BIOCHEMISTRY AND MOLECULAR BIOLOGY
BIOCHEMISTRY AND MOLECULAR BIOLOGY
the regulation of metabolism by external factors that provide communication between cells. The vital activity of each individual cell is subordinated to the needs of the organism as a whole. An animal cannot exist without a complex system of intercellular communications, which are carried out through a diverse system of commands.
Now it turned out that the external control of cells is much more difficult than one might imagine. The mechanisms that carry out individual types of regulation are known, but it is not completely clear how all regulatory factors are combined into a single system in the whole organism or at least in a single cell. Communication between cells is provided by signaling molecules. They are released from some cells and migrate to others equipped with receptors capable of receiving these signals. These are target cells. Binding of the signaling molecule to the receptor leads to a biochemical response of the cell.
The signal can be transmitted by direct contact between two cells. An example is the activation of a helper T cell by an antigen-presenting cell. The presence of gap junctions or regulated pores between neighboring cells allows molecules to migrate from one cell to another, carrying out direct communication between cytoplasms and coordinating cellular functions.
✵ What types of cellular activity are controlled by external signals?
✵ What types of signaling molecules are involved in chemical signaling?
✵ Which cells secrete signaling molecules. How is their release controlled and how do they reach their target cells?
✵ How do target cells recognize signals?
✵ How does the cell respond to the received signal?
What types of cellular activity are regulated by external signals?
For convenience, they can be divided into two groups.
Regulation that does not affect gene expression. Examples are: changes in the activity of enzymes involved in the metabolism of fats and carbohydrates
Regulation affecting gene expression. Most external control factors operate in this way. Since the regulation of gene expression affects almost all processes occurring in cells, the list of such processes is endless. In animals, gene expression is controlled primarily at the level of initiation of gene transcription, and this, in turn, depends on the activity of specific transcription factors
Thus, the bulk of this chapter deals with the mechanisms by which external control factors affect the transcription of specific genes. Most of the external signaling molecules, when delivering their signals, do not enter the cell, but interact with the external domains of membrane-bound receptors. Therefore, questions arise: how do signals perceived outside the cell membrane lead to certain events inside the cell (transmembrane signaling)? How is the signal generated on the cytoplasmic side of the membrane transmitted to the controlled site? In the case of gene control, this means signal transmission from the cytoplasmic membrane to the cell nucleus. In addition, a small number of lipid-soluble signaling molecules can pass through the lipid bilayer into the cell, where they contact intracellular receptors,
Receptor-mediated signaling. Water-soluble signaling molecules cannot pass through the lipid bilayer. They bind to the outer domains of the receptors. Lipid-soluble signaling molecules, such as steroids and thyroxine, enter the cell directly and bind to intracellular receptors. Some intracellular receptors are found in the nucleus; steroid receptors, after binding to a ligand, move into the nucleus. In the case of nitric oxide, the intracellular receptor produces cGMP, which can cause direct metabolic effects.
What are signal molecules?
From a chemical point of view, these are proteins, large and small peptides, steroids, eicosanoids, catecholamines, thyroxine and nitric oxide. The first six groups of substances include: insulin, glucagon, vasopressin, sex hormones, prostaglandins and adrenaline.
The biological classification includes the following groups of regulators:
1) hormones;
2) growth factors and cytokines;
3) neurotransmitters.
Hormones
These are “classic” signaling molecules, most of which have been known for a long time, probably because they are found in relatively large amounts in the body. Hormones are formed by cells that specialize in their production and are assembled into glands that secrete hormones directly into the bloodstream, and not into ducts that communicate with the external environment (as happens when pancreatic digestive enzymes are secreted into the intestines). Therefore, the endocrine glands are also called ductless glands. Hormones "find" their target cells with the help of the circulatory system, reaching tissues located at a considerable distance from the hormone-secreting gland. A large number of hormones are known and their biological effects are described in detail. The main hormones and their action are shown in table 26.1.
Growth Factors
A recent paper suggested that this class of signaling molecules would be more appropriately called developmental regulatory factors, as their role becomes clearer when viewed as developmental regulators rather than growth-promoting factors.
Growth factors are regulatory proteins secreted by cells of the same tissue to which they belong, for example, hepatocytes, lymphocytes, etc. Growth factors interact with the cell's external receptor. The platelet-derived growth factor ( PDGF) was the first to be discovered, which, as it turned out, is also formed in many other cells. Another classic example is the epidermal growth factor ( EGF; epidermal growth factor). Many different factors have been found. About 20 of them are involved in the regulation of the development of hematopoietic cells in the bone marrow. Many growth factors are colony-stimulating factors ( CSF; colony-stimulating factors), or interleukins. They got their name for stimulating the growth of colonies of certain leukocytes under experimental conditions. They are named interleukins "in honor" of leukocytes, which influence other leukocytes through secretion. Most growth factors were discovered due to their mitogenic (growth) effect on cells, but the action of growth factors is much more complicated. Under certain situations, the same factor can stimulate or inhibit cell differentiation, having a completely different effect on the same cell. That is why the term developmental regulator seems more appropriate to describe these regulators, but we will use the generally accepted term - growth factors.
The term cytokine is used by immunologists for growth factors involved in the immune response.
Despite the variety of biological effects, all growth factors interact with external cell receptors and indirectly affect the initiation of transcription of specific genes.
In addition to the characteristics mentioned above, how does a growth factor differ from a hormone? As already noted, hormones are released into the circulatory system and are carried throughout the body, reaching their target cells. Most growth factors are paracrine in their action: they diffuse over a short distance and act locally only on nearby cells. Although there are some that act autocrine, stimulating the cells that secrete them (for example, interleukin-2 stimulates T-cell proliferation), in a different way; The release of insulin and glucagon from the pancreas directly depends on the level of glucose in the blood.
Regulation of growth factor release
Relatively little is known about it, and it is possible that multiple mechanisms control the release of these factors. Damage to the epithelial layer of blood vessels leads to platelet rupture and release of PDGF, which stimulates cell division and repair; but PDGF is also produced by many other cells in the body. In the immune system, activation of B and T cells causes the release of growth factors (cytokines) that stimulate their proliferation. Growth factors can stimulate cells to release other growth factors. Briefly, growth factors are involved in cell development and repair processes and in defense mechanisms such as the immune response, which includes extensive control of cell proliferation and differentiation.
Regulation of neurotransmitter release
A nerve impulse passing along the axon of a nerve cell causes the release of neurotransmitters through exocytosis from the vesicles of the nerve endings. The mechanism for this release will be described later.
Removal of signaling molecules
The essence of control lies in its reversibility. The isolated signal molecules must be removed, otherwise, the original signal would last indefinitely. A classic example of the removal of signaling molecules is the destruction of acetylcholine by acetylcholinesterase present in nerve synapses. Signal removal is necessary to prepare the synapse for the perception of the next nerve impulse. For acetylcholine, this occurs through the following reaction:
Catecholamines released from nerve endings are taken up by neighboring cells or removed through metabolic destruction.
How are signals recognized by target cells?
As already noted, for each signal, target cells have receptors on the outer surface or inside the cell. Signal molecules bind to them, causing changes in the conformation of the receptor protein (or proteins), which leads to a specific response. Several hormones (steroids, thyroxine, and oxide
nitrogen) are lipid soluble and enter directly into the cell, directly through the lipid bilayer, binding to intracellular receptors. The structures of thyroid hormones and two steroid hormones are shown in Fig. 26.4. The remaining signaling molecules are water soluble and bind to receptors on the surface of the cell membrane.
Structures of hormones
Structures of Tyrosine and Thyroid hormone
Structures of two steroid hormones
How does ligand binding lead to a cellular response?
Intracellular receptor-mediated responses
Steroid hormones regulate the expression of specific target cell genes at the level of transcription initiation. These include glucocorticoids, estrogen, and progesterone. These lipid-soluble hormones easily pass through the lipid bilayer of the cell to their receptors.
The estrogen and progesterone receptors are located in the nucleus, and the glucocorticoid receptor is located in the cytoplasm. They all have zinc as their DNA-binding domain and are complexed with heat shock proteins (Hsp). As we remember, heat shock proteins are chaperones, involved in folding and maintaining a certain conformation of a number of proteins.
The “zinc finger” of the glucocorticoid receptor is the best studied, so we will describe it as an example. In the cytoplasm, the receptor is associated with a complex of heat shock proteins that shield the nuclear signaling label (a specific peptide sequence).
When the glucocorticoid hormone binds to a specific site on the receptor, the heat shock protein complex dissociates from it, exposing the signaling label, and the receptor is transported to the nucleus. There, it binds in the dimeric form to the glucocorticoid-responsive DNA element. The latter is represented by a palindrome, each half of which is connected to two "zinc fingers" of the receptor dimer molecule.
Gene activation by steroid hormones. As an example, the glucocorticoid receptor is taken from the superfamily of steroid hormone and thyroxin receptors, which form complexes with heat shock proteins (Hsp) and bind with zinc fingers to DNA regions.
In the case of other steroid hormone receptors, the nuclear signaling label is not shielded by the heat shock protein complex, so that the entire structure is transported to the nucleus. But until Hsp is released during hormone binding, DNA binding does not occur.
Membrane receptor-mediated responses
Numerous intercellular connections in the body are carried out through membrane receptors. On fig. two types of such receptors are given. In the first case, ligand binding activates the internal domain of the receptor; in the second case, ligand binding causes receptor dimerization, which is accompanied by activation of internal domains. How the signal generated on the cytoplasmic side of the membrane is transmitted to the control site is a complex issue, so we will first look at membrane receptor-mediated responses and then get acquainted with the individual signaling pathways leading to cellular responses.
Transmembrane signal transduction by receptors. a - Binding of the ligand to the receptor causes an allosteric change in the cytosolic domain of the receptor protein molecule; b - ligand binding leads to dimerization of membrane receptors. This mechanism is applicable to receptors with tyrosine kinase activity. The binding of two cytoplasmic domains results in a biochemical response
Summary of Membrane Receptor-Mediated Responses
The main subject of discussion in this section is protein phosphorylation by the action of an ATP-using protein kinase. It is difficult to exaggerate the importance of protein phosphorylation in the processes of cellular regulation: in animals, phosphorylation is involved in the implementation of hormonal control, regulation by growth factors, regulation of cell cycle phases by cyclins, and control of smooth muscle contraction. On fig. the basic principles of regulation by means of phosphorylation are shown. It is worth referring again to Fig. 12.8, illustrating the central role of phosphorylation in the conduction of external signals.
The basic principle of regulation for most external signals. Phosphorylation of a protein changes its properties, resulting in a specific cellular response. Note that phosphorylation can cause the activation or deactivation of various proteins. External signals ultimately affect the activity of protein kinase and protein phosphatase
From the foregoing, it clearly follows that in most cases, receptor-mediated signaling involves the activation of protein kinases. Dephosphorylation of phosphoproteins is catalyzed by protein phosphatases and this step can also be controlled. Summarizes the relationship of regulatory systems mediated by membrane receptors. The diagram also shows the reversibility of the action of the signal by destroying the second intermediary.
Repeated exposure of the signal to the target cell often results in a weaker response. In the case of one class of adrenaline receptors, regulation according to the principle
feedback is accompanied by phosphorylation of the receptor protein and a decrease in sensitivity (this is called desensitization ) of the receptor to the ligand. To avoid confusion, we note that for other types of receptors, which will be described later, phosphorylation is an integral part of the activation process. Another type of response modulation is to control the number of functional receptors in the membrane. In the case of the insulin receptor, the insulin-receptor complex is taken up into the cell during endocytosis. The process of returning the receptor back to the membrane is controlled by the number of receptors in it. This may contribute to a decrease in the cell's response to this signal and, thus, weaken the action of the regulatory system; the latter is known as down regulation, or downregulation (eng. downregulatiori).
Description of individual signaling pathways within the cell linking receptor activation to cellular response
cAMP mediated pathways
we were already familiar with the regulation of metabolism by altering protein kinase activity under the action of cAMP, in this section we will discuss how the level of cAMP itself changes in the cell and how cAMP regulates gene expression.
Many primary messenger hormones, including adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), gonadotropins, thyroid stimulating hormone (TSH), parathyroid hormone, glucagon, catecholamines (adrenaline norepinephrine), and somatostatin, use cAMP as a secondary intermediary.This list, by no means exhaustive, is provided only to demonstrate the extremely diverse effects of cAMP on different cells. The system works because the cAMP response in a given cell matches the signal that the cell is able to pick up on its specific receptors. Thus, in cell A, signal X increases the level of cAMP, which causes cellular responses corresponding to signal X. Cell B does not have receptors for signal X, but there is for signal Y; they also increase the level of cAMP, but in the B cell this causes responses corresponding to the Y signal.
We will discuss two cases associated with the manifestation of these effects: 1) how the binding of the signaling molecule to the receptor controls the level of cAMP in the cell; 2) how cAMP affects gene transcription.
Control of cAMP levels in cells
cAMP is formed in the cell from ATP by the action of the enzyme adenylate cyclase, localized on the inner side of the cell membrane
Let's look at a typical example of the activation of adenylate cyclase by adrenaline, resulting in the formation of cAMP in the liver and skeletal muscle. The receptor (in this case, β 2 -adrenergic receptor) is a protein whose polypeptide chain crosses the membrane 7 times. Each of the 7 transmembrane segments contains about 19 hydrophobic amino acids. The latter form an α-helical structure sufficient to span the hydrophobic lipid bilayer of the membrane. Adrenaline "falls" into the gap between the transmembrane coils.
The regulatory G-protein is associated with the inner (cytoplasmic) surface of the receptor protein, which has 3 subunits: α, β, and y. Since these are 3 different polypeptides, the protein is called a heterotrimeric G protein, or simply a trimeric G protein. The α-subunit has a site that can bind either GTP or GDP. When it is busy with GDP, nothing happens (Fig. 26.11, a).
When an adrenaline molecule binds to a receptor, the cytoplasmic domain of the latter undergoes a conformational change. This, in turn, causes a conformational change in the associated G-protein. The latter causes the G protein (named G s in this system ; "s" stands for stimulatory) to exchange GDP for GTP. The G protein cannot make this exchange until it is bound to a hormone-bound receptor. The α-subunit-GTP complex detaches from the G-protein, migrates to the adenylate cyclase molecule and activates it, which leads to the formation of ATP (Fig. 26.11, b, c).
Thus, the hormone stimulates the synthesis of cAMP, using the G-protein as an intermediary (see Fig. 26.11, c). Activation of cAMP formation by the α-GTP subunit must be limited in time: otherwise, a single hormonal stimulus will act indefinitely. If the receptor no longer binds the hormone, cAMP production should cease; hydrolysis of cAMP in the cell by phosphodiesterase completes the process (see Fig. 26.9, b).
The situation is similar to what happens on a staircase with a limited lighting time: when you press a button, the light comes on, and then the button starts to slowly return to its original position - and the light turns off after a minute or two. From time to time you have to press the button so that the light does not go out. The G-protein is precisely the "timing device" that limits the activation time of adenylate cyclase.
The α-subunit of the G protein in GTP-bound form (which activates adenylate cyclase) has enzymatic GTPase activity. It hydrolyzes GTP to GDP and Pi (Fig. 26.11d) . GTPase activity is low, so GTP hydrolysis does not occur immediately. As soon as GDP is formed during hydrolysis, the α-subunit returns to its original state. It dissociates from adenylate cyclase, which becomes inactive, and in the GDP-bound state it attaches to β-
and y-subunits, forming a G-protein-GDP complex in contact with the receptor (Fig. 26.11.6). If the latter still binds the hormone molecule, the whole cycle can start over (see Fig. 26.11, b). The place of GDP is taken by GTP, the α-subunit dissociates in order to activate the adenylate cyclase molecule. If the receptor no longer binds the hormone molecule (see Fig. 26.11, a), the process stops. Thus, in order to continue cAMP synthesis, the α-subunit
moves back and forth from the receptor to adenylate cyclase. The duration of its stay on adenylate cyclase is determined by the time required for the hydrolysis of the attached GTP molecule.
The system has an amplifying effect: one receptor-bound hormone molecule can activate G-protein molecules one after another, and, therefore, one hormone molecule, binding to the receptor, can lead to the activation of several adenylate cyclase molecules and the formation of a large number of cAMP molecules. The importance of GTP hydrolysis in the process of ATP synthesis can be illustrated by the example of cholera. Cells of the intestinal mucosa secrete Na + into the intestinal lumen , and cAMP stimulates this process. Cholera toxin irreversibly inhibits the GTPase activity of the α-subunit of the G-protein. Thus, the hormonal signal that activates adenylate cyclase cannot be turned off; it is "frozen" in the α-GTP state. Prolonged formation of cAMP leads to a massive loss of Na + ionsfollowed by water molecules, causing debilitating diarrhea and possible death from fluid and electrolyte loss.
In the case shown in Figure 26.11, the GTP-α subunit stimulates adenylate cyclase activity. Adrenoreceptors of another type (α 2 -adrenergic receptors) act in a similar way, but here a different α-GTP subunit (Gi) is used, which inhibits the activity of adenylate cyclase. α- and β-adrenergic receptors mediate the various effects of catecholamines. As we already know (see p. 167), if a sudden action is necessary, adrenaline stimulates the liver and skeletal muscles, which is realized through β 2 -adrenergic receptors that activate adenylate cyclase. In contrast, α 2 -adrenergic receptors inhibit adenylate cyclase. Another subtype - α 1 -adrenergic receptors -also have their differences: they do not use cAMP as a second messenger, but cause the release of Ca 2+ through the phosphoinositide pathway (see p. 352). Thus, one hormone can have quite the opposite effect, depending on the type of receptor.
How does cAMP affect gene transcription?
In Chapter 12, we described how cAMP activates protein kinase A (PKA). In addition to participating in the control of enzyme activity, PKA is part of an important mechanism for the regulation of gene expression. The promoters of several cAMP-inducible genes contain cAMP-responsive elements (CREs). When does PKA phosphorylate?
CRE-binding protein (CREB; from the English cAMP-responsive element binding protein), it becomes an active transcription factor of the "leucine lightning" type (Fig. 26.12). There is a family of CREB proteins that apparently perform various functions in the cell.
So, we said that cAMP is a second messenger (see p. 166) in the transmission of an external signal to functional elements both in the cytoplasm and in the cell nucleus. Another cyclic nucleotide - cGMP - performs the same function in other regulatory systems, which we will consider.
Signal transmission using cyclic GMP as a second intermediary
A number of signals cause an increase in cGMP levels within the cell, which in turn activates protein kinase (PKC). The formation of cGMP from GTP is catalyzed by
membrane-bound and soluble forms of the enzyme guanylate cyclase (Fig. 26.13). In the first case, guanylate cyclase is the intracellular domain of the membrane receptor protein. Binding of the hormone to the external receptor leads to the activation of the intracellular domain and an increase in the synthesis of cGMP. This pathway differs from the mechanism by which the adrenoreceptor triggers the formation of cAMP: in this case, the internal domain of the receptor is itself a guanylate cyclase and is activated directly during hormone attachment (Fig. 26.14). The resulting cGMP mediates the cellular response by activating a specific protein kinase (PKG). An example of this type of regulation is the atrial natriuretic factor produced by endothelial cells: it stimulates the secretion of Na +kidneys. The effects of cGMP are more specialized than cAMP. They involve smooth muscle relaxation and affect nerve cells and vision (see below).
Rice. 26.14. Schematic representation illustrating the basic principles of the action of cyclic nucleotides as second messengers in cell signaling. R 1 - receptor α 2 -adrenergic type; R 2 - receptor β 2 -adrenergic type; R 3 is the binding site of the atrial natriuretic peptide, one of the domains of which is embedded in the membrane, and the catalytic domain is oriented to the side. AC - plasma membrane adenylate cyclase, GC 1 - plasma membrane guanylate cyclase, GC 2 - cytoplasmic guanylate cyclase activated by nitric oxide (NO)
There is another, soluble form of guanylate cyclase in the cytosol. As a prosthetic group, it contains a heme molecule, with which, perhaps, the simplest intercellular signaling molecule, NO, or nitric oxide, binds. The heme molecule is an extremely sensitive NO detector; it sends an activation signal to the enzyme. NO is formed in the endothelial cells of the vascular system from the guanidine group of arginine under the action of the enzyme nitroxide synthase.NO diffuses into the musculature of the blood vessels, causing the formation of cGMP, which, in turn, promotes muscle relaxation and vascular relaxation. Acetylcholine stimulates the formation of nitric oxide, which suggests that it may be mediated by neural regulation. Nitric oxide is also synthesized in response to the mechanical action exerted by the bloodstream on the endothelial cells of the lining of blood vessels, leading to vasodilation (vasodilatation).
Trinitroglycerin, a drug long used to treat angina pectoris, nitric oxide is slowly formed from it, thereby causing vascular relaxation and reducing the workload on the heart. Since NO is oxidized to NO 2 and NO 3 in a matter of seconds , it is a signaling molecule of local action; being rapidly soluble in lipids, nitric oxide easily leaves the cells producing it and passes into neighboring ones. Nitric oxide is part of a complex regulatory system with multiple physiological effects. Rice. 26.14 summarizes the basic principles of the action of cyclic nucleotides as second messengers in intercellular signaling.
Hormones that transmit a signal using various second messengers; phosphoinositide cascade
So far, we have been familiar with a diverse group of hormones that use cyclic nucleotides, cAMP and cGMP, as second messengers. Another group of hormones and growth factors use other intracellular mechanisms for signaling. Potential hormones bind to specific cell receptors, which also interact with G proteins (i.e., GTP-binding proteins), but cyclic nucleotides are not involved in signal transmission. Examples of hormones using this mechanism are thyrotropin-releasing hormone, gonadotropin-releasing hormone, and growth factor PDGF.
Phospholipid phosphatidylinositol-4,5-diphosphate (PIP 2 ) is formed in the membrane by phosphorylation of phosphatidylinositol, the structure of which is given in Chapter 10. Note once again that the G-protein couples the receptor stimulus to the signaling pathway within the cell and acts as a "molecular timing device". ". The interaction of the hormone with the receptor causes the G-protein associated with the receptor to exchange GDP for GTP. The Gp-GTP complex migrates and activates the membrane-bound enzyme phospholipase C, which cleaves PIP 2 into inositol triphosphate (IP 3 ) and diacylglycerol (DAG) (Fig. 26.15). The G protein hydrolyzes GTP to GDP and P i , leading to self-inactivation of the enzyme.
IP 3 causes the release of Ca 2+ from the lumen of the endoplasmic reticulum (ER), which contains high concentrations of this ion. IP 3 opens ligand-dependent Ca 2+ channels in the membrane (see below), ensuring the entry of the ion into the cytoplasm. Under the action of Ca 2+ -ATPase, Ca 2+ ions constantly return to the lumen of the ER, and IP 3 and DAG are removed enzymatically. Thus, the combination of a hormone or any other agonist with a receptor involved in the phosphoinositide cascade leads to an increase in intracellular DAG and Ca 2+, and the process is reversed after dissociation of the hormone-receptor complex.
Rice. 26.16. Phosphatidylinositide cascade: the relationship of DAG, IP 3 and Ca 2+ as second messengers. The Gp protein binds to phospholipase C only in complex with GTP; process reversal occurs when GTP is hydrolyzed to GDP.
DAG is a physiological activator of a specific protein kinase that is distinct from PKA and PCG. Ca 2+ is also required for its maximum activation.
The DAG-activated protein kinase is called PKC; it is associated with phosphatidylserine molecules located on the cytosolic side of the cell membrane. PKC is involved in the regulation of numerous cellular processes by phosphorylation of various target proteins and some growth factors. The important role of PKC in the control of cell division is illustrated by the oncogenic effect of phorbol esters, which are the so-called tumor promoters. Phorbol esters are DAG analogs that activate PKC. Below are the structures of DAG and phorbol ester (it is not necessary to memorize the structure of the latter).
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