Chronic Disease - Copper Enzymes

Chronic Disease - Copper Enzymes

Chronic Disease

• Dr Jon Sellars, Newcastle University.

• Email: jon.sellars@newcastle.ac.uk

Learning Outcomes

• Understand metallobiology's general principles, emphasizing metal types and their incorporation in biological systems leading to downstream effects.

• Understand the relationship between metals and disease, focusing on:

  • Wilson’s disease (Cu overload)

  • Menkes disease (Cu deficiency)

  • Haemochromatosis (Fe overload)

Copper Enzymes Overview

Copper-containing enzymes:

• 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

• Cross-linking of elastin, collagen: lysyl oxidase

• Cross-linking of keratin: sulfhydryl oxidase

• Melanin production: tyrosinase

• Iron homeostasis: ceruloplasmin and hephaestin ferroxidases

• Implicated in myelination, regulation of the circadian rhythm, and may be necessary for angiogenesis.

Human Copper Homeostasis

Copper Destinations:

1. SOD1 in the cytosol

2. Mitochondria: incorporation into the respiratory chain

3. Cuproenzymes for excretion from the Golgi

Copper Chaperones:

1. CCS: delivery to SOD1

2. Unknown chaperone: delivery to the mitochondria

3. Atox1: movement into the Golgi

Copper and the Human Immune System

• ATP7A relocalizes from the Golgi to the phagosome.

• Mediates Cu uptake and downstream production of reactive oxygen species via the Fenton reaction.

• Bacteria have evolved Copper exporters to overcome this mechanism.

Copper Regulation

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

• Copper chelators show increasing mRNA levels produced (copper-specific effect).

Ctr1

• Reversible trafficking between the plasma membrane and intracellular vesicles.

• Yeast homologues of copper chaperone Atx1 and the target Ccc2a.

• The copper-binding domain is similar between Atx1 and Ccc2a, yet the charge and protein-protein interaction ensure copper transfer.

SOD1

• SOD1 acquires Cu from CCS.

• Protein-protein interaction to deliver Cu.

• The equation for this process is:

  $SH$
  $SH$
  $SH$
  $HS SH$
  $SH$
  $apo-SOD1$
  $Zn^{2+}$
  $SH$
  $SH$
  $Zn$
  $Zn(II)-SOD1$
  $Docked heterodimer$
  $HS SH$
  $O_2$
  $Disulphide formation$
  $SH$
  $Cu(I)-CCS$
  $HS SH$
  $SH$
  $SH$
  $Disulphide isomerization$
  $Cu^{+1}$
  $apo-CCS$
  $Zn$
  $Zn$
  $SH$

Mitochondrial Copper Chaperones

• Diagram showing unfolded protein, TOM OM, www, IMS Cox170, QCCS, IM, SOD1, Sco1, CUA, Cox2, CuA, CCO, Cox1, CuB, Mia40, Matrix, Cox11, ss Oss, Erv1, O(SH)2, Cox17

Regulation in Response to Oxygen (Hypoxia)

• Macrophage response: under hypoxia, CTR1 levels increase, thus Cu levels within the macrophage increase.

• CCS, SOD1, and CcO decrease in concentration, while an increase in ATP7A directs Cu to the Golgi.

• Ceruloplasmin (oxidase) is required for the mobilization of iron, part of hemoglobin.

Systemic Copper Regulation

• Cardiac hypertrophy in mouse heart.

• ATP7A expression levels are much higher in mouse with CTR1 deletion.

• Signal from the heart to other parts of the body, inducing ATP7A expression.

ATP7A/ATP7B

• Diagram of ATP7A/ATP7B structure, including TGN Retention Sequence, metal-binding sites (MBS), P-domain, Phosphorylation, N-domain, A-domain, Phosphatase domain, ATP-binding motif, Internalization motif, and PDZ motif

• Metal Binding Site (MBS) motif = MXCXSC

• Ion transduction domain = CONCPC

ATP7A

1. Cu+ transfer from ATOX1 to ATP7A/B

2. Cu+ loading/Initiation

3. Progression Cu+ translocation, Cu+ binding to CPC

4. Phosphorylation P domain /Closing entrance channel

5. Completion of Cu+ translocation

6. Dephosphorylation P domain /Setting basal conditions

ATP7A/ATP7B

• Members of a large family of P-type ATPases.

• Energy-utilizing cation pumps (Na+/K+, H+/K+ pumps, plasma membrane and sarcoplasmic reticulum Ca2+ pumps).

• ATP7A/ATP7B transport copper using the energy released from the hydrolysis of ATP.

• Catalytic activity involves domains specific for binding and hydrolysis of ATP, similar in all P-type ATPases:

  • Nucleotide-binding domain (N-domain)

  • Phosphorylation domain (P-domain)

  • Activation domain (A-domain)

• Transport and translocation of copper require special motifs and structures for recognition, binding, and translocation of the copper across the membrane.

• These motifs contain cysteine residues, which play an important role in copper binding.

• ATP7A/ATP7B have six copper-binding domains (MBD1–6) with a consensus MTXCXXC motif.

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

• There is a physical interaction between ATP7A/ATP7B and the copper chaperone ATOX1 through these domains and the CPC motif.

• The CPC motif within TMD6 binds copper during transport.

• The N- and P-domains reside between TMD6 and TMD7.

• The N-domain binds ATP, and the γ-phosphate of ATP is transferred to the aspartate residue in the P-domain, resulting in the formation of a transient phosphorylated intermediate.

ATP7A Trafficking

• When intracellular copper levels rise:

  • ATP7A traffics from TGN to vesicles near the basolateral membrane

  • ATP7B traffics to sub-apical membrane vesicles

• From these locations, ATP7A and ATP7B mediate copper export.

• When intracellular copper levels drop, ATP7A/7B recycle back to TGN.

• For both ATP7A/7B, metal binding domain 5 and 6 are sufficient to 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

Overview

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

• Documented by John Menkes in 1962.

• Symptoms:

  • Developmental delay

  • Brain degeneration

  • Sparse kinky hair

  • Low muscle tone

  • Low bone density

  • Seizures

  • Aneurysms

  • Gastrointestinal and cardiac defects

• Severity depends on the mutation of the ATP7A gene.

• Over 400 mutations – deletions, missense, splice site, exon duplications, and point mutations

Menkes Disease: Failure in Systemic Cu Absorption and Distribution

• Mutations in ATP7A.

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

• Low levels in the brain and other tissues.

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

• Neurons and glial cells are deprived of copper.

• Decreased supply of copper reduces the activity of copper-containing enzymes.

• Necessary for the structure and function of bone, skin, hair, blood vessels, and the nervous system.

• Signs and symptoms are caused by the reduced activity of these copper-containing enzymes and loss of the regulation of the NMDA receptor.

  • 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 Features and Progression

• Classical MD is the most severe form.

• Occipital horn syndrome (OHS) is the mildest form characterized by wedge-shaped calcium deposits in a bone at the base of the skull (the occipital bone).

• In general, patients with OHS have mutations that lead to a partially functional protein or reduced levels of normal protein.

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

• Initial development is normal up to 2–4 months of age, then the patient ceases to develop further with gradually loss of some of the previously developed skills.

• Most patients develop therapy-resistant seizures from about 2 to 3 months of age.

• Death typically occurs before the third year of life due to infection, vascular complications (such as sudden and massive cerebral hemorrhage due to vascular rupture), or from neurological degeneration.

Diagnosis and Treatment

• Diagnosis:

  • Initial diagnosis is suggested by clinical features (especially typical hair changes) and supported by reduced levels of serum copper and ceruloplasmin.

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

  • Genetic typing.

• Treatment:

  • Mainly symptomatic treatment, but copper administration may extend life span.

  • Oral administration of copper is ineffective as copper is trapped in the intestines – success is dependent on early initiation and presence of at least partially functional ATP7A.

Wilson's Disease

Overview

• An inherited disorder in which excessive amounts of copper accumulate in the body, particularly in the liver, brain, and eyes.

• Symptoms usually first appear between the ages of 6 and 45 - most often during the teenage years.

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

• Liver disease is typically the initial feature:

  • Jaundice

  • Fatigue

  • Loss of appetite

  • Abdominal swelling

• Nervous system or psychiatric problems are often the initial features in individuals diagnosed in adulthood or young adults:

  • Clumsiness

  • Tremors

  • Difficulty walking

  • Speech problems

  • Impaired thinking ability

  • Depression

  • Anxiety

  • Mood swings

• Kayser-Fleischer ring: copper deposits in the front surface of the eye forming a green-to-brownish ring.

Wilson Disease: Failure in Cu Excretion

• Diagram showing the process of copper accumulation and its effects.

• Includes effects such as:

  • Neurological disorders

  • Kayser-Fleisher rings

  • Haemolytic anaemia

  • Osteoarthritis

  • Cardiomyopathy

  • Hepatomegaly

  • Arthritis

  • Renal tubular dysfunction

Pathophysiology

• 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 – a large number in the N domain.

• ATP7B fails to transport copper into bile.

• Copper accumulates in liver cells.

• Damage to liver cells by Fenton chemistry leading to fibrosis and cirrhosis.

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

Symptoms

• Brain:

  • Copper is deposited in the basal ganglia and damaged by Fenton chemistry, leading to neurological/psychiatric problems.

  • Initial mild cognitive deterioration is followed by parkinsonism (tremor, rigidity, lack of balance).

  • Range of cognitive symptoms – impulsive behavior, apathy, loss of memory.

  • Psychiatric – depression and anxiety.

• Liver:

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

  • Jaundice.

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

  • Portal hypertension (increased pressure in the portal vein).

Diagnosis and Treatment

• Diagnosis:

  • Neurological symptoms

  • Kayser-Fleischer rings

  • Low ceruloplasmin level

  • Copper levels in urine (> $40$ mmol/24h)

  • Liver biopsy ($250$ mg copper g-1 dried liver)

  • Genetic testing

• Treatment:

  • If diagnosed early, Wilson’s disease can be successfully treated; however, it can be fatal if not diagnosed or treated properly.

  • Low copper diet.

  • Initial medication - drugs that chelate copper, which is then excreted in urine (Penicillamine, tetrathiomolybdate) for 6 months.

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

  • Liver transplant.