Microtubule assembly and disassembly is regulated by their interactions with associated proteins which are specific to cell types and locations. It is important this regulation takes place as microtubules play an important role in cytoskeleton stability, cell shape, and also for the transport of organelles, vesicles and chromosome movement when the cells needs to respond to external/internal stimuli. Two examples of proteins that regulate microtubules are tau and stathmin.
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Tau is a MAP (microtubule associated protein). It is coded by the tau gene on chromosome 17. It is found in neuronal cell bodies and specifically regulates microtubule growth out of the cell body and into the axons. It therefore plays an important role in neuritis growth and also forms the skeleton for vesicle/organelle transport in neurons which is important in signal transduction. It is also believed to stabilize the axonal core by increasing microtubule interactions with other intracellular components e.g. actin filaments. Tau comes in different forms which are found in different locations such as spinal cord, dorsal root ganglia and also foetal and adult brain. There are 6 different isoforms of tau in adult brain which is a result of alternative splicing of mRNA.
Tau is a hydrophilic, soluble unfolded protein which has a molecular mass of 36-45 kDa. It is 90nm in length and is rod shaped. It consists of up to 352-431 amino acids which join via peptide bonds to form the polypeptide. As it is a protein it has an N terminal (NH2 group) and a C terminal (COOH group) which contributes to its ability to promote microtubule assembly.
The C terminal contains a conserved binding domain which is 55nm in length and is composed of either 3 repeats of 18 positively charged amino acids (foetal brain) or 4 repeats (adult brain located between 237-367 amino acid). These repeats align alongside the microtubules and are separated by 13/14 amino acid linkers.
The linkers, who do not themselves bind to the microtubules, serve a function in positioning the binding repeats on the microtubule. They also give tau’s structure a degree of flexibility and could help limit microtubule assembly as it enables tau to displace from the microtubules more easily than if the whole region was bound to it.
The role of the repeats is to bind tau to the tubulin in microtubules. It is in these repeat regions where there is highest affinity and free energy is delocalised (-10.7kCal/mole) leading to a great degree of binding. The high affinity is due to weak interactions between small amino acid groups, such as van der walls forces and ionic attractions. On each residue addition, the binding energy increases by 0.336kj/mole as interactions increase. Therefore adult brain which is composed of 4 repeats is more strongly bound to microtubules that foetal brain which may increase microtubule assembly. They way in which it does this is not fully understood, but we know that microtubule assembly requires a critical concentration of tubulin to be above and that tubulin will only bind to the +ve end of the microtubules if it is capped by stable GTP. Thus tau may bind to the alpha beta tublin and prevent the hydrolysis of beta GTP bound tubulin to unstable GDP which would cause the depolymerisation of tubulin and for microtubule subunits to disassemble from the -ve end. Therefore as long as there is no hydrolysis of beta GTP bound tubule the cap tubulin can polymerize onto the end, leading to microtubule growth. Tau is therefore present in cell bodies so that microtubule assembly can occur so that it projects out to form the axonal core for vesicle and organelle transportation.
Further upstream towards the n terminal is a proline-gylcine-glycine rich region which causes a sharp turn in the polypeptide which causes the N terminal to project away from the microtubule. It also increases the flexibility and elasticity of tau which contributes to its thermodynamic stability.
There is also a site which is targeted by protein kinases in the cell which form part of the intracellular signalling cascade. The site is thus known as a phoshorylation site and is called KSP as it is rich in serine lysine and proline which undergo phosphorylation. When this occurs the structure of tau changes and it can no longer bind to microtubules, therefore assembly decreases and reduction in depolymerisation is no longer reduced. This could lead to possible shrinking of the microtubules as the stable beta GTP tubulin cap could be hydrolysed so no more tubulin polymerization can take place and subsequently subunits are lost at a greater rate and the microtubule disassembles. This could decrease cytoskeleton stability and transport within the cells.
The n terminal is a nonbinding short projection that points away from the microtubules. It binds the microtubule to other cell components such as other microtubules/actin. Therefore tau plays a role in increasing the cytoskeletons stability. The free energy of the total protein is 4.4 fold smaller than when just the binding domain is considered, indicating the n terminal region can modulate binding of tau to the microtubule therefore decreasing rate of assembly.
Over expression of the gene coding for tau has been reported to cause neurofribrilar aggregation in Alzheimers patients, possibly due to too much tau, thus too many interactions between fibrils in the brain leading to aggregation. Therefore tau also plays an important function in disease states.
Unlike tau, stathmin is a phosphoprotein coded by the stathmin gene on the p arm of chromosome 1 and is a moledcular weight of 17kDa. It targets free tubulin in cells and sequesters it, reducing tubulins ability to polymerize onto the end of microtubules, thus decreasing its ability to grow. Stathmin is therefore very important in controlling microtubule dynamics especially in cell cycle which will be explored later.
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Stathmin contains 60% helical structure at its c terminus which initiates at Lys 41- 75. The alpha helix is 14nm in and is composed of 90 amino acids forming a rod shape. Stathmin therefore has a secondary structure which does not appear to be present in tau. Based on knowledge of alpha helix, it is either right/left handed coil with a polypeptide backbone with the hydrophilic amino acid side chains extending out. The coil is stabilized by electrostatic attraction, disulphide bonds and van der walls forces. Most of all, hydrogen bonding that lye’s parallel to the axis and forms between the C=0 group and the N-H group of another amino acid 4 resides later, makes the helix stable. There are 3.6 residues per turn. The helix is the region (stathmin like domain SLD) that binds the protein to the microtubule. Upon binding a stable curved T2S complex is formed. This is a ternary complex formed from 1 mole of stathmin to 2 moles of tublin in a head to tail arrangement. The curved nature of the complex and the formation of the heterodimer, inhibits tubulins ability to add to the +ve end of the microtubule and polymerize, thus microtubule growth is reduced and rate of GTP hydrolysis could overtake, leading to microtubule shrinkage. Stathmins ability to do this means it plays an important role in the cell cycle as high levels of stathmin promotes rapid shrinking of the mitotic spindle formed from microtubles, enabling cell division. Thus stathmin plays a role in cell division
Intraceullar protein kinases also target stathmin by phosphorylating 4 residues ( SER 16, 25, 38, 63). Phosphorylation of this amino acids cause conformational change in the protein as the SLD region changes from a structures helix to a random coil, leading to a 10 fold decrease in binding to tubulin. Therefore there is more free tubulin which can add and ploymerize to the beta GTP stable +ve end of the microtubule, leading to mictrobule aseembly. The phosphoylated stathmin therefore has an important function in creating the mitotic spindle that radiate out from the centrosomes from either end of the cell so that daughter chromosomes can be pulled apart and separated so cell division and cell pliferation can take place.
In fact, it has been reported that over expression of this gene perhaps due to mutation,can increase risk of some caners such as breast cancer. In 50 primary breast tumours, 35 showed over expression of the gene, 5 of which showed 6 times the normal levels of stathmin. Unphosphorylated stathmin therefore can lead to uncontrollable microtubule turn over and cell proliferation which is seen in cancerous cells. However, if stathmin could be manipulated so that it either does / does not phosphorylate then it could be used as a therapeautic agent to treat cancer as microtubule assembley and disaasembly cant take place thus the mitotic spindles could not function properly leading to reduced uncontrollable cell proliferation.
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