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