Breakthrough findings on multiple sclerosis

Researchers from the National University of Singapore (NUS) have discovered a new type of immune T helper cells that may help develop treatment for multiple sclerosis.

T helper cells help the activity of other immune cells by helping, suppressing, or regulating immune response.

The researchers, led by NUS medical school professor Fu Xin-Yuan and Doctor Sheng Wanqiang from the Cancer Science Institute of Singapore, found that the immune cells play a crucial role in the immune system and the development of neuronal inflammation.

Together with Dr Zhang Yong-Liang from the department of microbiology at the NUS Yong Loo Lin School of Medicine, the team found that the newly discovered T helper cells is programmed by the STAT5 protein.

The T helper cells can recruit and activate other inflammatory cells to cause neuroinflammation, demyelination and nerve system damage.

The researchers’ work show that if doctors are able to block the STAT5 protein, patients suffering from the autoimmune disease will benefit greatly from it.

MECHANISMS OF GENETIC VARIATIONS

Allele

A DNA coding that occupies a specific place on a chromosome. Alleles hold genetic information such as the color of flower petals.

Genotype

The inheritable information carried in the cells of all living organisms. This stored information is used as a "blueprint" for building and maintaining the organism

Phenotype

The physical attributes of an organism. They are caused by both the organism’s genotype and its environment.

Latent

Present and capable of becoming active, but not yet visible or active.

Patent

Visible and active.

Morphology

What an organism looks like and what it is made of, also known as the form and structure of an organism.

Adaptive Radiation

The rapid diversification of a species to suit many different environments.

Physiology

The functions in living things, or the scientific study of those functions.

Preadaptation

When a creature uses a feature of their anatomy for a purpose seemingly unrelated to its original task.

Built-in Variation in the Gene Pool

For most character traits present in organisms, more than one allele exists. The different genes must have come about by chance alone, because we are dealing with genotype. The genotype of an organism includes both latent and patent genes. Only genes that have been activated are expressed in the phenotype. A new gene must first be expressed before natural selection comes into play.

As far as alleles are concerned, expression is governed by a complex system of dominance versus recessiveness. Furthermore, the frequency of genetic expression can also alter the phenotype. For example, the gene coding for growth hormone can influence the size of the organism. Variation in size does thus not necessarily require new genes, just differential expression of the same genes. An example of built-in variation in the gene pool can be seen in the differences between breeds of dogs. As to how the genes responsible for the variation came in to existence, chance or design are the only options given, since we are dealing with genotype.

By selecting from the built-in natural variation of the gene pool, various breeds of dogs and domestic cattle were produced. Great changes in physiology and morphology are involved, and evolution is here certainly excluded. Differences in dogs are greater than the differences in genera of the Canidae family.

Genetic expression is also influenced, so as to bring about differences in structural expression by the genes in terms of size. Differential hormonal modulation in response to environmental stimuli can alter the time and magnitude of response, effectively producing reproductively isolated communities which would be regarded as different species by evolutionists, but are in effect merely extremes of genetic expression within an existing gene pool. The vast numbers of latent genes would then be accounted for.

Evolutionists recognize that changes in genotype frequencies do occur to produce changes in gene distribution. They, however, explain most changes as resulting from chance mutations, and this is not tenable.

Even evolutionists admit that preadaptation must have played a major role in enabling organisms to survive environmental changes. Preadaptation, however, requires preexisting genes capable of responding to environmental stimuli—precisely what creationists claim. Where did these fully expressional genes come from? 

Homologous

Similarity in structure between parts of different organisms, such as the similarity between a human hand and a bat wing.

Genotype

The inheritable information carried in the cells of all living organisms. This stored information is used as a "blueprint" for building and maintaining the organism

Phenotype

The physical attributes of an organism. They are caused by both the organism’s genotype and its environment.

Genome

A full set of chromosomes. A regular cell contains two full genomes.

Ligase

An enzyme that joins two larger molecules together with a chemical bond.

DNA

Deoxyribonucleic acid (DNA) is a molecule that stores genetic make up, or code. All living things have DNA.

Chiasma

The point where two chromatids are interwoven in a cell.

Chromatid

One of the two identical halves of a replicated chromosome.

Nucleotide

Molecules that join together to make up the structure of DNA and RNA.

Reproductive Exchange

Through sexual reproduction, genetic material is exchanged. This induces genetic recombination. The significance of this is obvious: the exchange of material increases the variation. This holds particular advantages to populations and is considered by evolutionists to be an innovation that greatly enhances the evolutionary process.

We know what sexual reproduction achieves. It increases the variation. However, increased variation in the genotype is of no value until it is expressed in the phenotype. The new varieties must be expressed in the offspring before natural selection can feast on this increased variation. The process that brings about the variation (sexual reproduction) is not subject to selection, only the result thereof (the increased variation in the offspring) is subject to selection.

The exchange of gametes requires a modified form of cell division which is the process of meiosis. During meiosis, the number of chromosomes is halved, resulting in the gametes having half the chromosomes. Sexual fusion of two gametes then restores the number of chromosomes. Variation in the genome is greatly increased by two processes occurring during meiosis: independent assortment and crossing over. Both these processes are extremely complex, but in themselves are not subject to selection. They rearrange the genetic material, resulting in new combinations of the material. As this reshuffling occurs at the level of the genotype, it is not subject to natural selection until the new combinations have been expressed in the phenotype. 

i) Independent Assortment

Independent assortment is achieved when chromosomes line up in homologous pairs and move independently to the one pole or the other. The process is governed by complex enzyme systems which in turn must also have come about by chance. The possible variation that can be achieved by independent assortment depends on the number of chromosomes present. In humans, there are 46 chromosomes, which would arrange themselves in 23 homologous pairs. There are thus 80 trillion possible variations.

ii) Crossing Over

Crossing Over is an awe-inspiring process. When homologous chromosomes are lined up during meiosis, they can, in a very precise way, exchange genetic material. There are five steps in achieving this:

a) Enzymes open the double helix of DNA in the aligned chromosomes to permit intermolecular base pairing.

b) One strand of each helix is cut at equivalent positions.

c) The enzyme ligase joins them to form a half-chromatid chiasma (because only one strand of each chromatid cross over), resulting in a cross-shaped molecule.

d) The cross-shaped molecule is cut in half by an enzyme, leaving a break in one strand of each recombinant.

e) The break is sealed by ligase.

The process has to be extremely precise. If even one nucleotide is transferred incorrectly, the genetic message becomes useless.

Somehow the genetic material from one parental chromosome and the genetic material from the other parental chromosome are cut up and pasted together during each meiosis, and this is done with complete reciprocity. In other words, neither chromosome gains or loses any genes in the process. In fact, it is probably correct to say that neither chromosome gains or loses even one nucleotide in the exchange.

Plasmid

A DNA molecule that is separate from the chromosomal DNA, and can replicate without the chromosomal DNA.

Genome

A full set of chromosomes. A regular cell contains two full genomes.

DNA

Deoxyribonucleic acid (DNA) is a molecule that stores genetic makeup, or code. All living things have DNA.

Nucleotide

Molecules that join together to make up the structure of DNA and RNA.

Transposons

Sequences of DNA that can move around to different positions within the genome of a single cell. In the process, they can cause mutations and change the amount of DNA in the genome.

Transposons

Chunks of DNA that can move around within a genome.

Transposable Elements

Transposable elements are sometimes called "jumping genes." They consist of segments of DNA that can move from one position on a chromosome to another. In 1951, Nobel prize-winning Dr. Barbara McClintock proposed that genes are not fixed on chromosomes, but that they can move around on the chromosome. At first her findings were discarded because they contradicted the genetic concept of the day. Today, her discovery of what she calls transposable elements has an established place in science.

Transposable elements allow antibiotic resistance and increased variation. The genes move because they are part of a small circular auxiliary genome called a plasmid, which enters and leaves the main genome at a specific place where there is a nucleotide sequence that is also present on the plasmid. Other genes move within small fragments of the genome called transposons. Together, transposons and plasmids produce genetic recombinations.

Integration at a new position also transfers the gene to that new position. The repositioning may be random, but occurs at sequence-specific insertion points which means that the process is orderly. The splicing and repositioning is carried out by enzyme systems and involves the transfer of complete information. 

Speciation

The evolutionary process by which new species arise.

Genome

A full set of chromosomes. A regular cell contains two full genomes.

Recombination of Chromosomes

Changes in chromosomal structure have been cited as important contributing factors in providing variation, and as a mechanism for speciation.

Changes in chromosomes can include changes in chromosome number or arm number, deletions, duplications, inversions, or even radical reorganizations of the genome.

CIRCULAR BACTERIAL CHROMOSOME

A circular bacterial chromosome, showing DNA replication proceeding bidirectionally, with two replication forks generated at the "origin". Each half of the chromosome replicated by one replication fork is called a "replichore".

Circular bacterial chromosomes are the bacterial chromosomes contained in a circular DNA molecule. Unlike the linear DNA of vertebrates, typical bacterial chromosomes contain circular DNA.

Most bacterial chromosomes contain a circular DNA molecule - there are no free ends to the DNA. Free ends would otherwise create significant challenges to cells with respect to DNA replication and stability. Cells that do contain chromosomes with DNA ends, or telomeres (most eukaryotes), have acquired elaborate mechanisms to overcome these challenges. However, a circular chromosome can provide other challenges for cells. After replication, the two progeny circular chromosomes can sometimes remain interlinked or tangled, and they must be resolved so that each cell inherits one complete copy of the chromosome during cell division.

Replication of a circular bacterial chromosome

Bacterial chromosome replication is best understood in the well-studied bacteria Escherichia coli and Bacillus subtilis. Chromosome replication proceeds in three major stages: initiation, elongation and termination. The initiation stage starts with the ordered assembly of "initiator" proteins at the origin region of the chromosome, called oriC. These assembly stages are regulated to ensure that chromosome replication occurs only once in each cell cycle. During the elongation phase of replication, the enzymes that were assembled at oriC during initiation proceed along each arm ("replichore") of the chromosome, in opposite directions away from the oriC, replicating the DNA to create two identical copies. This process is known as bidirectional replication. The entire assembly of molecules involved in DNA replication on each arm is called a "replisome." At the forefront of the replisome is a DNA helicase that unwinds the two strands of DNA, creating a moving "replication fork". The two unwound single strands of DNA serve as templates for DNA polymerase, which moves with the helicase (together with other proteins) to synthesize a complementary copy of each strand. In this way, two identical copies of the original DNA are created. Eventually, the two replication forks moving around the circular chromosome meet in a specific zone of the chromosome, approximately opposite oriC, called the terminus region. The elongation enzymes then disassemble, and the two "daughter" chromosomes are resolved before cell division is completed.

Initiation

The E. coli bacterial replication origin, called oriC consists of DNA sequences that are recognised by the DnaA protein, which is highly conserved amongst different bacterial species. DnaA binding to the origin initiates the regulated recruitment of other enzymes and proteins that will eventually lead to the establishment of two complete replisomes for bidirectional replication.

DNA sequence elements within oriC that are important for its function include DnaA boxes, a 9-mer repeat with a highly conserved consensus sequence 5' - TTATCCACA - 3', that are recognized by the DnaA protein. DnaA protein plays a crucial role in the initiation of chromosomal DNA replication. Bound to ATP, and with the assistance of bacterial histone-like proteins [HU] DnaA then unwinds an AT-rich region near the left boundary of oriC, which carries three 13-mer motifs, and opens up the double-stranded DNA for entrance of other replication proteins.

This region also contains four “GATC” sequences that are recognized by DNA adenine methylase (Dam), an enzyme that modifies the adenine base when this sequence is unmethylated or hemimethylated. The methylation of adenines is important as it alters the conformation of DNA to promote strand separation, and it appears that this region of oriC has a natural tendency to unwind.

Elongation

When the replication fork moves around the circle, a structure shaped like the Greek letter theta Ө is formed. John Cairns demonstrated the theta structure of E. coli chromosomal replication in 1963, using an innovative method to visualize DNA replication. In his experiment, he radioactively labeled the chromosome by growing his cultures in a medium containing 3H-thymidine. The nucleoside base was incorporated uniformly into the bacterial chromosome. He then isolated the chromosomes by lysing the cells gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months. This Experiment clearly demonstrates the theta replication model of circular bacterial chromosomes.

As described above, bacterial chromosomal replication occurs in a bidirectional manner. This was first demonstrated by specifically labelling replicating bacterial chromosomes with radioactive isotopes. The regions of DNA undergoing replication during the experiment were then visualized by using autoradiography and examining the developed film microscopically. This allowed the researchers to see where replication was taking place. The first conclusive observations of bidirectional replication were from studies of B. subtilis. Shortly after, the E. coli chromosome was also shown to replicate bidirectionally.

The E. coli DNA polymerase III holoenzyme is a 900 kD complex, possessing an essentially a dimeric structure. Each monomeric unit has a catalytic core, a dimerization subunit, and a processivity component. DNA Pol III uses one set of its core subunits to synthesize the leading strand continuously, while the other set of core subunits cycles from one Okazaki fragment to the next on the looped lagging strand. Leading strand synthesis begins with the synthesis of a short RNA primer at the replication origin by the enzyme Primase (DnaG protein).

Deoxynucleotides are then added to this primer by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. Leading strand synthesis then proceeds continuously, while the DNA is concurrently unwound at the replication fork. In contrast, lagging strand synthesis is accomplished in short Okazaki fragments. First, an RNA primer is synthesized by primase, and, like that in leading strand synthesis, DNA Pol III binds to the RNA primer and adds deoxyribonucleotides.

When the synthesis of an Okazaki fragment has been completed, replication halts and the core subunits of DNA Pol III dissociates from the β sliding clamp [B sliding clap is the processivity subunit of DNA Pol III]. The RNA primer is remove and replaced with DNA by DNA polymerase I [which also possesses proofreading exonuclease activity] and the remaining nick is sealed by DNA ligase, which then ligates these fragments to form the lagging strand.

Termination

Termination is the process of fusion of replication forks and disassembly of the resplisomes to yield two separate and complete DNA molecules. It occurs in the terminus region, approximately opposite oriC on the chromosome. The terminus region contains several DNA replication terminator sites, or "Ter" sites. A special "replicaiton terminator" protein must be bound at the Ter site for it to pause replication. Each Ter site has polarity of action, that is, it will arrest a replication fork approaching the Ter site from one direction, but will allow unimpeded fork movement through the Ter site from the other direction. The arrangement of the Ter sites forms two opposed groups that forces the two forks to meet each other within the region they span. This arrangement is called the "replication fork trap."

Replication of the DNA separating the opposing replication forks, leaves the completed chromosomes joined as ‘catenanes’ or topologically interlinked circles. The circles are not covalently linked, but cannot be separated because they are interwound and each is covalently closed. The catenated circles require the action of topoisomerases to separate the circles [decatanation]. In E.coli, DNA topoisomerase IV plays the major role in the separation of the catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break.

Nicotinamide adenine dinucleotide

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. The compound is a dinucleotide, because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide. Nicotinamide adenine dinucleotide exists in two forms, an oxidized and reduced form abbreviated as NAD⁺ and NADH respectively.

NADH, also known as coenzyme 1, is a naturally occurring biological substance. The "H" after NAD stands for hydride, or "high-energy hydrogen".

In metabolism, nicotinamide adenine dinucleotide is involved in redox reactions, carrying electrons from one reaction to another. The coenzyme is, therefore, found in two forms in cells: NAD⁺ is an oxidizing agent – it accepts electrons from other molecules and becomes reduced. This reaction forms NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD. However, it is also used in other cellular processes, the most notable one being a substrate of enzymes that add or remove chemical groups from proteins, in posttranslational modifications. Because of the importance of these functions, the enzymes involved in NAD metabolism are targets for drug discovery.

In organisms, NAD can be synthesized from simple building-blocks (de novo) from the amino acids tryptophan or aspartic acid. In an alternative fashion, more complex components of the coenzymes are taken up from food as the vitamin called niacin. Similar compounds are released by reactions that break down the structure of NAD. These preformed components then pass through a salvage pathway that recycles them back into the active form. Some NAD is also converted into nicotinamide adenine dinucleotide phosphate (NADP); the chemistry of this related coenzyme is similar to that of NAD, but it has different roles in metabolism.

Although NAD⁺ is written with a superscript plus sign because of the formal charge on a particular nitrogen atom, at physiological pH for the most part it is actually a singly-charged anion (charge of minus 1), while NADH is a doubly-charged anion.

NAD is considered the "V factor" required for the growth of Haemophilus influenzae.

Physical and chemical properties

Nicotinamide adenine dinucleotide, like all dinucleotides, consists of two nucleotides joined by a pair of bridging phosphate groups. The nucleotides consist of ribose rings, one with adenine attached to the first carbon atom (the 1' position) and the other with nicotinamide at this position. The nicotinamide moiety can be attached in two orientations to this anomeric carbon atom. Because of these two possible structures, the compound exists as two diastereomers. It is the β-nicotinamide diastereomer of NAD⁺ that is found in organisms. These nucleotides are joined together by a bridge of two phosphate groups through the 5' carbons.

The redox reactions of nicotinamide adenine dinucleotide.

In metabolism, the compound accepts or donates electrons in redox reactions. Such reactions (summarized in formula below) involve the removal of two hydrogen atoms from the reactant (R), in the form of a hydride ion (H⁻), and a proton (H⁺). The proton is released into solution, while the reductant RH₂ is oxidized and NAD⁺ reduced to NADH by transfer of the hydride to the nicotinamide ring.

RH₂ + NAD⁺ → NADH + H⁺ + R;

From the hydride electron pair, one electron is transferred to the positively charged nitrogen of the nicotinamide ring of NAD⁺, and the second hydrogen atom transferred to the C4 carbon atom opposite this nitrogen. The midpoint potential of the NAD⁺/NADH redox pair is −0.32 volts, which makes NADH a strong reducing agent. The reaction is easily reversible, when NADH reduces another molecule and is re-oxidized to NAD⁺. This means the coenzyme can continuously cycle between the NAD⁺ and NADH forms without being consumed.

In appearance, all forms of this coenzyme are white amorphous powders that are hygroscopic and highly water-soluble. The solids are stable if stored dry and in the dark. Solutions of NAD⁺ are colorless and stable for about a week at 4 °C and neutral pH, but decompose rapidly in acids or alkalis. Upon decomposition, they form products that are enzyme inhibitors.

UV absorption spectra of NAD⁺ and NADH.

Both NAD⁺ and NADH strongly absorb ultraviolet light because of the adenine. For example, peak absorption of NAD⁺ is at a wavelength of 259 nanometers (nm), with an extinction coefficient of 16,900 M⁻¹cm⁻¹. NADH also absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 M⁻¹cm⁻¹. This difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assays – by measuring the amount of UV absorption at 340 nm using a spectrophotometer.

NAD⁺ and NADH also differ in their fluorescence. NADH in solution has an emission peak at 460 nm and a fluorescence lifetime of 0.4 nanoseconds, while the oxidized form of the coenzyme does not fluoresce. The properties of the fluorescence signal changes when NADH binds to proteins, so these changes can be used to measure dissociation constants, which are useful in the study of enzyme kinetics. These changes in fluorescence are also used to measure changes in the redox state of living cells, through fluorescence microscopy.

Concentration and state in cells

In rat liver, the total amount of NAD⁺ and NADH is approximately 1 μmole per gram of wet weight, about 10 times the concentration of NADP⁺ and NADPH in the same cells. The actual concentration of NAD⁺ in cell cytosol is harder to measure, with recent estimates in animal cells, ranging around 0.3 mM, and approximately 1.0 to 2.0 mM in yeast. However, more than 80% of NADH fluorescence in mitochondria is from bound form, so the concentration in solution is much lower.

Data for other compartments in the cell are limited, although, in the mitochondrion the concentration of NAD⁺ is similar to that in the cytosol. This NAD⁺ is carried into the mitochondrion by a specific membrane transport protein, since the coenzyme cannot diffuse across membranes.

The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD⁺/NADH ratio. This ratio is an important component of what is called the redox state of a cell, a measurement that reflects both the metabolic activities and the health of cells. The effects of the NAD⁺/NADH ratio are complex, controlling the activity of several key enzymes, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase. In healthy mammalian tissues, estimates of the ratio between free NAD⁺ and NADH in the cytoplasm typically lie around 700; the ratio is thus favourable for oxidative reactions. The ratio of total NAD⁺/NADH is much lower, with estimates ranging from 3–10 in mammals. In contrast, the NADP⁺/NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. These different ratios are key to the different metabolic roles of NADH and NADPH.

Biosynthesis

NAD⁺ is synthesized through two metabolic pathways. It is produced either in a de novo pathway from amino acids or in salvage pathways by recycling preformed components such as nicotinamide back to NAD⁺.

De novo production

Some metabolic pathways that synthesize and consume NAD⁺ in vertebrates. The abbreviations are defined in the text.

Most organisms synthesize NAD⁺ from simple components. The specific set of reactions differs among organisms, but a common feature is the generation of quinolinic acid (QA) from an amino acid—either tryptophan (Trp) in animals and some bacteria, or aspartic acid in some bacteria and plants. The quinolinic acid is converted to nicotinic acid mononucleotide (NaMN) by transfer of a phosphoribose moiety. An adenylate moiety is then transferred to form nicotinic acid adenine dinucleotide (NaAD). Finally, the nicotinic acid moiety in NaAD is amidated to a nicotinamide (Nam) moiety, forming nicotinamide adenine dinucleotide.

In a further step, some NAD⁺ is converted into NADP⁺ by NAD⁺ kinase, which phosphorylates NAD⁺. In most organisms, this enzyme uses ATP as the source of the phosphate group, although several bacteria such as Mycobacterium tuberculosis and a hyperthermophilic archaeon Pyrococcus horikoshii, use inorganic polyphosphate as an alternative phosphoryl donor.

Salvage pathways use three precursors for NAD⁺.

Salvage pathways

Besides assembling NAD⁺ de novo from simple amino acid precursors, cells also salvage preformed compounds containing nicotinamide. Although other precursors are known, the three natural compounds containing the nicotinamide ring and used in these salvage metabolic pathways are nicotinic acid (Na), nicotinamide (Nam) and nicotinamide riboside (NR). These compounds can be taken up from the diet, where the mixture of nicotinic acid and nicotinamide are called vitamin B₃ or niacin. However, these compounds are also produced within cells, when the nicotinamide moiety is released from NAD⁺ in ADP-ribose transfer reactions. Indeed, the enzymes involved in these salvage pathways appear to be concentrated in the cell nucleus, which may compensate for the high level of reactions that consume NAD⁺ in this organelle. Cells can also take up extracellular NAD⁺ from their surroundings.

Despite the presence of the de novo pathway, the salvage reactions are essential in humans; a lack of niacin in the diet causes the vitamin deficiency disease pellagra. This high requirement for NAD⁺ results from the constant consumption of the coenzyme in reactions such as posttranslational modifications, since the cycling of NAD⁺ between oxidized and reduced forms in redox reactions does not change the overall levels of the coenzyme.

The salvage pathways used in microorganisms differ from those of mammals. Some pathogens, such as the yeast Candida glabrata and the bacterium Haemophilus influenzae are NAD⁺ auxotrophs – they cannot synthesize NAD⁺ – but possess salvage pathways and thus are dependent on external sources of NAD⁺ or its precursors. Even more surprising is the intracellular pathogen Chlamydia trachomatis, which lacks recognizable candidates for any genes involved in the biosynthesis or salvage of both NAD⁺ and NADP⁺, and must acquire these coenzymes from its host.

Functions

Nicotinamide adenine dinucleotide has several essential roles in metabolism. It acts as a coenzyme in redox reactions, as a donor of ADP-ribose moieties in ADP-ribosylation reactions, as a precursor of the second messenger molecule cyclic ADP-ribose, as well as acting as a substrate for bacterial DNA ligases and a group of enzymes called sirtuins that use NAD⁺ to remove acetyl groups from proteins. In addition to these metabolic functions, NAD⁺ emerges as an adenine nucleotide that can be released from cells spontaneously and by regulated mechanisms, and can therefore have important extracellular roles.

Oxidoreductase binding of NAD

The main role of NAD⁺ in metabolism is the transfer of electrons from one molecule to another. Reactions of this type are catalyzed by a large group of enzymes called oxidoreductases. The correct names for these enzymes contain the names of both their substrates: for example NADH-ubiquinone oxidoreductase catalyzes the oxidation of NADH by coenzyme Q. However, these enzymes are also referred to as dehydrogenases or reductases, with NADH-ubiquinone oxidoreductase commonly being called NADH hydrogenase or sometimes coenzyme Q reductase.

When bound to a protein, NAD⁺ and NADH are usually held within a structural motif known as the Rossmann fold. The motif is named after Michael Rossmann who was the first scientist to notice how common this structure is within nucleotide-binding proteins. This fold contains three or more parallel beta strands linked by two alpha helices in the order beta-alpha-beta-alpha-beta. This forms a beta sheet flanked by a layer of alpha helices on each side. Because each Rossmann fold binds one nucleotide, binding domains for the dinucleotide NAD⁺ consist of two paired Rossmann folds, with each fold binding one nucleotide within the cofactor. However, this fold is not universal among NAD-dependent enzymes, since a class of bacterial enzymes involved in amino acid metabolism have recently been discovered that bind the coenzyme, but lack this motif.

3-D conformation of NAD⁺.

When bound in the active site of an oxidoreductase, the nicotinamide ring of the coenzyme is positioned so that it can accept a hydride from the other substrate. Since the C4 carbon that accepts the hydrogen is prochiral, this can be exploited in enzyme kinetics to give information about the enzyme's mechanism. This is done by mixing an enzyme with a substrate that has deuterium atoms substituted for the hydrogens, so the enzyme will reduce NAD⁺ by transferring deuterium rather than hydrogen. In this case, an enzyme can produce one of two stereoisomers of NADH. In some enzymes the hydrogen is transferred from above the plane of the nicotinamide ring; these are called class A oxidoreductases, whereas class B enzymes transfer the atom from below.

Despite the similarity in how proteins bind the two coenzymes, enzymes almost always show a high level of specificity for either NAD⁺ or NADP⁺. This specificity reflects the distinct metabolic roles of the respective coenzymes, and is the result of distinct sets of amino acid residues in the two types of coenzyme-binding pocket. For instance, in the active site of NADP-dependent enzymes, an ionic bond is formed between a basic amino acid side-chain and the acidic phosphate group of NADP⁺. On the converse, in NAD-dependent enzymes the charge in this pocket is reversed, preventing NADP⁺ from binding. However, there are a few exceptions to this general rule, and enzymes such as aldose reductase, glucose-6-phosphate dehydrogenase, and methylenetetrahydrofolate reductase can use both coenzymes in some species.

Role in redox metabolism

A simplified outline of redox metabolism, showing how NAD⁺ and NADH link the citric acid cycle and oxidative phosphorylation.

The redox reactions catalyzed by oxidoreductases are vital in all parts of metabolism, but one particularly important area where these reactions occur is in the release of energy from nutrients. Here, reduced compounds such as glucose and fatty acids are oxidized, thereby releasing energy. This energy is transferred to NAD⁺ by reduction to NADH, as part of beta oxidation, glycolysis, and the citric acid cycle. In eukaryotes the electrons carried by the NADH that is produced in the cytoplasm are transferred into the mitochondrion (to reduce mitochondrial NAD⁺) by mitochondrial shuttles, such as the malate-aspartate shuttle. The mitochondrial NADH is then oxidized in turn by the electron transport chain, which pumps protons across a membrane and generates ATP through oxidative phosphorylation. These shuttle systems also have the same transport function in chloroplasts.

Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions, the cell maintains significant concentrations of both NAD⁺ and NADH, with the high NAD⁺/NADH ratio allowing this coenzyme to act as both an oxidizing and a reducing agent. In contrast, the main function of NADPH is as a reducing agent in anabolism, with this coenzyme being involved in pathways such as fatty acid synthesis and photosynthesis. Since NADPH is needed to drive redox reactions as a strong reducing agent, the NADP⁺/NADPH ratio is kept very low.

Although it is important in catabolism, NADH is also used in anabolic reactions, such as gluconeogenesis. This need for NADH in anabolism poses a problem for prokaryotes growing on nutrients that release only a small amount of energy. For example, nitrifying bacteria such as Nitrobacter oxidize nitrite to nitrate, which releases sufficient energy to pump protons and generate ATP, but not enough to produce NADH directly. As NADH is still needed for anabolic reactions, these bacteria use a nitrite oxidoreductase to produce enough proton-motive force to run part of the electron transport chain in reverse, generating NADH.

Non-redox roles

The coenzyme NAD⁺ is also consumed in ADP-ribose transfer reactions. For example, enzymes called ADP-ribosyltransferases add the ADP-ribose moiety of this molecule to proteins, in a posttranslational modification called ADP-ribosylation. ADP-ribosylation involves either the addition of a single ADP-ribose moiety, in mono-ADP-ribosylation, or the transferral of ADP-ribose to proteins in long branched chains, which is called poly(ADP-ribosyl)ation. Mono-ADP-ribosylation was first identified as the mechanism of a group of bacterial toxins, notably cholera toxin, but it is also involved in normal cell signaling. Poly(ADP-ribosyl)ation is carried out by the poly(ADP-ribose) polymerases. The poly(ADP-ribose) structure is involved in the regulation of several cellular events and is most important in the cell nucleus, in processes such as DNA repair and telomere maintenance. In addition to these functions within the cell, a group of extracellular ADP-ribosyltransferases has recently been discovered, but their functions remain obscure. NAD⁺ may also be added onto cellular RNA as a 5'-terminal modification.

The structure of cyclic ADP-ribose.

Another function of this coenzyme in cell signaling is as a precursor of cyclic ADP-ribose, which is produced from NAD⁺ by ADP-ribosyl cyclases, as part of a second messenger system. This molecule acts in calcium signaling by releasing calcium from intracellular stores. It does this by binding to and opening a class of calcium channels called ryanodine receptors, which are located in the membranes of organelles, such as the endoplasmic reticulum.

NAD⁺ is also consumed by sirtuins, which are NAD-dependent deacetylases, such as Sir2. These enzymes act by transferring an acetyl group from their substrate protein to the ADP-ribose moiety of NAD⁺; this cleaves the coenzyme and releases nicotinamide and O-acetyl-ADP-ribose. The sirtuins mainly seem to be involved in regulating transcription through deacetylating histones and altering nucleosome structure. However, non-histone proteins can be deacetylated by sirtuins as well. These activities of sirtuins are particularly interesting because of their importance in the regulation of aging.

Other NAD-dependent enzymes include bacterial DNA ligases, which join two DNA ends by using NAD⁺ as a substrate to donate an adenosine monophosphate (AMP) moiety to the 5' phosphate of one DNA end. This intermediate is then attacked by the 3' hydroxyl group of the other DNA end, forming a new phosphodiester bond. This contrasts with eukaryotic DNA ligases, which use ATP to form the DNA-AMP intermediate.

Extracellular actions of NAD⁺

In recent years, NAD⁺ has also been recognized as an extracellular signaling molecule involved in cell-to-cell communication.NAD⁺ is released from neurons in blood vessels, urinary bladder, large intestine, from neurosecretory cells, and from brain synaptosomes, and is proposed to be a novel neurotransmitter that transmits information from nerves to effector cells in smooth muscle organs. Further studies are needed to determine the underlying mechanisms of its extracellular actions and their importance for human health and diseases.

Pharmacology and medical uses

The enzymes that make and use NAD⁺ and NADH are important in both pharmacology and the research into future treatments for disease. Drug design and drug development exploits NAD⁺ in three ways: as a direct target of drugs, by designing enzyme inhibitors or activators based on its structure that change the activity of NAD-dependent enzymes, and by trying to inhibit NAD⁺ biosynthesis.

The coenzyme NAD⁺ is not itself currently used as a treatment for any disease. However, it is being studied for its potential use in the therapy of neurodegenerative diseases such as Alzheimer's and Parkinson disease. Evidence on the benefit of NAD⁺ in neurodegeneration is mixed; some studies in mice have produced promising results whereas a placebo-controlled clinical trial in humans failed to show any effect.

NAD⁺ is also a direct target of the drug isoniazid, which is used in the treatment of tuberculosis, an infection caused by Mycobacterium tuberculosis. Isoniazid is a prodrug and once it has entered the bacteria, it is activated by a peroxidase enzyme, which oxidizes the compound into a free radical form. This radical then reacts with NADH, to produce adducts that are very potent inhibitors of the enzymes enoyl-acyl carrier protein reductase, and dihydrofolate reductase. In one experiment, mice given NAD for one week had improved nuclear-mitochrondrial communication.

Since a large number of oxidoreductases use NAD⁺ and NADH as substrates, and bind them using a highly conserved structural motif, the idea that inhibitors based on NAD⁺ could be specific to one enzyme is surprising. However, this can be possible: for example, inhibitors based on the compounds mycophenolic acid and tiazofurin inhibit IMP dehydrogenase at the NAD⁺ binding site. Because of the importance of this enzyme in purine metabolism, these compounds may be useful as anti-cancer, anti-viral, or immunosuppressive drugs. Other drugs are not enzyme inhibitors, but instead activate enzymes involved in NAD⁺ metabolism. Sirtuins are a particularly interesting target for such drugs, since activation of these NAD-dependent deacetylases extends lifespan. Compounds such as resveratrol increase the activity of these enzymes, which may be important in their ability to delay aging in both vertebrate, and invertebrate model organisms.

Because of the differences in the metabolic pathways of NAD⁺ biosynthesis between organisms, such as between bacteria and humans, this area of metabolism is a promising area for the development of new antibiotics. For example, the enzyme nicotinamidase, which converts nicotinamide to nicotinic acid, is a target for drug design, as this enzyme is absent in humans but present in yeast and bacteria.

History

Arthur Harden, co-discoverer of NAD.

The coenzyme NAD⁺ was first discovered by the British biochemists Arthur Harden and William John Young in 1906. They noticed that adding boiled and filtered yeast extract greatly accelerated alcoholic fermentation in unboiled yeast extracts. They called the unidentified factor responsible for this effect a coferment. Through a long and difficult purification from yeast extracts, this heat-stable factor was identified as a nucleotide sugar phosphate by Hans von Euler-Chelpin. In 1936, the German scientist Otto Heinrich Warburg showed the function of the nucleotide coenzyme in hydride transfer and identified the nicotinamide portion as the site of redox reactions.

A source of nicotinamide was identified in 1938, when Conrad Elvehjem purified niacin from liver and showed this vitamin contained nicotinic acid and nicotinamide. Then, in 1939, he provided the first strong evidence that niacin was used to synthesize NAD⁺. In the early 1940s, Arthur Kornberg made another important contribution towards understanding NAD⁺ metabolism, by being the first to detect an enzyme in the biosynthetic pathway. Subsequently, in 1949, the American biochemists Morris Friedkin and Albert L. Lehninger proved that NADH linked metabolic pathways such as the citric acid cycle with the synthesis of ATP in oxidative phosphorylation. Finally, in 1959, Jack Preiss and Philip Handler discovered the intermediates and enzymes involved in the biosynthesis of NAD⁺; consequently, de novo synthesis is often called the Preiss-Handler pathway in their honor.

The non-redox roles of NAD(P) are a recent discovery. The first of these functions to be identified was the use of NAD⁺ as the ADP-ribose donor in ADP-ribosylation reactions, observed in the early 1960s. Later studies in the 1980s and 1990s revealed the activities of NAD⁺ and NADP⁺ metabolites in cell signaling – such as the action of cyclic ADP-ribose, which was discovered in 1987. The metabolism of NAD⁺ has remained an area of intense research into the 21st century, with interest being heightened after the discovery of the NAD⁺-dependent protein deacetylases called sirtuins in 2000, by Shinichiro Imai and coworkers at the Massachusetts Institute of Technology.