Metals in Redox Biology

Transcription

1 Metals in Redox Biology I. Functional roles for metal ions II. Metal toxicity III. Molecular mechanisms underlying metal ion homeostasis IV. Therapeutic control of metals Jaekwon Lee Redox Biology Center and Department of Biochemistry University of Nebraska-Lincoln

2 Many Metal Ions Are Essential Cellular Components H He Li Be B C N O F Ne Na Mg Al Si P S Cl Ar K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As z Se Br Kr Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe Cs Ba Ln Hf Ta W Re Os Ir Pt Au Hg TI Pb Bi Po At Rn Fr Ra Ac Th Pa U Abundant biological elements Essential trace elements Toxic environmental contaminants Use as therapeutics Cisplatin - Anti-cancer drug H + O + C + N > 95 % of the human body S, P, Cl 1-0.1% K, Na, Ca, Mg Fe (3.5g/70kg BW) Zn Cu 2x10-4 (0.14g/70kg BW) Se, Mn, Ni 2x10-5 The Biological Chemistry of the Elements, 2nd Ed

3 Diverse Roles of Metal Ions 1/3 of Proteins Are Metalloproteins. Catalytic or structural cofactors Superoxide dismutase 1 (PDB ID 2SOD) Electron transfer Cytochrome C Oxygen carriers Hemoglobin, Myoglobin Cytochrome C oxidase (Lehninger Biochemistry 4th Ed) Sensing Oxygen, Redox Gene regulation Signal transduction Metabolism Membrane potential Neurotransmission Osmolality and ph control Synthesis of holo-metalloproteins : Metal uptake, distribution & incorporation

4 Oxidation and Reduction of Metals + e - Fe 3+ Fe 2+ - e - + e - Cu 2+ Cu + - e - Functional & detrimental roles The Biological Chemistry of the Elements J.J.R. Frausto Da Silva and R.J.P. Williams, 2nd Ed

5 Cys, Met, and His - Common Metal Binding Residues Copper-requiring enzymes Copper binding residues No reliable method identifying metal-binding proteins and sites in silico Cys and Met are sensitive to rodox à Affects metal binding à Inactivate metalloproteins & induce metal toxicity by releasing metals Rulisek L and Vondrasek J (1998) J. Inorg. Biochem. 71,

6 Metal-containing Prosthetic Groups Complicated mechanisms for synthesis (e.g., 30 enzymes for cobalamine) & Incorporation into proteins Heme Fe-S cluster Chlorophyll Cobalamine : Methyl transferase F 430 : Methane formation

7 Fe-S Cluster-based Sensing Inactive SoxS gene promoter SoxR - A sensor of O 2.- & NO. stress in E. coli O 2.- Active SoxS gene promoter SoxS expression SoxR [2F-2S] + SoxR [2F-2S] 2+ Transcription Regulation (e.g., Antioxidants, Fe metabolism) Aconitase - A sensor of oxidative stress & iron starvation in mammals [4Fe-4S] Green J and Paget MS (2004) Nat. Rev. Microbiol. 2:954-66

8 Metals in Redox Biology I. Functional roles for metal ions II. Metal toxicity III. Molecular mechanisms underlying metal ion homeostasis IV. Therapeutic control of metals

9 Inhibition of E. coli growth by metal ions Most Metal Ions Are Highly Toxic Environmental contamination 4% (250 mm) copper sulfate Copper door handles to kill superbugs Nies DH (1999) Appl Microbiol Biotechnol, 51,

10 Mechanisms of Metal Ion Toxicity ROS generation by redox-active metals Fe 2+ (Cu + ) + H 2 O 2 à HO. + HO - + Fe 3+ (Cu 2+ ) Fe 2+ (Cu + ) + O 2 à O Fe 3+ (Cu 2+ ) Correlation between metal toxicity & affinity to sulfur Cd 2+ Mn 2+ Zn 2+ Pb 2+ Cu 2+ Cu + Ag + Hg 2+ Fe 3+ (Cu 2+ ) + O 2.- à Fe 2+ (Cu + ) + O 2 Oxidation of cellular thiols RSH + Fe 3+ (Cu 2+ ) à RS. + Fe 2+ (Cu + ) + H + 2GSH + Cd 2+ à GS-Cd-SG Non-specific binding : Cys residues : Binding sites of other metals Toxic concentration E.coli R. metallidurans Nies DH (2003) FEMS Microbiol Rev. 27, Affinity to S

11 Metals in Host-Pathogen Interactions Use of copper for bactericidal effects : Copper transport into the phagosome - Up regulation of copper importers : Up regulation of Cu exporter in bacteria Limit metal availability to pathogens : Regulation of divalent metal transporter Cu Macrophage Cytoplasm Cytoplasm Fe, Mn, Zn Phagosome Cu ROS Mycobacteria Phagosome Mycobacteria SOD, CAT Cytokines limit bioavailable Fe Carrier-mediated metal transport to limit free metal Pathogenic microorganisms secrete toxin(s) to acquire metal ions.

12 Delicate Control of Metal Ion Metabolism To acquire enough amounts w/o toxicity or giving it to pathogens Uptake Distribution (carrier-mediated mechanisms) * * * * * * * * Utilization * * * * ** Storage Chelation * * * * * Export Excretion

13 Metals in Redox Biology I. Functional roles for metal ions II. Metal toxicity III. Molecular mechanisms underlying metal ion homeostasis : Iron, copper, cadmium IV. Therapeutic control of metals

14 Iron Uptake and Utilization in Mammals Body Fe pool Hemoglobin (70% of total body iron), myoglobin, ferritin (storage), transferrin (carrier), Fe-containing proteins (heme, Fe-S cluster, direct binding of Fe) Ribonucleotide reductase Fe-deficient Anemia - The most common nutritional problem Biochemistry (Voet and Voet, 3 rd ) Only 3-6 % of dietary Fe is absorbed. - Fe exists as oxidized and insoluble compounds in the environment

15 Molecular Factors for Iron Uptake and Distribution Intestine Blood Organs and Tissue Ferritin * * * * * * * * * Transferrin receptor Heme * Heme oxygenase Macrophages in the liver and spleen * Identified genetic defects * Redox Biochemistry Textbook (Chapter 4.5) Sheth S and Brittenham GM (2000) Annu. Rev. Med. 51:443 Andrews NC (2002) Curr. Top. Chem. Biol. 6:181

16 Heme Biosynthesis Requires multiple enzymes and molecular factor in the mitochondria and cytosol Schultz IJ et al. (2010) J Biol Chem. 285:26753.

17 Heme Trafficking (e.g., transporters, carriers) Is Poorly Understood NADPH oxidases Schultz IJ et al. (2010) J Biol Chem. 285:26753.

18 Heme Degradation: Heme oxygenases (HO-1 and HO-2) : Recycling of heme from the red blood cells, regulation of heme levels 3.5 Fe recycle from senescent red blood cell Cytoprotective response Biliverdin - physiological antioxidants (newborn jaundice) CO Signaling and regulation Stimulation of guanylate cyclase and/or MAP kinases

19 Post-transcriptional Regulation of Fe Metabolism Genes in Mammals Fe regulatory proteins 1 (IRP1) & 2 (IRP2) A. Regulation of IRP1 function by Fe, NO. and H 2 O 2 Fe Fe NO H 2 O 2 Annu, Rev. Nutr. (2000) 20:627-

20 B. Control of IRP2 levels by an Fe and O 2 sensor and ubiquitin ligase High iron High oxygen FBXL5 IRP2 E3 ligase Ub IRP2 degradation by the proteasome FBXL5 IRP2 Binding to mrnas of Fe metabolism genes : Translation and stability control Low iron Low oxygen FBXL5 E3 ligase FBXL5 degradation by the proteasome Ub Vashisht AA et al. (2009) Science 326:718; Salahudeen et al. (2009) Science, 326:722. Rouault TA (2009) Science, 326:676.

21 Iron regulatory proteins (IRPs) control the translation and stability of target mrna G U A G C U A U A U C G U A U A C C G U U C G C G U A U A 1 G C 2 Eisenstein R. S. (2000) Annu, Rev. Nutr. 20,627-

22 Iron Metabolism in Bacteria Extract metals from the environment : Lowering external ph (reduction of Fe 3+ to Fe 2+ ) : Secrete siderophores - Less than1 KDa MW - High affinity to Fe 3+ - ~500 have been characterized. - Maintain solubility of Fe Uptake through siderophore importers : Could be targets of new antibiotics Acquire from various Fe sources : Siderophore (Fe 3+ ), Fe 2+, transferrin, lactoferrin, heme

23 Direct Fe Sensing by Fur to Repress Target Gene Expression High Fe à Repression Holo-Fur Off Fur-binding site Fe-acquiring genes Low Fe à Derepression Apo-Fur On Andrews SC et al. (2003) FEMS Microbiol Rev. 27: 215.

24 Transcriptional Regulation of Fur by Oxidative Stress OxyR H 2 O 2 à OxyR activation à H 2 O 2 scavengers & Fur Oxidative Stress (e.g., excess free Fe) OxyR & SoxS Anti-oxidants Fur Inactive SoxS gene promoter SoxR Active SoxS gene promoter Fe binding Repression of Fe uptake & Metabolic adaptation O 2.- à SoxR activation à SoxS expression à SodA (MnSOD, DldA(flavodoxin), Zwf (glucose 6-phosphate dehydrogenase) & Fur

25 Iron Storage & Detoxification Bacterioferritin (Bfr) and Ferritin in mammals : 24-mer, 500 kda, store Fe +++ Dps in bacteria: 12-mer, 250 kda, 500 Fe +++ : Non-specific DNA-binding protein : Use H 2 O 2 as a Fe oxidant : Up regulated by OxyR Ferritin Andrews SC et al. (2003) FEMS Microbiol Rev. 27: 215.

26 Copper Homeostasis in Eukaryotes Cu chaperones (e.g., Atx1, CCS1): Escort Cu for the safe delivery Ctr1 Cu + Ccs1 CCO Cox17 Sco1 Sco2 Atx1 CCC2 (ATP7A/B) Superoxide dismutase 3 Fe oxidases (Cp, Hp) Lysyl oxidases Dopamine hydroxylase SOD1 ** MT Redox Biology Textbook (Chapter 4.5)

27 Metallothioneins (MTs) Cu, Zn, Hg, Cd sequestration 61 amino acid peptide (20 cysteins) Many (~17) isoforms in human Induced by metals and oxidative stress Annu. Rev. Pharamcol. Toxicol. (1999) 39:267 PNAS (1998) 95:3333

28 Cadmium Detoxification in Eukaryotes GSH (Glutathione) MT (Metallothionein) Exporters P-type ATPases, ABC transporters Antioxidants Ycf1 Yeast S. cerevisiae GS-Cd-SG GS-Cd-SG Cd Vacuole Cd-MT Yap1, Ace1,Met4 Cd Nucleus Pca1 Divalent metal (Zinc, Iron, Calcium) importers Cd export? Mammals Cd Cd-MT GS-Cd-SG MTF1, Nrf2 Nucleus Cd

29 Cd responsive Regulation of Pca1 by the ER-associated Degradation (ERAD) System Cd < 5 min t 1/2 of Pca1 protein No change of mrna Plasma " membrane Cd - + Cd min " ñ Proteasome ñ Pca1" PGK" Ub ñ ERAD" (Doa10," Ubc7," Cue1)" ñ Pca1 Cd ñ Cd ER membrane Adle, D. et al. (2009) PNAS, 106,10189.

30 Cd-dependent Degradation Signal in Pca1 Mapping of a Cd regulatory motif in Pca1 392 C424 C421 C P X P N Plasma membrane COOH 350 Protein degradation machinery (e.g., molecular chaperones, ubiquitination enzymes) NH 3 Cys-rich è Degradation 250-SCEKRTFKGSTNVGISGSSST DSLSEKFFSEQYSRMYNRYSSILKN LGCICNYLRTLGKESCCLPKVRFCS GEGASKKTKYSYRNSSGCLTKKKTH GDKER Cd Degradation signal : hydrophobic aa : Amphipathic helix è Stabilization CC Smith, N. et al. (2016) J. Biol. Chem. 291: Smith, N. et al. (2016) J. Biol. Chem. 291:

31 Metals in Redox Biology I. Functional roles for metal ions II. Metal toxicity III. Molecular mechanisms underlying metal ion homeostasis : Iron, copper, cadmium IV. Therapeutic control of metals 1. Copper homeostasis as a target of anticancer therapy 2. Ascorbate for a cancer therapeutic 3. Metal chelators as new antibiotics

32 I. Therapeutic Control of Copper to Combat Cancer Higher Tissue Copper Levels in Cancer Patients Reference Cancer type Control group Cancer patients N Cu-dependent cell growth : HeLa and other cancer cells and non-cancer cells : Mechanisms? Unpublished data Cell number (relate to control) * * µm BCS Cu chelator µm CuCl 2 Gupte A and Mumper RJ (2009) Cancer Treat Rev

33 Copper-related Therapeutics Copper chelators : FDA-approved for Wilson disease (genetic defect in Cu exporter in the liver) : Clinical trials as cancer therapeutics Inhibition of HeLa cell growth Penicillamine Trientine hydrochloride Tetrathiomolybdate (TTM) Cell number (% control) TTM (Cu chelator) Gefitinib (RGFR inhibitor) µm Synergistic anticancer effects Unpublished data Cell number (1X10 6 ) TTM µm Gefitinib µm

34 Increase Copper Import to Induce Its Toxicity : Antibiotics & anti-cancer therapeutics Promote Cu transport & catalyze redox reactions (e.g., Disulfirm) Light-activated release of caged copper Cu + ROS Modified from Helsel ME and Franz KJ (2015) Dalton Trans. Ciesienski KL et al., (2008) JACS

35 II. Ascorbate Promotes Metal-catalyzed ROS Generation Pharmacologic doses of ascorbate act as a pro-oxidant & decrease tumor growth Chen Q et al. (2008) Proc Natl Acad Sci U S A. 105: Kill cancer cells but induce moderate damage to normal cells Normal cells Murine cancer cells Human cancer cells >25 Ascorbate EC 50 (mm) >25 Ascorbate EC 50 (mm)

36 Redox-active Metals Might Be the Mediators of Ascorbic Acid-induced Cancer Cell Death High Cu and Fe levels in cancer cells AA : Ascorbic acid (reductant) M n : Oxidized metal M n-1 : Reduced metal M n-1 HO. + M n Chen Q et al. (2008) Proc Natl Acad Sci U S A. 105:11105.

37 III. Metal Chelation as a Mechanism of Innate Immunity Metal Chelation Inhibits Bacterial Growth in Tissue Abscesses (Corbin BD et al, Science. 2008, 319:962) Identification of a protein enriched in S. aureus infected tissue S100A8 : A component of calprotectin (S100A8/S100A9 complex) : Ca 2+, Mn 2+, Zn 2+ binding : Abundant at neutrophil cytosol

38 Calprotectin (S100A8/S100A9) Chelates Metals to Reduce Bacterial Growth Growth inhibition S. aureus Detoxification ROS Superoxide dismutase Mn 2+ and Zn 2+ chelation from the media ROS Mn + Calprotectin Mn +, Zn + chelation Neutrophil Corbin BD et al, Science. 2008, 319:962

39 Summary I. Functional roles for metal ions Enzyme cofactors Electron transfer Sensing, signaling, transcription regulation, and innate immunity II. Metal toxicity ROS generation Correlated with affinity to sulfur III. Metal acquisition, distribution, and detoxification Minimize free metals Iron, Copper, and Cadmium IV. Metals in human diseases Control of metal homeostasis for health benefits