found to be âˆ¼18.8â€…mM. The structure of PurE is described in comparison to that of human class II PurE. It is confirmed biochemically that His38 is essential for function. These studies aim to provide foundations for future structure-based drug-discovery efforts against S. aureus purine biosynthesis.
Binding of ReO4(-) with an Engineered MoO4(2-)-binding Protein: Towards a New Approach in Radiopharmaceutical Applications Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry. Jan, 2012 | Pubmed ID: 21861186 Radiolabeled biomolecules are routinely used for clinical diagnostics. (99m)Tc is the most commonly used radioactive tracer in radiopharmaceuticals. (188)Re and (186)Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO(4)(-)) ion as a new way to label proteins. We found that a molybdate (MoO(4)(2-))-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetry measurements, we determined the dissociation constant of ModA for ReO(4)(-) to be 541 nM and we solved a crystal structure of ModA with a bound ReO(4)(-). On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K(d) = 104 nM). High-resolution crystal structures of ModA (1.7 Ã…) and A11C/R153C mutant (2.0 Ã…) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond.
The Oxidation-sensing Regulator (MosR) is a New Redox Dependent Transcription Factor in Mycobacterium Tuberculosis The Journal of Biological Chemistry. Sep, 2012 | Pubmed ID: 22992749 Mycobacterium tuberculosis thrives in oxidative environments such as the macrophage. In order to survive the bacterium must sense and adapt to the oxidative conditions. Several antioxidant defenses including a thick cell wall, millimolar concentrations of small molecule thiols and protective enzymes are known to help the bacterium withstand the oxidative stress. However, oxidation-sensing regulators that control these defenses have remained elusive. In this paper, we report a new oxidation-sensing regulator, Rv1049 or MosR (Mycobacterium tuberculosis oxidation-sensing Regulator). MosR is a transcriptional repressor of the MarR family, which, similarly to B. subtilis OhrR and S. aureus MgrA, dissociates from DNA in the presence of oxidants, enabling transcription. MosR senses oxidation through a pair of cysteines near the N-terminus (Cys10 and Cys12) that upon oxidation forms a disulfide bond. Disulfide formation rearranges a network of hydrogen bonds which leads to a large conformational change of the protein and dissociation from DNA. MosR has been shown previously to play an important role on survival of the bacterium in the macrophage. In this study we show that the main role of MosR is to upregulate expression of rv1050 (a putative exported oxidoreductase that has not yet been characterized) in response to oxidants and propose that it is through this role that MosR contributes to the bacterium survival in the macrophage.
Molecular Mechanism of Quinone Signaling Mediated Through S-quinonization of a YodB Family Repressor QsrR Proceedings of the National Academy of Sciences of the United States of America. Mar, 2013 | Pubmed ID: 23479646 Quinone molecules are intracellular electron-transport carriers, as well as critical intra- and extracellular signals. However, transcriptional regulation of quinone signaling and its molecular basis are poorly understood. Here, we identify a thiol-stress-sensing regulator YodB family transcriptional regulator as a central component of quinone stress response of Staphylococcus aureus, which we have termed the quinone-sensing and response repressor (QsrR). We also identify and confirm an unprecedented quinone-sensing mechanism based on the S-quinonization of the essential residue Cys-5. Structural characterizations of the QsrR-DNA and QsrR-menadione complexes further reveal that the covalent association of menadione directly leads to the release of QsrR from operator DNA following a 10° rigid-body rotation as well as a 9-Å elongation between the dimeric subunits. The molecular level characterization of this quinone-sensing transcriptional regulator provides critical insights into quinone-mediated gene regulation in human pathogens.
Pharmaceutical Integrated Stress Response Enhancement Protects Oligodendrocytes and Provides a Potential Multiple Sclerosis Therapeutic Nature Communications. Mar, 2015 | Pubmed ID: 25766071 Oligodendrocyte death contributes to the pathogenesis of the inflammatory demyelinating disease multiple sclerosis (MS). Nevertheless, current MS therapies are mainly immunomodulatory and have demonstrated limited ability to inhibit MS progression. Protection of oligodendrocytes is therefore a desirable strategy for alleviating disease. Here we demonstrate that enhancement of the integrated stress response using the FDA-approved drug guanabenz increases oligodendrocyte survival in culture and prevents hypomyelination in cerebellar explants in the presence of interferon-γ, a pro-inflammatory cytokine implicated in MS pathogenesis. In vivo, guanabenz treatment protects against oligodendrocyte loss caused by CNS-specific expression of interferon-γ. In a mouse model of MS, experimental autoimmune encephalomyelitis, guanabenz alleviates clinical symptoms, which correlates with increased oligodendrocyte survival and diminished CNS CD4+ T cell accumulation. Moreover, guanabenz ameliorates relapse in relapsing-remitting experimental autoimmune encephalomyelitis. Our results provide support for a MS therapy that enhances the integrated stress response to protect oligodendrocytes against the inflammatory CNS environment.