Structural Biochemistry/Metal Levels in Eukaryotes

Overview
Transition metals like zinc, iron, and copper are common components in a many different types of proteins. These metals are crucial for living; however, surplus amounts of these metals are detrimental to cell growth and viability. Luckily there are many mechanisms that help regulate this excess of metals when required. In response to these changes of metal levels, certain genes encodes transporting of metals and storage proteins that help maintain ideal levels of each metal. Deficiencies of these metals within cells can also cause health problems. Through many different types of complementary mechanisms, cellular metal homeostasis is achieved. Although not all transcriptional factors have been understood completely, they give researchers a sense of idea that could potentially explain the linkage between metal levels, health, and diseases. Metal levels can also be altered by different types of cancers, and even by neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Thus imbalanced metal levels can further cause severe implications when diagnosed with these illnesses.

Different Types of Transition Metals
Zinc Zinc one of the vital transition metals in organisms, is important for the accurate folding of certain protein structures. It is the cofactor for hundreds of enzymes; however, in excess, zinc is very toxic to growth, and can wrongfully bind to unfitting locations in proteins and damage their function. Iron Iron is another type of transition metal that is vital to life, acting as an electron acceptor or donor in physiological reactions. Like zinc, iron is also used as cofactors in enzymes required for oxygen transportation, DNA synthesis, ribosome biosynthesis, photosynthesis and many other functions. Like other transition metals, in excess, iron is very hazardous to growth and viability. Thus many organisms have evolved specialized mechanisms that regulate cellular iron levels. Copper Found in a variety of eukaryotes, copper is a fundamental metal that is necessary for life in many organisms. Being a redox active metal, copper is a cofactor in many different proteins, including Cu/Zn superoxide dismutase 1 and cytochrome c oxidase. Like zinc and iron, when copper is in excess, it is highly toxic to cells and viability. Thus in order to maintain ideal level of copper, many eukaryotes developed transcription factors that regulate copper levels by controlling the genes that encodes for copper uptake and elimination.

Zinc Deficiency
Changes in gene expression do not only occur in excess of zinc, but in deficiencies as well. In a variety of eukaryotic species, different sequence-specific DNA-binding factors have been found that are necessary for the variations in gene expression. Studying these zinc-regulated factors can provide a means to obtain certain types of proteins and different mechanisms that can be used to detect cellular zinc limitations in other organisms not known.

Zap1
Found in budding yeast Saccharomyces cerevisiae, the transcription factor Zap1, a zinc-responsive protein, can detect zinc levels. When zinc deficiency occurs, Zap1 can initiate the expression of about 80 genes, targeting genes that are require for zinc uptake and genes that can help survival during extreme zinc deficiencies. Zap1 can detect cellular zinc levels by a variety of domains. Zinc finger pair is a regulatory domain that overlaps with AD2, or activation domain 2. During zinc deficiencies, zinc binds to the zinc fingers within the AD2, which allows AD2 to undergo a conformational change causing exposures of residue that initiates the domain function. The zinc can rapidly switch from one of the zinc finger pair to the other, which allows the possibility of Zap1 to detect cellular zinc level. AD1, another activation domain within Zap1, is independently controlled by cellular zinc levels-activating gene expression in response to deficiencies. When AD1 is combined with AD2, it allows Zap1 to recruit more coactivators under precise stress situations.

bZip19 and bZip23
In another type of organism, more than one transcription factor are required for regulating genes during zinc deficiencies. Found in Arabidopsis thaliana, bZip19 and bZip23 activates the expression of genes required for zinc uptake.

Iron Deficiency
Changes in gene expression do not only occur in excess of iron, they also occur in response to iron limitations as well and have been observed in many different eukaryotes such as green algae, fungi and plants. Most of the information that is known about gene transcription that has been affected by iron limitation is by the studying of yeasts.

Aft1 and Aft2
Found in Saccharomyces cerevisiae, Aft1 and Aft2, are transcription factors that responds to iron deficiencies by increasing the expression of about 40 genes. These genes can encode for certain proteins that require iron uptake, iron searching, or intracellular iron transport. Aft1 and Aft2 also regulate the expression of CTH1 and CTH2, genes which are iron-dependent, which help cells to conserve iron. Aft1 and Aft2 senses cellular iron deficiency by using a signal produced by the mitochondrial Fe-S cluster machinery. This signal however is still presently unknown, but studies revealed that Aft1 has the ability to sense these iron signals due to certain cytoplasmic proteins.

Php4
Found in fission yeast Schizosaccharomyces pombe, Php4, is another iron-responsive transcription factor that helps cells fight against iron deficiency. Similar to CTH1 and CTH2, Php4 controls the flux of iron through iron-depending pathways when iron is present. This is due to Php4 regulating genes that encodes for proteins that bind to iron or those found in metabolic pathways that needs iron. Transcription factor Grx4 is necessary to deactivate Php4 in high levels of iron concentration.

Copper Deficiency
In many different eukaryotes such as yeast, plants, green algae, and fruit flies, cells can activate certain genes that encodes copper uptake when deficiency of copper occurs. Many of the transcription factors required for gene activation during copper limitation have been identified; however, how they function to detect deficiency have not been identified yet. Although only a few is shown here, there are often homologs of these transcription factors that provides the same role in a variety of other eukaryotes. Thus, there are many different proteins that have evolved using the equivalent copper responsive domain to detect multiple levels of coppers needed for homeostasis.

Mac1
Mac1 is a transcription factor found in the yeast Saccharomyces cerevisiae, becoming active when copper deficiency occurs and regulating the expression of genes required for copper uptake. In order for Mac1 to become activated, it requires both the Mac1 DNA-binding and transactivation domains. The transactivation domain comprise of cysteine-rich domains that are able to bind four copper ions. In copper excess, over binding of copper to this domain disables the Mac1 transcription factor. Mac1 DNA binding activity can become maximized with addition of Sod1, a rich copper-binding protein, during copper cellular deficiency. Thus Sod1 might have some unknown ability in copper sensing.

Crr1
In another species, Chlamydomonas reinhardtii, the green algae uses a different transcription factor that regulates copper limitation called Crr1. When copper levels are low, Crr1 can activate the expression of over 60 genes to achieve needed copper levels. Like Mac1, Crr1 also contains copper regulated domains, one is a cysteine-rich metal-responsive domain, and the other is SBP DNA binding domain.

SPL7
In another species, A. thaliana, copper levels are balanced by transcription factor SPL7, which is a homolog or descendant of the same ancestor, of Crr1. In order for copper homeostasis to become obtained, SPL7 expresses genes that are required for copper uptake and intracellular copper organization during copper deficiency. Different miRNAs can become activated by SPL7 that allows targeting of mRNAs that encodes for copper-binding proteins.

Zinc Excesses
In many multicellular organisms, there are distinct systems that detect zinc excess and deficiencies. MTF1 is a transcription factor that provides protection against zinc excess, which is very toxic to cells.

MTF1
MTF1 is a transcription factor found in mammals and fishes that helps protects cells from excess of zinc, activated when high concentration is present. It binds to metal-response elements which activates the target gene expression. Similar to Zap1, MTF1 senses cellular zinc excess by using regulatory zinc-finger domains. In contrast, the zinc fingers controls the binding of MTF1- only when zinc are in excess.

Iron Excess
Studies obtained in a range of eukaryotes such as yeast, nematodes, flies and mammals, have noticed a variety of changes in gene expression due to excess of iron. Only in fungi however, have researchers identified transcription factors that are able to detect high levels of iron. Iron homeostasis is usually controlled at a post-transcription level within multicellular organisms.

Yap5
Found in Saccharomyces cerevisiae, transcription factor Yap5 protects cells from toxicity by expressing CCC1 activation when iron excess is present. Once Yap5 binds to the CCC1 prometer, CCC1 provides protection by transporting iron into an iron storage vacuole. CCC1 isn’t active unless high iron levels have been reached. Yap5 also comprises of cysteine-rich domains that are required for regulation of iron.

Fep1
This transcription factor is expressed when excess levels of iron have been reached. Found in S. pombe, Fep1 protects cells by stopping the expression of genes that acquires for iron when iron are at high concentration. Fep1 also regulates the expression of transcription factor Php4, controlling the flow of iron through metabolic pathways. Transcription factor Grx4 is required to deactivate Fep1 when iron levels are low, which is also required to deactivate Php4 when iron levels are high, which is likely the same signal that determine the switch of cells between both iron levels.

Copper Excess
In order to protect eukaryotes species such as fungi and flies from copper toxicity, certain transcriptional factors are able to detect this high level of copper, and express regulatory mechanisms that can help protect cells. These factors can directly bind to copper and gene expressions are regulated.

Ace1
Found in Saccharomyces cerevisiae, Ace1 is a transcription factor that can bind directly to copper ions when copper is in excess. The binding to copper allows the activation of genes that help defend cells from toxicity that arises due to surplus of copper. A variety of other fungi have homologs of Ace1 that can also provide the same function by protecting against excess of copper.

dMTF-1
dMTF-1, a homolog of MTF1, regulates the expression of genes that are required for copper levels to become balanced when copper levels are in excess or in deficiency. dMTF-1 also have a cysteine-rich domain that functions in sensing copper toxicity by binding to four Cu+ ions.