Chronic Disease: Metallobiology and Metal-Related Disorders

Chronic Disease: Metallobiology and Metal-Related Disorders

Introduction

  • Chronic diseases related to metal imbalances are a significant area of study in metallobiology.

  • Dr. Jon Sellars (jon.sellars@newcastle.ac.uk) at Newcastle University focuses on this subject.

Learning Outcomes

  • Understand the general principles of metallobiology.

    • Emphasis on types of metals and their incorporation in biological systems, leading to downstream effects.

  • Understand the relationship between metals and disease.

    • Focus on:

      • Wilson’s disease (Copper overload)

      • Menkes disease (Copper deficiency)

      • Hemochromatosis (Iron overload)

Copper Enzymes and Their Functions

  • Copper-containing enzymes are crucial for various biological processes:

    • Cellular respiration: Cytochrome c oxidase

    • Neurotransmitter biosynthesis: Dopamine β-hydroxylase (dopamine to norepinephrine)

    • Maturation of peptide hormones: Peptide-amidating enzyme

    • Free radical scavenging: Superoxide dismutase (SOD1)

    • Cross-linking of elastin and collagen: Lysyl oxidase

    • Cross-linking of keratin: Sulfhydryl oxidase

    • Melanin production: Tyrosinase

    • Iron homeostasis: Ceruloplasmin and hephaestin ferroxidases

  • Copper is also implicated in:

    • Myelination

    • Regulation of the circadian rhythm

    • Angiogenesis

Human Copper Homeostasis

  • Three main destinations for copper (Cu):

    1. SOD1 in the cytosol

    2. Mitochondria (for incorporation into the respiratory chain)

    3. Cuproenzymes (for excretion from the Golgi)

  • Three copper chaperones:

    1. CCS (delivery to SOD1)

    2. Unknown chaperone (delivery to mitochondria)

    3. Atox1 (movement into the Golgi)

Copper and the Human Immune System

  • ATP7A relocalizes from the Golgi to the phagosome during an immune response.

    • Mediates copper uptake and downstream production of reactive oxygen species (ROS) via the Fenton reaction.

  • Bacteria have evolved copper exporters to overcome and counteract this mechanism.

Copper Regulation in Response to Copper Levels

  • CTR1 mRNA levels decrease with increasing copper concentration, but not with iron (Fe).

  • Copper chelators increase mRNA production, showing a copper-specific effect.

Ctr1 and Copper Chaperones

  • Ctr1 undergoes reversible trafficking between the plasma membrane and intracellular vesicles.

  • Yeast homologues:

    • Copper chaperone Atx1

    • Target Ccc2a

  • The copper-binding domain is similar between Atx1 and Ccc2a, but the charge ensures proper copper transfer.

SOD1 and Copper Delivery

  • SOD1 acquires copper from CCS.

  • Protein-protein interaction facilitates copper delivery.

  • Disulfide formation and isomerization are important steps in this process.

The chemical equation of SOD1 acquiring copper from CCS

apo−SOD1+Cu(I)−CCS→Cu+1+apo−CCSapo-SOD1 + Cu(I)-CCS \rightarrow Cu^{+1} + apo-CCS

Mitochondrial Copper Chaperones

  • Chaperones (Cox17, Sco1, Cox11) facilitate copper insertion into cytochrome c oxidase (CcO) subunits (Cox1, Cox2).

Regulation in Response to Hypoxia

  • Macrophage response to hypoxia:

    • CTR1 levels increase, raising copper levels within the macrophage.

    • CCS, SOD1, and CcO concentrations decrease.

    • ATP7A directs copper to the Golgi.

  • Ceruloplasmin (oxidase) is required for iron mobilization.

    • Part of hemoglobin production.

Systemic Copper Regulation and Cardiac Hypertrophy

  • In a mouse model of cardiac hypertrophy:

    • ATP7A expression levels are higher in mice with CTR1 deletion.

    • A signal from the heart induces ATP7A expression in other parts of the body.

ATP7A and ATP7B

  • ATP7A/ATP7B are transmembrane proteins with specific domains and motifs:

    • TGN Retention Sequence

    • Metal Binding Site (MBS) motif: MXCXSC

    • Phosphorylation domain (P-domain)

    • ATP-binding domain (N-domain)

    • Phosphatase domain (A-domain)

    • Ion transduction domain

  • Key residues:

    • F37, D1044, G1300, D1497

Mechanism of Copper Transport by ATP7A

  1. Copper transfer from ATOX1 to ATP7A/B

  2. Copper loading/initiation

  3. Progression of Cu+1Cu^{+1} translocation; copper binding to CPC

  4. Phosphorylation of P domain/closing entrance channel

  5. Completion of copper translocation

  6. Dephosphorylation of P domain/setting basal conditions

ATP7A/ATP7B as P-type ATPases

  • ATP7A/ATP7B are members of the P-type ATPase family, which includes:

    • Na+/K+Na^+/K^+ pumps

    • H+/K+H^+/K^+ pumps

    • Plasma membrane and sarcoplasmic reticulum Ca2+Ca^{2+} pumps

  • They transport copper using ATP hydrolysis.

    • The catalytic activity involves:

      • Nucleotide-binding domain (N-domain)

      • Phosphorylation domain (P-domain)

      • Activation domain (A-domain)

  • Copper binding domains (MBD1-6) with a consensus MTXCXXC motif.

    • Copper binds in the reduced form, Cu(I)Cu(I).

  • Physical interaction between ATP7A/ATP7B and copper chaperone ATOX1 through these domains and the CPC motif.

Trafficking of ATP7A and ATP7B in Response to Copper Levels

  • High intracellular copper levels:

    • ATP7A traffics from TGN to vesicles near the basolateral membrane.

    • ATP7B traffics to sub-apical membrane vesicles.

    • Both mediate copper export.

  • Low intracellular copper levels:

    • ATP7A/7B recycle back to TGN.

  • Metal-binding domains 5 and 6 mediate trafficking to the cell periphery.

  • C-terminus leucine repeats are required for retrograde trafficking.

  • ATP7A TGN retention is mediated by a 38 amino acid sequence within transmembrane domain three.

  • ATP7B TGN retention is mediated by a nine amino acid region within the amino terminus.

Menkes Disease

  • X-linked recessive disorder caused by mutations in ATP7A (approximately 1 in 100,000).

  • Documented by John Menkes in 1962.

  • Clinical features:

    • Developmental delay

    • Brain degeneration

    • Sparse, kinky hair

    • Low muscle tone (hypotonia)

    • Low bone density

    • Seizures

    • Aneurysms

    • Gastrointestinal and cardiac defects

  • Severity depends on the specific ATP7A mutation.

    • Over 400 mutations known (deletions, missense, splice site, exon duplications, and point mutations).

Pathophysiology of Menkes Disease

  • Failure in systemic copper absorption and distribution.

  • Copper accumulates in some tissues (small intestine, kidneys).

  • Brain and other tissues have low copper levels.

  • Copper is trapped in the blood-brain barrier and blood-cerebrospinal fluid barrier.

  • Reduced activity of copper-containing enzymes:

    • Cytochrome c oxidase (cellular respiration): CNS degeneration, ataxia, muscle weakness, respiratory failure

    • Superoxide dismutase (free radical scavenging): CNS degeneration

    • Ceruloplasmin/Hephaestin (iron transport): Anemia

    • Tyrosinase (pigment formation): Hypopigmentation

    • Dopamine β-hydroxylase (catecholamine production): Ataxia, hypothermia

    • Lysyl oxidase (collagen and elastin cross-linking): Loose skin and joints, osteoporosis

    • Sulfhydryl oxidase (cross-linking of keratin): Abnormal hair

Clinical Presentation and Diagnosis of Menkes Disease

  • Classical Menkes disease (MD) is the most severe form.

  • Occipital horn syndrome (OHS) is the mildest form, characterized by wedge-shaped calcium deposits in the occipital bone.

    • OHS patients have partially functional protein or reduced levels of normal protein.

    • Gene deletions result in severe classical MD, with death in early childhood.

  • Initial development is normal up to 2–4 months of age, followed by developmental arrest and loss of skills.

  • Therapy-resistant seizures develop around 2–3 months of age.

  • Death typically occurs before the third year of life due to infection, vascular complications, or neurological degeneration.

  • Diagnosis involves:

    • Clinical features (typical hair changes)

    • Reduced serum copper and ceruloplasmin levels

    • Analysis of DOPA to dihydroxyphenylglycol ratio (dopamine β-hydroxylase activity)

    • Genetic typing

Treatment of Menkes Disease

  • Mainly symptomatic treatment

  • Copper administration may extend lifespan.

  • Oral administration of copper is ineffective, as copper is trapped in the intestines.

    • Success depends on early initiation and presence of at least partially functional ATP7A.

Wilson's Disease

  • Inherited disorder characterized by excessive copper accumulation in the body, particularly in the liver, brain, and eyes.

  • Symptoms usually appear between ages 6 and 45 (most often in the teenage years).

  • Features include a combination of liver disease and neurological/psychiatric problems.

Clinical Manifestations of Wilson's Disease

  • Liver disease:

    • Jaundice

    • Fatigue

    • Loss of appetite

    • Abdominal swelling

  • Neurological/psychiatric problems:

    • Clumsiness

    • Tremors

    • Difficulty walking

    • Speech problems

    • Impaired thinking ability

    • Depression

    • Anxiety

    • Mood swings

  • Kayser-Fleischer rings: Green-to-brownish ring in the front surface of the eye caused by copper deposits.

Pathophysiology of Wilson's Disease

  • Caused by mutations in ATP7B (autosomal recessive).

  • Occurs in 1 in 30,000 people.

  • Usually presents at a young age (< 20 years).

  • Over 700 mutations known (many in the N domain).

  • ATP7B fails to transport copper into bile.

  • Copper accumulates in liver cells.

  • Damage to liver cells occurs via Fenton chemistry, leading to fibrosis and cirrhosis.

  • Copper is then released into the blood and deposits in the kidneys, eyes, and brain.

Neurological and Hepatic Symptoms of Wilson's Disease

  • Brain:

    • Copper is deposited in the basal ganglia.

    • Damage occurs via Fenton chemistry.

    • Neurological/psychiatric problems:

      • Mild cognitive deterioration

      • Parkinsonism (tremor, rigidity, lack of balance)

      • Impulsive behavior, apathy, loss of memory

      • Depression and anxiety

  • Liver:

    • Lipid peroxidation, DNA damage, and loss of respiratory chain function.

    • Jaundice

    • Hepatic encephalopathy (build-up of waste products in the blood, such as ammonia)

    • Portal hypertension (increased pressure in the portal vein)

Diagnosis and Treatment of Wilson's Disease

  • Diagnosis:

    • Neurological symptoms

    • Kayser-Fleischer rings

    • Low ceruloplasmin level

    • Elevated copper levels in urine (>40 mmol/24h)

    • Liver biopsy (250 mg copper/g dried liver)

    • Genetic testing

  • Treatment:

    • Early diagnosis and treatment are crucial.

    • Low copper diet

    • Initial medication: copper chelators (penicillamine, tetrathiomolybdate) for 6 months to excrete copper in urine.

    • Zinc acetate induces metal-binding proteins (metallothionein) within cells.

    • Liver transplant