PDE3
Encyclopedia
Phosphodiesterase
A phosphodiesterase is any enzyme that breaks a phosphodiester bond. Usually, people speaking of phosphodiesterase are referring to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below...
. The PDEs belong to at least eleven related gene families
Gene family
A gene family is a set of several similar genes, formed by duplication of a single original gene, and generally with similar biochemical functions...
, which are different in their primary structure
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...
, substrate affinity, responses to effector
Effector
Effector may refer to:*An actuator, in robotics*Effector , a music album by the Experimental Techno group Download*Effector cell, a type of lymphocyte in immunology...
s, and regulation mechanism.
Most of the PDE families are composed of more than one gene. PDE3 is clinically significant because of its role in regulating heart muscle, vascular smooth muscle and platelet aggregation. PDE3 inhibitors have been developed as pharmaceuticals, but their use is limited by arrhythmic effects and they can increase mortality in some applications.
Structure
The mammalian PDEs share a common structural organization and contain three functional domains, which include the conserved catalytic core, a regulatory N-terminus, and the C-terminus. The conserved catalytic core is much more similar within PDE families, with about 80% amino acidAmino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
identity, than between different families. It is believed that the core contains common structural elements that are important for the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
and cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
phosphodiester bonds. It is also believed that it contains family-specific determinants for differences in affinity for substrates and sensitivity for inhibitors.
The catalytic domain of PDE3 is characterized by a 44-amino acid insert, but this insert is unique to the PDE3 family, and is a factor when determining a structure for a potent
Potent
Potent may refer to:*Vair#Potent for the heraldic fur*Warren Potent for the Australian Olympic medalist in shootingSee also:*Potency...
and selective PDE3 inhibitor
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...
.
The crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
of the catalytic domains of several PDEs, including PDE3B, have shown that they contain three helical subdomains :
- N-terminal cyclin fold region
- Linker region
- C-terminal helical bundle
At the interface of these domains a deep hydrophobic pocket is formed by residues that are highly conserved among all PDEs. This pocket is the active site and is composed of four subsites :
- Metal binding site (M site)
- Core pocket (Q pocket)
- Hydrophobic pocket (H pocket)
- Lid region (L region)
The M site is at the bottom of the hydrophobic binding pocket and contains two divalent
Divalent
In chemistry, a divalent ion or molecule has a valence of two and thus can form two bonds with other ions or molecules. An older term for divalent is bivalent....
metal binding sites. The metal ions that can bind to these sites are either zinc or magnesium. The zinc binding site has two histidine and two aspartic acid residues that are absoulutely conserved among those PDE's studied to date.
The N-terminal portions of PDEs are widely divergent and contain determinants that are associated with regulatory properties specific to different gene families. For PDE3, those determinants are the hydrophobic membrane association domains and cAMP-dependent protein kinase phosphorylation sites.
Substrate affinity
At first, the PDE3s were purified and described as enzymes that hydrolyse both cGMPCyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
and cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
with Km values between 0.1 – 0.8 µM. However the Vmax for cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
is 4 - 10 times higher than Vmax for cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
hydrolysis.
When different PDEs were first identified, two types of PDEs (PDE3 and PDE4) that exhibited high affinities for cAMP were isolated. PDE3 exhibited high affinity for both cGMP and cAMP, but PDE4 had high affinity for only cAMP. For that reason, the PDE3 was called the cGMP-inhibited PDE to distinguish it from PDE4.
The 44-amino acid insertion in the catalytic domain of PDE3s is believed to be involved in PDE3's interaction with its substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
and inhibitors, but that remains to be established.
The proposed molecular mechanism of cyclic nucleotide specificity of PDEs is the so-called glutamine switch mechanism.
In the PDEs that have had their structure solved, there seems to be an invariant glutamine residue that stabilizes the binding of the purine ring in the active site (binding pocket). The g-amino group of the glutamine residue can alternatively adopt two different orientations:
- The hydrogen bond network supports guanine binding – cGMP selectivity
- The hydrogen bond network supports adenine binding – cAMP selectivity.
In PDEs that can hydrolyse both cGMP and cAMP (PDE3s), the glutamine can rotate freely and therefore switch between orientations.
PDE3 isoforms
The PDE3 family in mammals consists of two members, PDE3A and PDE3B. The PDE3 isoforms are structurally similar, containing an N-terminal domain important for the localization and a C-terminus end. The 44-amino acid insertion in the catalytic domain differs in the PDE3 isoforms, and the N-terminal portions of the isoforms are quite divergent. PDE3A and PDE3B have strikingly similar pharmacological and kinetic properties, but the distinction is in expression profiles and affinity for cGMP.Localization of PDE3
PDE3A is mainly implicated in cardiovascular function and fertility but PDE3B is mainly implicated in lipolysis. Table 1 is an overview of localization of the PDE3 isoforms.| PDE3A | PDE3B | |
|---|---|---|
| Localization in tissues | - Heart Heart The heart is a myogenic muscular organ found in all animals with a circulatory system , that is responsible for pumping blood throughout the blood vessels by repeated, rhythmic contractions... * - Vascular smooth muscle Vascular smooth muscle Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that is responsible for the... * - Platelets - Oocyte Oocyte An oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic... - Kidney Kidney The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and... |
- Vascular smooth muscle Vascular smooth muscle Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that is responsible for the... - Adipocytes - Hepatocytes - Kidney Kidney The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and... - β cells - Developing sperm Spermatozoon A spermatozoon is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote... - T-lymphocytes - Macrophages |
| Intracellular localization | - Membrane Cell membrane The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell... -associated or cytosolic |
- Membrane Biological membrane A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content... -associated (predominantly) |
| Table 1: Overview of PDE3 isoform localization. *Variants of PDE3A have differential expression in cardiovascular tissues |
||
In general, PDE3 can be either cytosolic or membrane-bound and has been associated to plasma membrane, sarcoplasmic reticulum, golgi
Golgi
Golgi may refer to:*Camillo Golgi , Italian physician and scientist after which the following terms are named:**Golgi apparatus , an organelle in the eukaryotic cell...
, and nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
envelope.
PDE3B is predominantly membrane-associated ,and is localized to endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
and microsomal
Microsome
In cell biology, microsomes are vesicle-like artifacts re-formed from pieces of the endoplasmic reticulum when eukaryotic cells are broken-up in the laboratory; by definition, microsomes are not ordinarily present in living cells....
fractions.
PDE3A can be either membrane-associated or cytosolic, depending on the variant and the cell type it is expressed in.
The gene profile
The PDE3 family is composed of two genesGênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
, PDE3A and PDE3B. In cells expressing both genes, PDE3A is usually dominant. Three different variants of PDE3A (PDE3A1-3) are products of alternate startcodon usage of the PDE3A gene. The PDE3B encodes a single isoform only.
In their full-length both PDE3A and PDE3B contain two N-terminal hydrophobic membrane association regions, NHR1 and NHR2 (figure 2). The difference of the PDE3A1-3 variants lies in whether they include:
- both NHR1 and NHR2
- only NHR2
- neither NHR1 nor the NHR2.
The last can be predicted to be exclusively on soluble/cytosolic form.
Regulation
PDE3A and PDE3B activity is regulated by several phosphorylationPhosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
pathways. Protein kinase A and Protein kinase B both activate PDE3A and PDE3B via phosphorylation at two different phosphorylation sites (P1 and P2) between NHR1 and NHR2 (figure 2). Hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
by PDE3 isoforms is also directly inhibited by cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
, although PDE3B is only ≈10% as sensitive to cGMP
Cyclic guanosine monophosphate
Cyclic guanosine monophosphate is a cyclic nucleotide derived from guanosine triphosphate . cGMP acts as a second messenger much like cyclic AMP...
inhibition as PDE3A.
The PDE3B has been extensively studied for its importance in mediating the antilipolytic and antiglycogenlytic effect of insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
in adipose and liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
tissues
Biological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
. The activation of PDE3B in adipocytes is associated with phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
of serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
residue
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....
by an insulin-stimulated protein serine kinase
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
(PDE3IK). By blocking insulin activation of PDE3IK, and in turn phosphorylation/activation of PDE3B, the antilipolytic effect of insulin can be antagonized. Activation of PDE3B decreases concentrations of cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
, which in turn reduces Protein kinase A activity. Protein kinase A is responsible for activation of lipase
Lipase
A lipase is an enzyme that catalyzes the formation or cleavage of fats . Lipases are a subclass of the esterases.Lipases perform essential roles in the digestion, transport and processing of dietary lipids in most, if not all, living organisms...
, which induces lipolysis
Lipolysis
Lipolysis is the breakdown of lipids and involves the hydrolysis of triglycerides into free fatty acids followed by further degradation into acetyl units by beta oxidation. The process produces Ketones, which are found in large quantities in ketosis, a metabolic state that occurs when the liver...
as well as other physiological pathways.
Whether phosphorylation pathways, which regulate activity of PDE3A or PDE3B, could serve as potential drug
Drug
A drug, broadly speaking, is any substance that, when absorbed into the body of a living organism, alters normal bodily function. There is no single, precise definition, as there are different meanings in drug control law, government regulations, medicine, and colloquial usage.In pharmacology, a...
targets rather than the catalytic domain of the PDE3 enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
itself is unclear and beyond the scope of this text.
Function of PDE3
PDE3 enzymes are involved in regulation of cardiac and vascular smooth muscleVascular smooth muscle
Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that is responsible for the...
contractility. Molecules that inhibit PDE3 were originally investigated for the treatment of heart failure, but, because of unwanted arrhythmic side-effects
Adverse effect
In medicine, an adverse effect is a harmful and undesired effect resulting from a medication or other intervention such as surgery.An adverse effect may be termed a "side effect", when judged to be secondary to a main or therapeutic effect. If it results from an unsuitable or incorrect dosage or...
, they are not studied for that indication
Indication
Indication can refer to:* A synonym for Sign.* Human interface Highlighting the single object pointed to as a cursor is moved, without any other user action such as clicking, is indication....
any longer. Nonetheless, the PDE3 inhibitor milrinone
Milrinone
Milrinone is a phosphodiesterase 3 inhibitor. It potentiates the effect of cyclic adenosine monophosphate .Milrinone also enhances contraction of the left ventricle by increasing Ca2+-ATPase activity on the cardiac sarcoplasmic reticulum...
is approved for use in heart failure in intravenous form.
Both PDE3A and PDE3B are expressed in vascular smooth muscle
Vascular smooth muscle
Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that is responsible for the...
cells and are likely to modulate contraction. Their expression in vascular smooth muscle is altered under specific conditions such as elevated cAMP and hypoxia
Hypoxia (medical)
Hypoxia, or hypoxiation, is a pathological condition in which the body as a whole or a region of the body is deprived of adequate oxygen supply. Variations in arterial oxygen concentrations can be part of the normal physiology, for example, during strenuous physical exercise...
.
PDE3 inhibitor
PDE3 inhibitor
A PDE3 inhibitor is a drug which inhibits the action of the phosphodiesterase enzyme PDE3. They are used for the therapy of acute heart failure and cardiogenic shock. Approved PDE3 inhibitors include amrinone, cilostazol, milrinone and enoximone...
s:
- antagonize plateletPlateletPlatelets, or thrombocytes , are small,irregularly shaped clear cell fragments , 2–3 µm in diameter, which are derived from fragmentation of precursor megakaryocytes. The average lifespan of a platelet is normally just 5 to 9 days...
aggregation - block oocyteOocyteAn oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic...
maturation - increase contractility of the heartHeartThe heart is a myogenic muscular organ found in all animals with a circulatory system , that is responsible for pumping blood throughout the blood vessels by repeated, rhythmic contractions...
- enhance vascular smooth muscleVascular smooth muscleVascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that is responsible for the...
relaxation - enhance airway smooth muscle relaxation
It has been demonstrated that PDE3A inhibition prevents oocyte
Oocyte
An oocyte, ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis. The female germ cells produce a primordial germ cell which undergoes a mitotic...
maturation in vitro and in vivo. For example, when mice are made completely deficient of PDE3A, they become infertile.
Aggregation of platelets is highly regulated by cyclic nucleotides. PDE3A is a regulator of this process, and PDE3 inhibitors effectively prevent aggregation of platelets. Cilostazol is approved for treatment of intermittent claudication
Intermittent claudication
Intermittent claudication is a clinical diagnosis given for muscle pain , classically in the calf muscle, which occurs during exercise, such as walking, and is relieved by a short period of rest.Claudication derives from the Latin verb claudicare, "to limp".-Signs:One of the hallmarks of arterial...
and is thought to involve inhibition of platelet aggregation and also inhibition of smooth muscle proliferation and vasodilation.
The most studied roles of PDE3B have been in the areas of insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
, IGF1, and leptin
Leptin
Leptin is a 16 kDa protein hormone that plays a key role in regulating energy intake and energy expenditure, including appetite and metabolism. It is one of the most important adipose derived hormones...
signaling. When PDE3B is overexpressed in β-cells
Beta cell
Beta cells are a type of cell in the pancreas located in the so-called islets of Langerhans. They make up 65-80% of the cells in the islets.-Function:...
in mice, it causes impaired insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
secretion and glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
intolerance.
The involvement of PDE3B in regulation of these important pathways has inspired researchers to begin studying the possible roles of this enzyme in disorders such as obesity
Obesity
Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems...
, diabetes, and cellulite
Cellulite
Cellulite is a topographic skin change that occurs in most postpubertal females. It presents as a modification of skin topography evident by skin dimpling and nodularity that occurs mainly in women on the pelvic region, lower limbs, and abdomen, and is caused by the herniation of subcutaneous fat...
.
SAR (structure-activity relationships)
From early studies an initial model of PDE, active site topography was derived. This early model can be summarized into the following steps concerning cAMPCyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
active site topography:
- cAMP substrate with its adenineAdenineAdenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
and ribose moieties in an “anti” relationship - The phosphate atom in cAMP binds to PDE active site, using an arginineArginineArginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
residue and a water molecule, which was initially associated with Mg2+. A second arginine residue and the Mg2+ may also play roles during binding and/or play roles in the next step - SN2 attack of phosphorus by H2O with formation of a trigonal bipyramid transition stateTransition stateThe transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
- 5´-AMP is formed as an “inverted” product. Electronic charges conserve the net charge overall and across the transition stateTransition stateThe transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...