Structural Biochemistry/Proteins/Protein in Signal Transduction
- 1 Signal-Transduction Pathways
- 1.1 Introduction to Signal-Transduction Pathways
- 1.2 Ligand Binding
- 1.3 Signal-Transduction Cascade
- 1.4 Heterotrimeric G Proteins
- 1.5 Phosphoinositide-specific phospholipase C(PLC)
- 1.6 Serine/Threonine Kinase Transmembrane Receptors TGF beta RI and II
- 1.7 Insulin Signaling
- 1.8 Two-Component Systems
- 1.9 Connected Hubs of Signal Transduction Pathways
- 1.10 Sirtuin Regulation of Mitochondria
Introduction to Signal-Transduction Pathways
Signal transduction is the chain of events that occurs when a cell converts a message to ultimately a physiological response. The message comes in the form of a particular molecule in the cell’s environment. Some examples of signal transduction are as follows. Upon release of epinephrine in response to stress, the cells in the body receive the message, then responds by preparing to use stored energy and improving cardiac function. After a meal, insulin is released into the bloodstream, indicating to cells to take in the glucose. In a wound, epidermal growth factor is released and simulates certain cells to proliferate.
Signal transduction is an important process since it occurs through the communication between difference domains and coupling of information. It is also important to know that ligand induced conformational changes are important to many aspects of protein function. Its importance is demonstrated when an enzyme binds a substrate because the act of binding causes changes in the protein’s structure to enable catalysis.
Signal transduction is an important process since it occurs through the communication between difference domains and coupling of information. It is also important to know that ligand induced conformational changes are important to many aspects of protein function. The importance of these conformational changes is demonstrated when an enzyme binds a substrate; the process of binding causes changes in the protein’s structure to enable catalysis. In addition, the binding of allosteric ligands can lead to the occurrence of more conformational changes as well.
Ligand binding often leads to changes in both the structure and dynamics with changes in the dynamics often occurring at different locations on the same proteins. The binding of a ligand to one site can often influence the structure, dynamics, and binding affinity at another site on the same protein. This demonstrates that coupling can often be achieved through many different ways. Many times, the effects of ligand binding on protein activity can be explained in terms of more common thermodynamic concepts; the fact that the transition of an active versus resting state depends on several factors such as the free energy difference between the two states, the protein affinity of the ligand, and the ability of a ligand to induce a transition once fully bound to the protein. In the case when a ligand binds to both an activated and resting conformation, incomplete activation often results. A ligand is considered to trigger activation when it binds with higher affinity to an active versus an inactive state.
The free energy difference, ΔGgap, can also be modified by environmental factors such as membrane composition. An example of this is demonstrated by the fact that the voltage dependent potassium channels exist in equilbrium only in an “on” or “off” conformations. These differ not only in their conductance characteristics but also in the number of charged groups on the two sides of the electrically impermeable membrane.
The K+ channels and M2 proton channels both share some similarities in their thermodyamic and structural coupling properties. For example, they are similar in the sense that both channels have dual gates to regulate the ion diffusion into the channel versus through the channel. In addition, they are able to achieve a high selectivity by binding multiple copies of permeablet ions. The entry of ions through channels is also very tightly regulated to prevent leakage; leakage can be harmful to the life of the organism.
Signal transduction relays a message to a certain physiological response by way of certain key steps. In the first step, an event or condition stimulates the release of the signal molecule, otherwise known as the primary messenger. Generally, primary messengers do not enter cells, and thus work by binding to the cell’s membrane protein on the extracellular side. In the second step, the primary messenger binds to a receptor protein that spans across the membrane, causing conformational change. In the third step, the signal is relayed into the cell by way of conformational change on the receptor protein. This initiates a change in concentration of certain small molecules inside the cell. The small molecules are called second messengers, and they relay the signal inside the cell by activating other receptor-ligand complexes within the cell. In the fourth step, the secondary messengers activate the effectors that directly produce a physiological response. This physiological response can be activation/inhibition of enzymes, membrane channels, or gene-transcription factors. In the final step, after the physiological response is completed, the signal is terminated
Second messengers provide certain advantages for the signal transduction. A signal can be amplified significantly by generating second messengers. Small amounts of membrane receptors can be activated, but large amounts of second messengers can be generated. Each activated receptor can produce many secondary messengers. Low concentrations of primary messengers in the extracellular environment can give rise to a large signal due to amplification by secondary messengers. Also, second messengers are able to influence other processes in the cell by diffusing to other compartments. In addition, a common secondary messenger can signal for multiple pathways. This is called cross talk.
Heterotrimeric G Proteins
Epinephrine produces signals by binding as a ligand to a membrane protein called β-adrenergic receptor, which from a class of receptors called the seven-transmembrane-helix receptors. These receptors contain seven helices that cross the membrane seven times, thus also referred to as the serpentine receptors. The binding of a ligand on the extracellular side induces a conformational change on the seven-transmembrane-helix receptor on the cytoplasmic side. The conformational change on the intracellular side of the receptor activates G protein. Activated G proteins then binds to and promotes the activity of adenylate cyclase, which is a membrane bound enzyme that converts ATP to cAMP. cAMP can then move across the cell as the secondary messenger to initiate physiological response. cAMP activates protein kinase A (PKA). Activated PKA activates other proteins that directly produce a physiological response.
Unactivated G protein is bound to GDP and exists as a heterotrimer protein, consisting of the α-, β-, and γ- subunit. The GDP is bound to the α-subunit. To activate the G protein, the GDP is released and GTP binds to the α-subunit. Once GTP binds, the α-subunit dissociates from the βγ dimer. The activated α-subunit then binds and activates adenylate cyclase. The α-subunit has an intrinsic GTPase that slowly hydrolyzes the bound GTP to GDP. Once hydrolyzed to GDP, the α-subunit is deactivated and reassociates with the βγ dimer. The deactivation of G protein is a time dependent process, based on the kinetics of the intrinsic GTPase. Aside from activating the cAMP cascade, the seven-transmembrane-helix receptor can also activate the phosphoinositide cascade. There are different types of G proteins. The β-adrenergic receptor functions with the Gs protein. The angiotensin II receptor activates the Gq protein. The mechanism of activating the G protein is the same in both cases. However, with the Gq protein, the α-subunit activates the enzyme phospholipase C, which catalyzes the cleavage of phosphatidylinositol bisphosphate on the membrane. Inositol trisphosphate and diacylglycerol is formed. Inositol trisphosphate diffuses away from the cell membrane and bind to the endoplasmic reticulum membrane. The calcium ion channels are opened and calcium ions enter the cytoplasm. Calcium ions are signaling molecules and ultimately stimulate release of vesicles and contraction of smooth-muscles. Diacylglycerol remains in the cell membrane, where it helps activates protein kinase C. Calcium ions are also needed to activate the protein kinase. Once activated, protein kinase C activates certain proteins by phosphorylation to produce physiological responses.
Calcium ion is a secondary messenger in many signaling processes because of several properties. The changes in calcium ions in the cell are easily detected. The calcium ion concentration inside cells is kept at a low level to avoid precipitation. Once calcium ions are released from the endoplasmic reticulum, the concentration of calcium ion in the cell increases by several orders of magnitude. This increase is readily felt by the cell. Calcium ions also bind tightly to proteins and induce a conformational change. Calcium ion can coordinate with several negative charged amino acid residues and thereby inducing a conformational change to activate proteins.
Phosphoinositide-specific phospholipase C(PLC)
PLC is an type of enzyme that binds to the inositol phospholipids in eukaryotes by hydrolyzing lipid phosphatidylinositol 4,5-bisphosphate and creating inositol 1,4,5-trisphosphate and diacylglycerol (DAG). The importance of PLC is its ability to stimulate hosphoinositide metabolism and calcium signaling.
PLC is complex and are capable of covering a wide domain of protein. There are three subtypes of PLC: β, γ, and δ. Studies have shown that the DNA structure of δ were first found in single-celled eurkaryotes which are now similar to yeast, fungi, and mold. On the other hand, the β and γ were found to be more similar between plants and animals.
These enzymes are used for catalysis and since PLC have modular domains, they form catalyic α/β barrels from the X and Y regions. At the end of the barrel, there is catalytic and hydrophobic residue that allow substrates to come in and out of the mouth of the barrel. PLC hydrolyzes oxygen and phosphate bonds that contribute to binding phosphoinositol to DAG. This is done by the substrate forming the cyclic 1,2-phosphodiester intermediate and from here, catalysis begins.
PLC regulates cellular activity such as the binding of G protein subunits, the Rho and Ras of the GTPases, lipids, and tyrosine kinases. These enzymes have certain properties that allow them to regulate protein. Its structure was made to target PLC isozymes which led to the ability to solely control what the PLC does during protein-protein or protein-lipid interaction. During this process,
Serine/Threonine Kinase Transmembrane Receptors TGF beta RI and II
Serine/Threonine kinase's are enzymes that catalyze the addition of a phoaphate group to a serine or threonine (which have similar side-chains). Many of these receptors are vital to signal pathways that result in the alteration of gene expression. These receptors have two parts that are separated when they are not in contact with their extracellular. Once the signaling molecule complexes to the correct part of the receptor, a conformational change in this part of the receptor enables it to complex to the second separated part of the receptor. This form of the receptor-signal complex also activates the enzymatic activity of the cytoplasmic part of the receptor which results in a signaling cascade. Figure 1 shows these steps for the particular example of Serine/Threonine Kinase Transmembrane Receptors TGF beta RI and II.
One particular set of Serine/Threonine Kinase Transmembrane Receptors are TGF beta receptors I and II (TGF beta RI and II). The signal for these transmembrane receptors is Transformation Growth Factor beta (TGF beta), a cytokine that contols many numerous cellular responses like proliferation, differentiation, apoptosis and migration. In this case, before the signaling molecule can bind its receptor it needs to be activated. Activation of TGF beta is necessary since it is usually secreted from the source cells as an inactive complex that is referred to as the large latency complex (LLC) composed of TGF beta, latency associated peptide (LAP), and latent TGF beta binding protein (LTBP). When TGF beta is within this complex it is not able to bind to its receptors, TGF beta RI and II.
One mechanism by which TGF beta is released from the LLC involves the aid of integrins and the extracellular membrane (ECM) component fibronectin (Figure 2). In this mechanism the LTBP anchors to fibronectin in the ECM. Then, the LAP portion of the LLC anchors to an integrin. After this, a pulling force generated by a part of the cytoskeleton that is associated to the integrin protein results in a conformational change of the part of the LLC that is bound to the integrin. This results in TGF beta to be released from the LLC and will eventually find its receptors TGF beta RI and II.
There is a class of signal-transduction cascade that uses a receptor that intrinsically contains a protein kinase. One example of this type of signal-transduction cascade is insulin. The insulin receptor is composed to two identical chains connected by disulfide bonds. The receptor has an α-subunit on the extracellular side of the plasma membrane. This receptor extends across the membrane, where the β-subunit lies in the intracellular side. Insulin binds to its receptor by interacting with the α-subunit. With the two ‘arms’ made from identical chains, the α-subunit essentially wraps around insulin. The β-subunit on the cytoplasmic side primarily consists of a tyrosine kinase, which transfers a phosphoryl group from ATP to tyrosine residues. The tyrosine kinase is intrinsic to the receptor, and thus the insulin receptor is often referred to as the receptor tyrosine kinase.
The insulin receptor is activated when the α-subunit wraps around insulin. When the α-subunit closes around insulin, it causes the β-subunits in the intracellular side to come together. When the two ‘arms’ of the β-subunits come to close proximity, the intrinsic tyrosine kinase becomes active. The tyrosine residues on the β-subunit become phosphorylated, causing a dramatic conformational change on the intracellular end of the receptor. Phosphorylating tyrosine on the receptor also serve to generate docking sites for other substrates, such as insulin-receptor substrates (IRS). Upon docking, the tyrosine residues on IRS are phosphorylated by the receptor. In this form, IRS works as an adapter protein, where IRS binds to lipid kinases and moves them to the membrane. The lipid kinase phosphorylates phosphoinositol bisphosphate to generate phosphatidylinositol trisphosphate. This phosphorylated lipid then activates a protein kinase PDK1, which then also activates another protein kinase: Akt. All of the kinases mentioned above are either anchored to the receptor or the membrane, except for Akt. Akt can move across the cell and cause the movement of glucose transporter GLUT4 to the cell membrane. Once at the membrane, GLUT4 can transport glucose from the extracellular environment into the cell.
To terminate the signal, the activated receptor is returned to its deactivated state. Specifically, the phosphorylated tyrosine residues on the receptors need to be have the phosphoryl group removed. However, the phosphorylated residues are stable and do not spontaneously hydrolyze back to their original form. Specific enzymes are used to hydrolyze phosphorylated proteins and convert the protein back to their inactive form.
Bacteria and archea utilize a two-component system for signal transduction. These systems are absent in animals, and serve as an interesting source for developing antibacterials. The two component system mainly contains a membrane-bound sensor histidine kinase in addition to a response regulator that targets which genes the bacteria expresses in response to certain stimuli. Furthermore, they are linear signal transducers, modifying and amplifying transductions by adding extra modules and using extra proteins called connector proteins. Signal transduction occurs with the phosphorylation of a histidine kinase residue. The first step in phosphorylation is histidine's autophosphorylation. The γ-phosphate is attacked by the exposed histidine in the DHp domain, and forces the CA domain to undergo different positions with respect to the histidine residue. Also, histidine phosphorylation is prevented with the binding of the RR to the ATP lid. This fixes the lid's position in between the nucleotide and histidine, preventing phosphorylation. In the entire histidine kinase, there are 2 mobile phosphorylating domains (otherwise known as CA domains) and 2 phosphoacceptors histidine residues. Evidence for both cis-autophosphorylation and trans-autophosphorylation have been seen, showing that this reaction and undergo both types. Current assumptions on determining cis or trans is based on the length of the hinge between the DHp and CA domains and the connection between the helices in the DHp domain. This knowledge can be obtained by studying phosphotransfer systems lacking autokinase activity, also called histidine phosphotransferases (HPts). Observations studying this system can transfer over to histidine autophosphorylation because the active center for phosphoryl transfer is very similar in both systems. Therefore, the data obtained from this system can be applied to any two-component system. The phosphatate reaction is when the histidine kinase component catalyze the dephosphorylation of the P~RR, essentially the reserve of RR-phosphorylation. Phosphotransfer and phosphotase reactions occur in different complexes due to their opposite nature. If they were to react in the same complex, they essentially cancel each other out and render the signal ineffective. Not all histidine kinases can catalyze this dephosphorylation, and those that cannot rely on a protein to assume this role. Histidine kinases have antiparallel transmembranes helices that aids in signaling from the membrane to the cytoplasm. Signal receptors on the membrane can trigger the helices into a combination of movements that relays the signal further deeper into the cell. Many complexes also have additional domains such as PAS and HAMP modules in between. For example, the HAMP module is composed of 4 helices in a bundle that contributes to the movement of signals by altering its structure in helical rotation and twisting. There remains much to be learned about two component systems, such as determining a fine definition of what causes cis and trans autophosphorylation. The roles of many different proteins in their structure also remains a mystery. In order to reach these discoveries, more detailed imaging techniques needs to be developed to view the structures on an atomic level.
Connected Hubs of Signal Transduction Pathways
Components of signal transduction pathways have become connected hubs, which bind to specific partners depending on factors such as affinity. One of the hubs is Ras and it is affected by change (no matter the pace and size of the change) which allows information to be carried out. Ras is very complex in recognizing its structure and it makes it difficult to create a model of Ras joining to its effector. By understanding its model, we are to understand the biological what Ras does such as how it specific about binding domains of its effectors.
Sirtuin Regulation of Mitochondria
A sirtuin is a very conserved group of proteins that can increase the life span of simple organisms, and control the metabolic and stress pathways. In mammals there are seven sirtuins of which 3 are located in the mitochondria.
What is a mitochondrion?
Mitochondria are very versatile organelles that function as the primary site of oxidative phosphorylation and plays a role in apoptosis and intracellular signaling. Mitochondria can modify their functions, morphology and cellular proliferation depending on extracellular conditions. The mitochondria have their own set of DNA which is referred to as mtDNA and codes for proteins that are involved in electron transport and ATP synthesis.
Sirtuins and the mitochondria
Sir 2 or Silent information regulator and its orthologs SIRTS 1-7 are called sirtuins. The seven in mammals have been conserved in the sirtuin domain in DNA. The sirtuins are used as regulatory proteins in the mitochondria that bind to NAD+ which is a co-factor in the for the proteins the sirtuins are bound to. The activity level of sirtuins is directly related to the increase in levels of NAD+
Sirtuins and apoptosis
“Apoptosis is the cellular process of programmed cell death. Mitochondria play an important role in apoptosis by the activation of mitochondrial outer membrane permeabilization, which represents the irrevocable point of no return in committing a cell to death (article).” There is no clear line on how sirtuins controls apoptosis but it can seen that when a cell does not have SIRT 3 then there is less likely that the cell will be stress induced apoptosis, showing that sirtuins are important to apoptosis.
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