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Metals in Redox Biology - University of...
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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
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 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
The Biological Chemistry of the Elements, 2nd Ed
Abundant biological elements
Essential trace elements
H + O + C + N > 95 % of the human body S, P, Cl 1 - 0.1% K, Na, Ca, Mg 1 - 0.1 Fe 0.005 (3.5g/70kg BW) Zn 0.003 Cu 2x10-4 (0.14g/70kg BW) Se, Mn, Ni 2x10-5
Toxic environmental contaminants
Use as therapeutics
Cisplatin - Anti-cancer drug
z
Catalytic or structural cofactors
Oxygen carriers Hemoglobin, Myoglobin
Sensing – Oxygen, Redox Gene regulation Signal transduction Metabolism
Membrane potential Neurotransmission
Osmolality and pH control ……
Superoxide dismutase 1 (PDB ID 2SOD)
Synthesis of holo-metalloproteins : Metal uptake, distribution & incorporation
Electron transfer Cytochrome C
Cytochrome C oxidase (Lehninger Biochemistry 4th Ed)
Diverse Roles of Metal Ions 1/3 of Proteins Are Metalloproteins.
Oxidation and Reduction of Metals
Fe3+ Fe2+
Cu2+ Cu+
The Biological Chemistry of the Elements J.J.R. Frausto Da Silva and R.J.P. Williams, 2nd Ed
Functional & detrimental roles
+ e-
- e-
+ e-
- e-
Cys, Met, and His - Common Metal Binding Residues
Copper-requiring enzymes Copper binding residues
Rulisek L and Vondrasek J (1998) J. Inorg. Biochem. 71, 115-127
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
Metal-containing Prosthetic Groups
Complicated mechanisms for synthesis (e.g., 30 enzymes for cobalamine) & Incorporation into proteins
Heme
Fe-S cluster
Cobalamine : Methyl transferase
Chlorophyll
F430 : Methane formation
Fe-S Cluster-based Sensing
SoxR - A sensor of O2.- & NO. stress in E. coli
SoxS expression
Transcription Regulation
(e.g., Antioxidants, Fe metabolism)
Green J and Paget MS (2004) Nat. Rev. Microbiol. 2:954-66
SoxR [2F-2S]+
Inactive SoxS gene promoter
Active SoxS gene promoter
Aconitase - A sensor of oxidative stress & iron starvation in mammals [4Fe-4S]
SoxR [2F-2S]2+
O2.-
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
Nies DH (1999) Appl Microbiol Biotechnol, 51, 730-750.
Inhibition of E. coli growth by metal ions
Most Metal Ions Are Highly Toxic
Copper door handles to kill superbugs
4% (250 mM) copper sulfate
Environmental contamination
Mechanisms of Metal Ion Toxicity
Non-specific binding
: Cys residues : Binding sites of other metals
ROS generation by redox-active metals
Fe2+ (Cu+) + H2O2 à HO. + HO- + Fe3+(Cu2+)
Fe2+ (Cu+) + O2 à O2.- + Fe3+(Cu2+)
Fe3+ (Cu2+) + O2.- à Fe2+ (Cu+) + O2
Oxidation of cellular thiols
RSH + Fe3+ (Cu2+) à RS. + Fe2+ (Cu+) + H+
2GSH + Cd2+ à GS-Cd-SG
Affinity to S
Toxi
c co
ncen
trat
ion
E.coli
R. metallidurans
Nies DH (2003) FEMS Microbiol Rev. 27, 313-339.
Hg2+ Ag+ Cu2+ Pb2+ Cd2+
Mn2+ Zn2+
Correlation between metal toxicity & affinity to sulfur
Cu+
Metals in Host-Pathogen Interactions
Macrophage
Limit metal availability to pathogens : Regulation of
divalent metal transporter
Cytoplasm
Phagosome
SOD, CAT
Fe, Mn, Zn
Mycobacteria
Cytokines limit bioavailable Fe Carrier-mediated metal transport to limit free metal
Pathogenic microorganisms secrete toxin(s) to acquire metal ions.
Use of copper for bactericidal effects : Copper transport into the phagosome - Up regulation of copper importers
: Up regulation of Cu exporter in bacteria
Cytoplasm
Phagosome Cu
Mycobacteria
ROS
Cu
Delicate Control of Metal Ion Metabolism
To acquire enough amounts w/o toxicity or giving it to pathogens
Utilization *
* * * *
*
Storage Chelation
Export Excretion
*
Uptake Distribution (carrier-mediated mechanisms)
* *
*
*
*
* *
*
*
* * * *
* *
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
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)
Iron Uptake and Utilization in Mammals
Fe-deficient Anemia - The most common nutritional problem
Only 3-6 % of dietary Fe is absorbed. - Fe exists as oxidized and insoluble compounds in the environment
Ribonucleotide reductase
Biochemistry (Voet and Voet, 3rd)
Intestine Blood
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
Molecular Factors for Iron Uptake and Distribution
Organs and Tissue
Transferrin receptor
Ferritin
Heme
Macrophages in the liver and spleen
Heme oxygenase
* Identified genetic defects
*
*
* *
*
* *
* * *
*
Schultz IJ et al. (2010) J Biol Chem. 285:26753.
Heme Biosynthesis
Requires multiple enzymes and molecular factor in the mitochondria and cytosol
Schultz IJ et al. (2010) J Biol Chem. 285:26753.
Heme Trafficking (e.g., transporters, carriers) Is Poorly Understood
NADPH oxidases
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
Heme Degradation: Heme oxygenases (HO-1 and HO-2)
: Recycling of heme from the red blood cells, regulation of heme levels
Annu, Rev. Nutr. (2000) 20:627-
Post-transcriptional Regulation of Fe Metabolism Genes in Mammals
Fe regulatory proteins 1 (IRP1) & 2 (IRP2)
Fe
Fe NO H2O2
A. Regulation of IRP1 function by Fe, NO. and H2O2
B. Control of IRP2 levels by an Fe and O2 sensor and ubiquitin ligase
Vashisht AA et al. (2009) Science 326:718; Salahudeen et al. (2009) Science, 326:722. Rouault TA (2009) Science, 326:676.
High iron High oxygen
Low iron Low oxygen
IRP2
IRP2
Ub
IRP2 degradation by the proteasome
FBXL5
FBXL5 E3 ligase
FBXL5 E3 ligase
FBXL5 degradation by the proteasome
Binding to mRNAs of Fe metabolism genes : Translation and stability control
Ub
Iron regulatory proteins (IRPs) control the translation and stability of target mRNA
A G U
G C U A U A U C G U A U A
C G C G U A U A G C
U G C C
U
1 2
Eisenstein R. S. (2000) Annu, Rev. Nutr. 20,627-
Extract metals from the environment
: Lowering external pH (reduction of Fe3+ to Fe2+)
: Secrete siderophores - Less than1 KDa MW - High affinity to Fe3+ - ~500 have been characterized. - Maintain solubility of Fe+++
- Uptake through siderophore importers : Could be targets of new antibiotics
Acquire from various Fe sources
: Siderophore (Fe3+), Fe2+, transferrin, lactoferrin, heme
Iron Metabolism in Bacteria
Andrews SC et al. (2003) FEMS Microbiol Rev. 27: 215.
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
H2O2 à OxyR activation à H2O2 scavengers & Fur
O2.- à SoxR activation à SoxS expression
à SodA (MnSOD, DldA(flavodoxin), Zwf (glucose 6-phosphate dehydrogenase) & Fur
Transcriptional Regulation of Fur by Oxidative Stress
Oxidative Stress (e.g., excess free Fe) OxyR & SoxS Anti-oxidants Fur Fe binding
Repression of Fe uptake & Metabolic adaptation
Inactive SoxS gene promoter
Active SoxS gene promoter
OxyR
SoxR
Bacterioferritin (Bfr) and Ferritin in mammals : 24-mer, 500 kDa, store 2000-3000 Fe+++
Dps in bacteria: 12-mer, 250 kDa, 500 Fe+++
: Non-specific DNA-binding protein : Use H2O2 as a Fe oxidant : Up regulated by OxyR
Iron Storage & Detoxification
Andrews SC et al. (2003) FEMS Microbiol Rev. 27: 215.
Ferritin
Superoxide dismutase 3 Fe oxidases (Cp, Hp) Lysyl oxidases Dopamine hydroxylase ……
Copper Homeostasis in Eukaryotes
Ctr1
Cu+
Ccs1
SOD1
Atx1
CCC2 (ATP7A/B)
Cox17 Sco1 Sco2 …
CCO
**** MT Redox Biology Textbook (Chapter 4.5)
Cu chaperones (e.g., Atx1, CCS1): Escort Cu for the safe delivery
Annu. Rev. Pharamcol. Toxicol. (1999) 39:267 PNAS (1998) 95:3333
Metallothioneins (MTs)
Cu, Zn, Hg, Cd sequestration
61 amino acid peptide (20 cysteins)
Many (~17) isoforms in human
Induced by metals and oxidative stress
Divalent metal (Zinc, Iron, Calcium)
importers
Cd Cd
Cd-MT
GS-Cd-SG
Cd-MT GS-Cd-SG
Ycf1
Cd Cd Cd export ?
Yeast S. cerevisiae
Mammals
GSH (Glutathione) MT (Metallothionein)
Exporters – P-type ATPases, ABC transporters Antioxidants
Vacuole
GS-Cd-SG
Nucleus
MTF1, Nrf2
Nucleus
Yap1, Ace1,Met4
Pca1
Cadmium Detoxification in Eukaryotes
Adle, D. et al. (2009) PNAS, 106,10189.
Cd
Cd
Plasma "membrane
Ub Cd Cd
Pca1
ERAD"(Doa10, "
Ubc7,"Cue1)"
ER membrane
ñ
ñ
ñ
ñ
Proteasome ñ
Cd responsive Regulation of Pca1 by the ER-associated Degradation (ERAD) System
< 5 min t1/2 of Pca1 protein
No change of mRNA
PGK" Pca1"
0 15 30 60 0 15 30 60 min "- + Cd
250-SCEKRTFKGSTNVGISGSSST DSLSEKFFSEQYSRMYNRYSSILKNLGCICNYLRTLGKESCCLPKVRFCS GEGASKKTKYSYRNSSGCLTKKKTHGDKER-350
Cd-dependent Degradation Signal in Pca1
N
CPX
392
Cys-rich
PC424C421
NH3
COOH
Mapping of a Cd regulatory motif in Pca1
Cd
Degradation signal : hydrophobic aa : Amphipathic helix
Protein degradation machinery (e.g., molecular chaperones, ubiquitination enzymes)
è
è
Degradation
Stabilization
350
250
250-350
CC
http://zhanglab.ccmb.med.umich.edu/I-TASSER/
Smith, N. et al. (2016) J. Biol. Chem. 291:12420. Smith, N. et al. (2016) J. Biol. Chem. 291:15082.
Plasma membrane
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
Higher Tissue Copper Levels in Cancer Patients Reference Cancer type Control group Cancer patients N
Gupte A and Mumper RJ (2009) Cancer Treat Rev
I. Therapeutic Control of Copper to Combat Cancer
Unpublished data 0
0.5
1
1.5 1.5 1 .5 0
Cel
l num
ber
(rel
ate
to c
ontro
l)
0 100 0 µM BCS Cu chelator 0 0 1 µM CuCl2
* * Cu-dependent cell growth
: HeLa and other cancer cells and non-cancer cells
: Mechanisms?
Copper-related Therapeutics Copper chelators
: FDA-approved for Wilson disease (genetic defect in Cu exporter in the liver) : Clinical trials as cancer therapeutics
Penicillamine Trientine hydrochloride Tetrathiomolybdate (TTM)
Cel
l num
ber (
% c
ontro
l)
0 20 40 60 µM
125 100 75 50 25 0
TTM (Cu chelator) Gefitinib (RGFR inhibitor)
Inhibition of HeLa cell growth
TTM 0 20 0 20 µM Gefitinib 0 0 5 5 µM
Cel
l num
ber (
1X10
6 )
2 1.5 1 .5 0
Synergistic anticancer effects
Unpublished data
Increase Copper Import to Induce Its Toxicity
: Antibiotics & anti-cancer therapeutics
Promote Cu transport & catalyze redox reactions
(e.g., Disulfirm)
Modified from Helsel ME and Franz KJ (2015) Dalton Trans.
Light-activated release of caged copper
Ciesienski KL et al., (2008) JACS
Cu+
ROS
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. 105:11105-9.
Kill cancer cells but induce moderate damage to normal cells
Ascorbate EC50 (mM)
Murine cancer cells
Normal cells
0 5 15 20 >25
Human cancer cells
Ascorbate EC50 (mM) 0 5 15 20 >25
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) Mn : Oxidized metal Mn-1 : Reduced metal
Chen Q et al. (2008) Proc Natl Acad Sci U S A. 105:11105.
Mn-1 HO. + Mn
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) : Ca2+, Mn2+, Zn2+ binding : Abundant at neutrophil cytosol
III. Metal Chelation as a Mechanism of Innate Immunity
Calprotectin (S100A8/S100A9) Chelates Metals to Reduce Bacterial Growth
Growth inhibition
Mn2+ and Zn2+ chelation from the media
S. aureus
Neutrophil
Calprotectin Mn+, Zn+ chelation
Mn+
Superoxide dismutase
ROS
ROS
Detoxification
Corbin BD et al, Science. 2008, 319:962
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
Summary