Zinc fingers fulfil ______ purposes.
Zinc binds to _ residues to help shape the zinc finger.
Zinc isn’t used to ___1___ effect, it simply enables the protein to take its particular shape (___2___ shape) to help facilitate_____-3-_____ interactions.
Zinc-fingers are prevalent in the control of ____1____ ____2____associated with ____3____into multiple cell types.
Metal ions form the ____1____ ____2____of some enzymes.
Aconitase contains a ____-1-____cluster at its active site.
The enzymes substrate, ____2____, binds to this exposed cluster, catalyzing the reaction.
Aconitase catalyses the conversion of ____2____ and____3____.
There are two forms in humans:
____4____ (Aco1) and mitochondrial (Aco2)
Redox acting metals, such as Fe and Cu, can also form the active site of the enzyme. Their redox ability (e.g. Fe2+ to Fe3+) allows the movement of ____1____.
E.g. In ____ __2__ ____this movement of electrons completes the last step in the electron transport chain.
2. Cytochrome c oxidase
Both E.coli and S.cerevisiae contain ____ 1 ____(SOD).
SOD is an enzyme found in all living cells that ____2____up certain chemical reactions.
The E.coli version contains ____3____.
The yeast version contains ____4____and Zn.
Why are they different yet do the same job?
You can take the E.coli version (SODB) and you can express it in yeast that lacks its own SOD1. The replacement SODB works perfectly fine.
You pick the metal that will do the best job.
(i.e. No point having Zn somewhere you need to move electrons around, as Zn is not redox active.)
Which metal is picked can depend on how the metal binds to ____5____
Some prefer to bind 6 amino-acids, some prefer 4.
1. Superoxide dismutase
Binding sites of proteins involved in metal homeostasis typically allow metal ____1____, but other sites can be buried, with the metal being kinetically trapped and safe from replacement with an incorrect metal. Even so, the correct metal must somehow become trapped in the first place. A subset of E.coli manganese superoxide dismutase, MnSOD, is known in vivo to acquire iron, which is catalytically ____2____. Recombinant MnSODs from other organisms, expressed in E. coli, mis-populate with iron, cobalt or nickel. Eukaryotic MnSOD, in the mitochondrial matrix, acquires catalytically ____2____iron when mitochondrial manganese and/or iron homeostasis is perturbed.
A metal ions suitability for a protein is governed by ____1____ and ____2____.
Proteins are flexible, so you can get metal ions joining other metal ions’ protein sites if the 2 share ____3____ characteristics.
Primary co-ordination sphere = the immediate ligands (primary residues) the metal binds to. I.e.. Above: Ni binds 6. Zn binds 4.
How many primary residues do the following metal ions bind to?
Co = _1_
Ni = _2_
Cu = _3_
Zn = _4_
Secondary co-ordination sphere = ____5____ that bind to the primary residues.
These 2 layers govern how the metal ion will act within the enzyme.
At catalytic centres, metals increase ____1____, electrophilicity and/or nucleophilicity of reacting species, promote heterolysis, or receive and donate ____2____. The protein’s primary and secondary metal ____3____ spheres tune the properties of the metal to optimize reactivity and influence metal selection. Donor ligands (S, O or N) can impart bias in favour of the correct metal. The metal-binding pocket can exclude ions with the wrong ____4____. Coordination geometry (octahedral, tetrahedral, square pyramidal, trigonal bipyramidal, square planar, trigonal or linear) can impart bias either in folded apoproteins if the preformed site is rigid or during folding if favourable energetics is coupled to the correct ____5____.
However, because proteins have flexibility, steric selection between metals is imperfect, especially in nascent polypeptides. Under these conditions, the relative affinities of metals for proteins are significantly governed by the ligand field stabilization energies of the metals themselves. This creates the universal orders of preference, which for divalent metals is the ____-6-____series. There is ambiguity about the position of zinc, which is either at the ____7____of the series or somewhere above cobalt. This ambiguity is attributed to the nephelauxetic effect. Cuprous ions, expected to dominate in more reducing cell environments, are also competitive, and some exceptionally tight ferric complexes are known. Crucially, such affinity series underpin calculations that each metal’s relative abundance in the biological locality is paramount in governing selective metal binding by proteins, highlighting the vital contribution of cell biology to the selection of metals by metalloproteins.
Metal availability has changed over time.
Originally Fe & Ni readily available in the environment due to ____1____ activity releasing it from the earths core.
The environment was reducing but over time changed to an ____2____ environment.
In the reducing environment there was lots of ____3____ (soluble) ions in the sea. Organisms were dependent on this.
As oxygen producing organisms flourished O2 was pumped into the atmosphere.
This oxygenated the environment, oxidizing ____3____ to Fe3+ (insoluble).
Organisms are unable to utilize Fe3+ so once the oxidation occurred it fell to seabed and therefore was no longer bio-available.
Ni bio-availability followed suit due to the reduction in ____1____ activity causing usable stocks to empty.
In today’s open oceans, the poor availability of ____1____ , but the presence of some copper, has led organisms to replace the former and exploit the latter. The oceanic diatom Thalassiosira oceanica has switched from using ____1____-containing cytochrome c6 to using copper-containing plastocyanin which was otherwise only known in some cyanobacteria and ____2____organisms containing chlorophyll b. The sulphur isotope record also implies that ancient oceans were dominated by sulphide and, hence, sulphides.
The exploitation of metals varies between species, and trends exist in the superkingdoms. The slope of plots comparing the number of zinc-binding domains with the total number of protein domains encoded by each genome is greater in ____3____ than in archaea or bacteria; the reverse trend is true for ____1____-, manganese- and cobalt-binding domains. An implication is that zinc was increasingly recruited as eukaryotic genomes became more complex, and this is especially true for multicellular ____3____ with a point of inflection detected at the intersection between unicellular and multicellular species. This reflects the eukaryotic diversification of structural zinc-binding domains, notably zinc-finger and RING-finger domains, which constitute ~3% and ~1% of the human proteome, respectively.
Archaea and bacteria have, as a proportion, more iron–sulphur proteins but fewer haem proteins than eukaryotes, and within the bacteria aerobic species have fewer iron–sulphur cluster proteins and more haem proteins than anaerobic bacteria.
Metal availability can be governed by the environment.
Quarry has ____1____ metal availability.
Ocean has ____2____ metal availability.
If the bio-availability of a metal suddenly changes, what can the organism do?
It can’t just change the metal used as it has ____1____to use the metal ion.
Helicobacter pylori is found in the human ____2____.
Helicobacter mustelae is found in a ferrets ____2____.
Pylori survives the acidic environment of the ____2____ by producing urease which breaks down urea to form ammonia. Ammonia is basic and therefore ____3____the pH of the bacterium’s immediate surroundings. Pylori’s urease contains a Ni ion and is very efficient in its job.
A typical human diet consists of much Ni (through ____4____).
Ferrets however only eat ____5____which contains little or no Ni. It does however contain much Fe ions.
Mustelae also expresses the same Ni urease but also expresses an almost identical enzyme that binds and uses Fe.
The Fe enzyme is less efficient but carries out the same job. Due to expected Ni deficiency, the Fe urease acts as a back up for Mustelae.
If the bio-availability of a metal suddenly changes, what can the organism do?
Organisms can evolve new systems to acquire the metal
Some cells express Fe reductases (Fe3+ insoluble --> Fe2+ soluble). This is called the ____1____ strategy.
Some cells produce an Fe3+PS (soluble complex) which is then able to be taken up by Fe transporters. This is called the ____2____ strategy.
If unable to adapt, the consequences of changes in metal ion availability = ____ __1__ ____.
Methanogens were abundant in ancient seas when Ni was also plentiful. They evolved ____2____that were dependent on Ni so when the Ni levels dropped they were unable to ____3____. They are now only found in extreme environments.
Limiting metal levels cause the switching on of genes coding for metal reductases and metal transporters leading to ____4____ metal uptake.
1. Loss of viability / Death
Too much metal is toxic.
Too little and there will not be enough to form the enzymes.
Therefore cells must perform metal ______ to regulate its concentrations.
Regarding metal toxicity:
Non biological metals (cadmium, lead, mercury, silver) and biological metal in excess of need or misdirected (copper, zinc, iron) have what negative effects? (2)
1. Bind to protein and inhibit its function
2. Displace another metal from binding site
Regarding metal toxicity:
Some metals can cause the ______ reaction.
______ reaction= H2O2 --> OH- + OH(radical) OH(radical)= Highly reactive hydroxyl radical (toxic!)
What does the Irving-Williams series rank?
Strength of metal-protein binding
From weak to tight binding, rank the Irving-Williams series.
Cells must find a way to ensure metallo-proteins do not come into contact with metal ions higher up the Irving-Williams series as this could lead to ____1____ and loss of function.
In bacterial cells one way to control this is to use the metals ____2____ on the series as importers only and the metals ____3____ on the series as both importers & exporters. This limits the level of the ions high in the series in the cell therefore reducing competition of these ions with the lower series ions.
Metals can displace other ions higher up the Irving-Williams series, but only when they are in a sufficiently ______ ______.
In a healthy person, ferrochelatase catalyses insertion of iron into protoporphyrin IX. But zinc protoporphyrin forms in patients with____1____. This insertion is the last step of ____2____synthesis. The Zn displaces Fe with ferrochelatase when Fe levels ____3____. Used as a medical diagnostic tool.
Cobalt binds to cryptococcal urease
Cryptococcus produces spores which we ____1____. In healthy individuals these spores are destroyed by ____2____ within the lungs. In immunocompromised individuals the spores germinate and the yeast disseminates around the body. They end up infecting the ____3____, which if not treated is universally fatal. The way the yeast gains access to the ____3____ is that it gets trapped within the small capillaries within the ____3____ and it sits there and metabolizes urea (plenty circulating) by use of urease, to produce ____4____ (which it uses as an energy source). This reaction causes a ____5____ change that causes damage that kills the surrounding cells, allowing entry of the yeast to the ____3____. Cryptococcal cells that are urease negative cannot cause the disease.
What kind of metals does the Fenton rection occur in?
Redox active metals.
What do the product of the Fenton reaction, oxygen radicals, do to the cell?
Damage cellular components due to causing oxidative stress in cells.
The human body actually uses metal ____1____ as a form of defence in macrophages.
A macrophage will phagocytose bacteria and break them down by combining them with a ____2____.
In addition to that, 2 transporters are localised to the phago-____2____.
1.NRAMP1 – pumps ____3____ out
2.ATP7A – pumps ____4____ in
High ____4____ levels which is very toxic in itself (high in IW series) combined with low ____3____ is used to help kill the pathogen.
There is also systemic regulation of Fe – (once the body senses a pathogen it restricts the amount of Fe that is flowing around the body to limit the Fe available to the ____5____).
We need sensors to sense both toxicity and limitation and to trigger the correct homeostatic action (exporters, importers) to reach the safe ion level.
Toxicity – Export metal. Repress metal import. The metal can also be sequested so it is not harmful to the cell. This is done by ____1____ proteins binding the metal and delivering the metal from one part of the cell to another whilst preventing it from ____2____ with anything.
Limitation – Import metal. Repress metal export. Release from metal stores. Change the ____3____ to utilize other pathways that do not require the sparse metal (E.g. pylori example, switch Zn with Fe in the enzyme. Leads to a drop in efficiency).
All of these cellular mechanisms are used to what?
Limit metal concentrations
Bacteria contain a range of transcription factors that sense individual metal ions.
They sense the metal ion by binding the metal and undergo ____1____ change that alters there ability to bind to ____2____ and causes either the switching on or off of genes.
Example. – In bacteria under high Cu there will be transcription factors that bind Cu and repress the transcription of Cu importers.
Also under low Cu there will be transcription factors that lead to the upregulation of transcription factors of Cu importers.
The Cu binding transcription factors must bind Cu with a ____3____ affinity than any of the other metal ions. This is because you don’t want, thinking of IW series, your Ni binding transcription factors to bind Cu as they would no longer work.
So the Cu transcription factor must be the first to bind the free Cu preventing it from coming into contact with any of the lower IW proteins/txn factors.
In vitro systems to measure when the Cu factor becomes activated discovered that only one Cu atom is needed to activate this system to ensure that no free Cu is able to bind anywhere it shouldn’t. The other metal ion transcription factors are in order of the IW series within the cell to ensure no ____4____ occurs at all.
In humans we know only a couple of examples where metal directly binds to a transcription factor to regulate its function.
MTF1 switches on the gene that encodes the protein MT that binds cadmium and Zn to ____1____ them (so they do no harm). What happens is: If cadmium is present it displaces Zn from MT. The freed Zn then binds to MTF1 which binds to ____2____ leading to transcription.
In ____3____ all metals are sensed for by transcription factors, in humans only 2 pathways exist.
In eukaryotes, instead of transcriptional regulation being important, membrane transporter turnover and localization is controlling how much metal ____1____ the cell.
So as metal ions are imported some transporters can undergo conformational changes that cause them to interact with proteins that regulate endocytosis and ____2____.
ATP7A&B Cu proteins are not broken down but localized somewhere else depending on Cu levels passing through the plasma membrane, to the ____3____, and back again.
Fe is regulated by the protein Hepicidin interacting with the Fe transporter internalizing it ready for ____2____.
Another way eukaryotes regulate protein expression in response to metal ions is by controlling the rate of translation.
Example – Yeast
Yeast contain transcription factors called Aft1 and Aft2 that sense low Fe levels (by interacting with proteins etc.). When Fe levels are low these factors ____1____ genes involving the uptake of Fe (transporters etc.) and also cause the recovery of Fe ____2____ in the vacuole.
They also produce the protein CTH2 (by activating its gene) by binding to certain transcripts of genes involved in the pathways that use Fe proteins. CTH2 ____3____ these transcripts so there are fewer of them. Therefore the cell is modulating its ____4____ in response to the amount of Fe present in that Fe is diverted to the more important Fe requiring processes.
Another major mechanism of cellular metal limitation is the use of chaperones to deliver metals to specific places with the cell.
Most metals are imported and they will bind to components within the cell at low ____1____ sites and exchange between these many sites within the cell until they become incorporated into the correct ____2____. How this happens is not well understood. This is fine for Zn, Fe for e.g., as it wont cause much if any displacement (low on IW). But this cannot be allowed to happen with e.g. Cu or Ni( ____3____ on IW) as it would cause havoc around within the cell. The way the cell circumnavigates this issue is by using chaperones (small ____4____) that specifically bind the free metal and deliver it to the target.
Example of metal chaperones in cellular metal limitation
Example – Ni and urease
UreE is a small peptide (chaperone) that dimerises and ____1____ to Ni (as it enters the cell). (It is presumed that Ni does this directly without binding to anything else first). This UreE dimer chaperone then protects the Ni (by preventing it from ____2____ with other parts of the cell) and delivers it to a urease complex before inserting into the protein to complete urease.
The chaperone ensures that it docks with the correct protein via protein-protein interaction (the chaperone is specific for the target). When the chaperone binds with the target there will be a slight ____3____ change allowing the metal to move from the chaperone to the target protein. This ensures that regardless of Ni’s high ____4____ towards many other proteins, it will only ever interact with urease within the cell.
Cu chaperones also exist in ____5____ organisms.
Compartmentalisation in cellular metal limitation
Example – Bacteria – Mnca & Cuca
Structurally Mnca and Cuca look identical. The only difference is that Mnca is a manganese protein and Cuca is a copper protein. If you take Mnca in vitro it will bind Cu with a ____1____ affinity (expected) than Mn. So when it folds it will bind Cu and that Cu will be trapped. So how does the cell ensure the Cu doesn’t get into that protein?
Gram negative bacteria have a ____2____ and (like all cells) retain a very low level of free Cu in the cytoplasm (in turn Mn levels are high). Mnca is translated and folds within the cytoplasm when there is Mn present but no Cu. Once folded it is transported through a transporter protein that imports only ____3____ proteins. Once it has crossed the ____2____ (that includes free Cu) the Mn ion is protected due to the folding and thus no ____4____ takes place.
Cuca however, is translated as an unfolded protein straight into the ____2____ (where there is Cu) ensuring that when that Cuca folds it will preferentially bind Cu.
So by controlling where the protein folds, the cell ensures the right metal binds to the correct protein.
If you have mutations in ATP7A, Cu is blocked from ____1____ the body at enterocytes and is also blocked from ____1____ the brain. This leads to a Cu ____2____ disease.
If you have mutations that effect the function of ATP7B, the transport of excess Cu out of the body is ____3____. This leads to Cu accumulation in the liver leading to a Cu ____4____ disease.
Cu enters the cell through ____1____.
At high Cu levels the Cu will be internalized by ____2____ and transported back to the membrane ready for excretion.
There are only 3 targets of Cu in the cell:
1. ____3____ – to be incorporated into the respiratory chain.
2. ______ 4 ______ – in the cytoplasm
3. ____5____ – for incorporation into Cu enzymes that are then excreted into the cell
In each case there is a chaperone that delivers Cu to its target:
1. Small unknown Cu ligand that delivers Cu to mitochondria
2. CCS delivers the Cu to ______ 4 ______
3. Atox1 delivers Cu to ATP7A&B
When Cu levels are high, ATP7B in hepatocytes relocates to the ____6____ membrane for excretion of Cu into the bile.
In other cells (when Cu levels are high) ATP7A relocates to the blood adjacent membrane to pump Cu-enzymes out of the cell.
4. Superoxide dismutase/SOD1
There is some Cu responsive transcription is humans. If we look at transcripts of CTR1 (Cu transporter), it is regulated in response to Cu by a transcription factor called ______.
The main way Cu is regulated is the localization and degradation of the ____1____.
High Cu levels lead to CTR1’s ____2____ and degradation (to prevent more Cu import).
ATP7A contains Cu binding motifs within its __1__-terminal region. It’s localization to the plasma membrane under ____2____ Cu levels is dependent on 2 of the binding motifs, 5 and 6 (MBS5 & MBS6).
Each motif is a repeated ____3____ that binds 1 Cu atom.
If you mutate either 5 or 6, you ____4____ the translocation from the ER to the plasma membrane.
So somehow binding of the Cu to 5 & 6 signals to the protein that there is too much Cu in the cell and causes it to move to the membrane and pump Cu-enzymes out.
In the yeast chaperones the Cu is bound by 2 cysteine ____1____. The difference between Ccc2A and Atx1 is the charge of the protein domains on the outside.
Ccc2a = - charge Atx1 = + charge
This opposite charge is what ____2____ the 2 together. They bind together in such a way that the Cu binding site of the dimer chaperone is opposite the receiving site of the transporter. As the chaperone comes into contact with the transporter the Cu is transferred from one to another.
It is therefore this physical ____3____ between the 2 chaperones that ensures the Cu reaches its target.
SOD is usually found as a dimer. It contains a structural __1__ site and a catalytic __2__ site. Once it is methylated with Zn (by an unknown mechanism) it will bind as a homodimer to the chaperone CCS. CCS contains 2 domains, 1 & 3, each of which contain __3__ cysteine residues which bind metals with high affinity. They bind one Cu atom between them and then dock with a monomer of SOD1 to form a heterodimer of SOD1 and CCS. The Cu is then transferred from domain 3 to SOD1 and then a ____4____ bond is formed to activate SOD and then the monomer will dimerize with another enzyme.
Therefore again it is the physical interaction between the chaperone and the SOD and target that ensures the metal transfers to the correct place. Structurally CCS and SOD are very similar and this is what enables them to bind to each other instead of forming homodimers of themselves.
The Cu ligand that transfers Cu to the electron respiratory chain is known to exist within the mitochondria and ____1____ but what it is remains unknown. It mops up Cu within the cytoplasm and transports it to the mitochondrial matrix. Once in the matrix it transfers the Cu to a chaperone protein called ____2____ in the intermembranal space which then transfers the Cu to 2 targets, Sco1 and Cox11, which are ____3____ bound proteins. They will then deliver the Cu to the CuA and CuB sites of cytochrome c oxidase.
Under hypoxia – less respiration required and less need for the activity of SOD1.
But there will be greater need for Fe in the body because under hypoxia Fe is mobilized to ____1____ red blood cell production (haem groups) and therefore supply more ____2____ to the body.
One of the proteins that is important for mobilization of Fe is ____3____.
Therefore cells need to produce more ____3____ for export to travel around the body and mobilize Fe. ____3____ also represses SOD1 and CCO.
The immune system makes use of the properties of metal to attack pathogens.
____1____ is usually found within the ER and Golgi relocates to the phago-lysosome (phagosome) within ____2____ in response to pathogens.
ATP7A/ATP7B are members of a large family of P-type ATPases that are energy-utilizing ____1____ pumps that includes the Na+/K+, H+/K+ pumps and the plasma membrane and sarcoplasmatic reticulum Ca2+ pumps.
ATP7A/ATP7B transport Cu using the energy released by ____2____ of ATP. This catalytic activity involves domains specific for the binding and ____2____ of ATP that are similar in all P-type ATPases. These are the ____3____-binding domain (N-domain), ____4____ domain (P-domain), and ____5____ domain (A-domain).
Transport and translocation of copper also requires special motifs and structures for recognition, binding, and translocation of the metal across the membrane. These motifs contain cysteine residues, which play an important role in copper binding.
ATP7A has six Cu binding domains (MBD1–6) with a consensus MTXCXXC ____1____. Cu binds to these domains in the ____2____ form, Cu(I).
There is a physical interaction between ATP7A and the Cu chaperone ATOX1 through these domains and the CPC motif.
The CPC motif within TMD6 binds Cu during ____3____.
The N- and P-domains reside between TMD6 and TMD7. _4_-domain binds ATP and the γ-phosphate of ATP is transferred to the aspartate residue in the DKTG motif (_5_-domain) resulting in the formation of a transient phosphorylated intermediate.
Following translocation of the Cu through the membrane, the _5_-domain is dephosphorylated. The A-domain is located between TMD4 and TMD5, and includes the TGE motif, where the glutamate residue has a key role in dephosphorylation of the phosphorylated intermediate.
It was once thought that ATOX1 delivered the Cu to the 6 domains and that the Cu would then move to the CPC domains and be transported from there. This has been disproven. The methylation of these 6 domains is for regulation (not transport).
Name the disease of Cu limitation.
Menkes Disease results in low activity of copper enzymes, which has the following results:
Cytochrome c oxidase - cellular respiration (CNS degeneration, ataxia, muscle weakness, respiratory failure)
Superoxide dismutase -free radical scavenging (CNS degeneration)
Ceruloplasmin/Hephaestin - iron transport (anaemia)
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)
X-linked recessive disorder - mutations in ATP7A (1 in 300,000)
Classical MD is the most severe form, while occipital horn syndrome (OHS) is the mildest form. In general patients with a milder phenotype (like OHS) have mutations that lead to a partially functional protein or reduced amounts of normal protein. Gene ____1____ result in the severe classical form of MD, with death in early childhood.
Classical MD: Progressive neurodegeneration and marked connective tissue dysfunction. Hair striking feature: unusual sparse and hypopigmented, lusterless scalp hair.
Initial development is normal up to 2–_2_ months of age then patients cease to develop further and gradually lose of some of 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 ____3____, vascular complications (such as sudden and massive cerebral haemorrhage due to vascular rupture), or from the neurological degeneration.
ATP7A – pumps Cu from the ____1____ into the blood, and from the blood into the brain.
What type of Menkes Disease you have simply depends on how functional your ATP7A is.
Some mutations stop any Cu being imported - ____2____ MD
Some mutations let less Cu being imported - ____3____
All tissues except for the ____1____ and brain accumulate Cu to abnormal levels.
Although high, the Cu level is not toxic as Cu absorption is lower, due to defective Cu ____2____ from the mucosal epithelium, and partly due to the scavenger role of metallothionein.
Cu is trapped in both the blood–brain barrier and the blood – cerebrospinal fluid barrier so that neurons and ____3____ cells are deprived of Cu.
Initial diagnosis is suggested by clinical features (especially typical hair changes) and supported by reduced levels of ____4____ Cu and ceruloplasmin.
Analysis of ratio of DOPA to dihydroxyphenylglycol (indicative of dopamine β-hydroxylase activity). Genetic typing.
Mainly symptomatic treatment but Cu administration may extend life span.
____5____ administration of Cu is ineffective as Cu is trapped in the intestines - success is dependent on early initiation and presence of at least partially functional ATP7A.
Name the disease of Cu toxicity.
Name the Cu ring seen in the eyes of sufferers of Wilson's Disease.
Caused by mutations in ____1____ (autosomal recessive)
Occurs in 1 in 100,000 people
Usually presents young age (< __2__ years)
~300 mutations known
____1____ fails to transport copper into bile
Copper accumulates in liver cells
Damage to liver cells by ____3____ chemistry leading to fibrosis and cirrhosis
Copper is then released into the blood by liver. This deposits in kidneys, ____4____ and brain.
Copper is deposited in the basal ____1____. Damaged by Fenton chemistry neurological/psychiatric problems. Initial mild cognitive ____2____ is followed by parkinsonism (tremor, rigidity, lack of balance).
Range of cognitive symptoms – impulsive behaviour, apathy, loss of memory
Psychiatric – depression and anxiety.
Hepatic encephalopathy – build up of ____3____ products in blood such as ammonia. Portal hypertension – increased ____4____ in portal vein.
Neurological symptoms, Kayser-Fleischer rings, low ceruloplasmin level.
Copper levels in urine (>40 mmol/24h)
Liver biopsy (250 mg copper g-1 dried liver)
____5____ copper diet
Initial medication - drugs that chelate copper which is then excreted in ____6____ (Penicillamine, tetrathiomolybdate) for 6 months.
Zinc acetate. Induces metal ____7____ proteins within cells (metallothionein).
Hereditary autosomal recessive ____1____ overload disease caused by loss-of-function mutations in the ____2____ (Cp) gene, resulting in impaired iron efflux from the cells.
____2____ is copper ferroxidase. There are two forms: serum (Cp) and glycosylphosphatidylinositol (GPI)-linked form (Cp-GPI) on cell membranes.
Patients present with hepatic iron overload, iron-refractory anaemia, retinal pigment degeneration, diabetes mellitus, and various neurological disorders due to parenchymal iron ____3____.
Cp-GPI is required to maintain ____4____ (iron exporter) at cell surface.
Hepcidin and Cp-GPI compete for interaction with ____4____. Lack of Cp-GPI results in internalization and degradation of ____4____ resulting in iron accumulation in cells. High iron results in Fenton chemistry and cell damage.
Iron ____5____ therapy (Deferoxamine).
Bedlington Terriers (bred to catch rats in coal mines) suffer from a Wilson’s Disease like disease. The mutation in MURR1 leads to less ____1____ being excreted and therefore less Cu being excreted. This leads to a build up of Cu to toxic levels.
Breeders are currently trying to breed this mutation out of the line.
“Iron ____1____ is the most common and widespread nutritional disorder in the world. As well as affecting a large number of children and women in developing countries, it is the only nutrient ____1____ which is also significantly prevalent in industralized countries. The numbers are staggering: 2 billion people – over 30% of the world’s population – are anaemic, many due to iron ____1____, and in resource-poor areas, this is frequently exacerbated by infectious diseases.”
“In developing countries every second pregnant woman and about 40% of preschool children are estimated to be anaemic. In many developing countries, iron ____1____ anaemia is aggravated by worm infections, ____2____ and other infectious diseases such as HIV and tuberculosis. The major health consequences include poor pregnancy outcome, impaired physical and cognitive development, increased risk of morbidity in children and reduced work productivity in adults. Anaemia contributes to 20% of all maternal deaths.”
World Health Organisation
Unlike Cu, there are many Fe containing enzymes in humans. They are involved in all sorts of processes.
Unlike Cu, Fe is regulated at the point of ____1____ (import). There is no mechanism to excrete Fe from the body. We lose a small amount of Fe from shedding dead ____2____ cells. Therefore no excretion in bile etc.
Most of the Fe is used in the formation of red blood cells and that Fe is circulated in the system when ____3____ break down dead RBCs.
Fe is stored in the liver.
It is transported around the body bound to a molecule called ____4____.
Fe regulation occurs at the point of export from the liver, ____3____ and enterocytes in the gut by a transporter called ferroportin. Ferroportin pumps Fe into the blood from these 3 sources.
Baring in mind there is no way of excreting Fe, you can see that if there is a defect in the regulation of ferroportin leading to too much Fe being pumped into the ____5____ system, there is no way of getting rid of it.
This whole process is regulated by Hepicidin.
In enterocytes – Fe from our diet is reduced by the reductase Dcytb that converts Fe_1_ to Fe2+. The Fe2+ is then soluble and is transported into the cell by ____2____.
Haem in our diet is also taken up by another transporter, HCP1, and that haem is broken down Haem oxygenase (HO1) to release the Fe.
Fe can then be bound in ____3____ to store it.
Or Fe can be transported out by ferroportin (if ferroportin is localized to the membrane).
Fe2+ is then transported into the serum before one of the Cu oxidases oxidises Fe2+ to Fe_1_ enabling the Fe_1_ form to bind to transferrin which then transports it around the body to where it is required.
When the transferrin-Fe3+ complex reaches its target cell the complex binds to a transferrin ____1____ that becomes endocytosed.
There is an endosomal __2__ change that causes the release of the Fe into the endosome. The Fe3+ is then reduced by the reductase Steap3 to Fe2+ is then pumped out of the ____3____ into the cytosol by DMT1.
The transferrin receptor now lacking Fe moves back to the membrane before releasing the now Fe-free transferrin.
A process occurs in macrophages where ____ 1 ____ cells are engulfed, the haem is released and Haem ____2____ then breaks down the haem to release the Fe.
The Fe is then pumped out by ferroportin before ceruloplasmin then oxidizes the Fe2+ to Fe3+ to allow the Fe to bind ____3____ and be transported around the body.
Macrophages also have proteins on their ____4____ (Hpx and Hp) that will bind any free haem in the blood and pump it into the cell to be recycled as above explained.
1. Red blood
Aconitase – Humans have 2 forms; ____1____ and cytosolic aconitase. Both bind 4Fe-4S clusters. The cytosolic form though, also acts as a sensor for Fe.
When there is a lot of Fe present 4Fe-4S is formed in the mitochondria, pumped out into the cytoplasm and then incorporated in IRP1 and forms the ____2____ site of aconitase (the protein wraps around the cluster).
If there is low Fe, 4Fe-4S is not made in the mitochondria, is not pumped into the cytoplasm and will not bind to IRP1 and IRP1 as a result retains a more ____3____ structure.
This ____3____ structure can bind to iron responsive elements (IRE) within certain mRNA’s. These IRE’s form loop structures either at the 5’ or 3’ end of mRNA’s involved in Fe ____4____.
The IRP in its Fe and non-Fe form will bind to an IRE.
If the IRE is at a 5’ it will block translation.
If the IRE is at the 3’ it will bind and stabilize that mRNA by preventing its ____5____ by mRNAases. Translation then occurs.
By this the cell regulates the translation of certain mRNA’s.
To sum up:
When Fe levels are low the mRNA of transferrin receptor 1 (TFR1) is protected leading to more translation --> more TFR1 --> more Fe reaching the cells. At the same time you repress the translation of mRNAs involved in e.g. Fe storage.
When Fe levels are high the 4Fe-4S binds to IRP1 stopping IRP1 from being able to bind to the IRE loops and so translation of the upstream mRNAs but ____5____ of the mRNAs that contain IREs in their 3’ end. Under these conditions translation of ferritin (storage) is up-regulated but TFR1 is down-regulated because not as many Fe ____6____ are needed.
Transcriptional regulation occurs with the cell.
The ____1____ factor HIF2 regulates genes such as the Fe reductase and Fe importer. HIF2 will induce the transcription of these genes under ____2____ Fe conditions.
When Fe levels become ____3____, the enzymes PHD1, 2 & 3 become active because they are Fe containing enzymes. They add an ____4____ group to HIF which causes is ubiquitination and degradation.
At ____2____ Fe levels PHD1, 2 & 3 remain inactive and in turn HIF is stable and can activate transcription.
Fe homeostasis back up system:
4Fe-4S clusters can be degradaded by oxidative stress or the binding of __1__.
So the 4Fe-4S cluster can be disassembled if there is much H202 around.
In cases where Fe levels are still ____2____ and the 4Fe-4S cluster cannot bind IRP1 because the cluster is being degraded by oxidative stress, this Fe containing complex binds to IRP and leads to the degradation of IRP1 to ensure that it is not able to bind IRE.
The protein that regulates Fe ____1____ is called Hepcidin.
Hepcidin is produced in the ____2____. It binds to ferroportin within macrophages and enterocytes in the ____2____ causing the internalization and degradation of the ferroportin.
In ____3____ Fe, Hepcidin (____3____ levels) binds to ferroportin and degrades it, thus preventing Fe importation.
In ____4____ Fe, Hepcidin (____4____ levels) doesn’t bind ferroportin, ferroportin is free to import Fe through the membrane into the cell.
What regulates Hepcidin? Signal from the liver?
Signal must be from the liver as although Fe is absorbed by the ____1____, Hepcidin regulates its uptake from the liver.
Hepcidin is regulated at the ____2____ level. There are 2 pathways that regulate the ____2____ of Hepcidin.
2.Via growth factor BMP6 (bone morphogenetic protein 6)
1. At ____3____ Fe, the Fe-transferrin complex will bind to transferrin receptor 2 (TFR2) which is predominantly only expressed in liver cells. This TFR2-Fe-tansferrin complex then binds to HFE (haemochromatosis protein). This activates the signaling molecule ERK1,2 which is phosphorylated and through an unknown mechanism activates the ____2____ of Hepcidin.
2. BMP6 binds to BMPR in conjunction with the protein HJV and together they lead to the ____4____ of the protein SMAD1,5,8 which then binds to the protein SMAD4 and then both SMAD proteins move into the nucleus and activate ____2____ of Hepcidin.
The ERK1,2 pathway affects the SMAD1,5,8 pathway.
In low Fe levels, where you don’t want much Hepcidin, Apo-TF does not bind, and instead of binding to TFR2 HFE binds to TFR1 leading to the ____1____ of the ERK1,2 pathway.
2 proteases break down HJV, either the ____2____ bound form which is broken down by Matriptase-2, or just the soluble part of HJV is released by the protease Furin to bind BMP6 and mop it up and prevent it from binding to the receptor. Both processes down-regulate Hepcidin ____3____.
Iron deficiency anaemia
Anaemia - ____1____ in haemoglobin or red cell concentration in blood.
Increased demand for iron
- ____2____ during childhood
- Treatment with erythropoiesis stimulating drugs to increase RBC resulting from chronic kidney failure, chemotherapy, certain treatments for HIV.
Limited supply of iron
- Poor ____3____
- Poor absorption – gastric resection, Helicobacter pylori infection, Crohn disease, Celiac disease, drug interference.
Increased loss of iron
- Blood ____4____, dialysis, surgery, trauma, bleeding (gastrointestinal, genitourinary, respiratory tract).
Hypochromic (reduced haemoglobin) microcytic (reduced red cell size) anaemia. Anaemia is the most common clinical presentation in general practice and hospitals.
Haemoglobin (men < 13g/dl and women <12g/dl), TSAT (<20%) mean cell volume (MCV) and mean cell haemoglobin (MCH), ferritin concentration (<30 ng/ml) but no signs of ____5____.
Disorder of iron absorption and ____1____ within the body, with a characteristic pattern of tissue damage resulting from excess iron deposition.
Most common inherited metabolic disorder in the Western world (1 in 250 of the northern European population)
Clinical presentation is variable and not confined to the classic triad of cirrhosis, ____2____, and skin pigmentation. Predominantly men (ratio of 9 to 1) between the ages of 40 and 60 years present with lethargy, weakness, and sleep disturbance or with diabetes. Early symptoms are often subtle and easily overlooked.
Increased iron uptake causes increased iron deposition in tissues:
General—skin pigmentation, lethargy, and malaise
Diagnosed on the basis of ____1____ iron stores.
Serum ____2____ concentration accurately reflects total body iron stores and is raised in the disease (> 200 ng/ml women 300 ng/ml men). Serum iron concentration and transferrin saturation are also raised (TSAT >45%).
Further assessment includes magnetic resonance ____3____ of the liver (to measure iron concentrations), liver biopsy (22 mg/g liver dry weight threshold for hepatocellular injury), and genotyping (in practice if not C282T or H63G then considered wild type as others are extremely rare).
Regular phlebotomy to lower iron stores (every week with maintenance treatments 4-8 times a year). Also, Deferoxamine as an iron ____4____.
Life expectancy normal if treatment before the development of diabetes or cirrhosis.
Hepatic fibrosis improved with iron removal.
Hepcidin levels are low due to ____5____ in: HFE (T1), HJV (T2a), Hepcidin (T2b), Transferrin receptor 2 (T3), Ferroportin (T4).
What causes Friedreich’s Ataxia?
The reduced expression of the protein Frataxin (an Fe chaperone in mitochondria) that is involved in 4Fe-4S cluster formation.
Most prevalent inherited ataxia (autosomal ____1____) affecting 1 in 50,000 (USA).
Ataxia is a loss of the ability to coordinate ____2____ movement.
Presents in young (before _3_ years). Nerve and muscle cells are particularly sensitive.
Degenerative atrophy of the posterior columns of the spinal cord which become thinner and nerve cells lose some of their ____4____ sheath contributing to progressive ataxia, sensory loss and muscle weakness.
Heart, pancreas and skeleton are also affected. The majority of patients present hypertrophic cardiomyopathy with thickened ventricular and inter-ventricular septum walls. Diabetes results from insulin deficiency as islet ____5____ cells are lost.
Frataxin gene (Chromosome 9q13) contains GAA trinucleotide repeat expansions in the first intron of the FXN gene. Therefore, does not influence protein structure but reduces the expression levels of the gene. The Frataxin protein is localized in the ____6____.
In yeast cells, low frataxin levels result in slow growth phenotype, ____1____ respiration, loss of mtDNA, sensitivity to oxidants, mitochondrial iron ____2____, low cytosolic iron, and high expression of iron ____3____ system.
FRDA patients have iron deposits in heart and deficiencies in mitochondrial complexes I, II, and III, and aconitases. Conditional knockout mice show iron sulphur enzyme deficiencies in the frataxin depleted tissues before iron ____2____ in mitochondria.
Mitochondrial iron ____2____ is a common feature of impaired iron sulphur synthesis.
Human frataxin interact with ISCU in presence of iron frataxin leading to hypothesis that frataxin is a ____4____ that delivers iron to the NSF1/ISCU complex.
Frataxin deficiency results in reduced iron sulphur biosynthesis, free iron deposits that causes ______ 5 ______ (Fenton reaction) which causes inactivation of existing iron sulphur clusters and damage to mtDNA.
5. Oxidative stress